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Evaluation of Infertility, Ovulation Induction and Assisted Reproduction

ABSTRACT

Infertility is a prevalent condition among men and women and can be emotionally devastating. The cause of a couple’s infertility may be female, male, a combination, or unexplained. This chapter serves to outline the evidence-based evaluation of infertility and treatments including ovulation induction and assisted reproductive technologies.

Reproduction is a mandate of nature.  The continuity of species demands promulgation.  However, as humans have attempted to control nature, reproduction has evolved into an option.  But, preventing conception is quite often easier than reproduction on demand.  Indeed, 12 months of intercourse without contraception and without conception is defined as infertility.  Although infertility has plagued humans throughout history, its face has changed throughout by economics of large families, women’s role in the workplace, smaller families born later in life and effective contraception.  Advanced technology has aided the diagnosis of infertility and revolutionized the treatment.  This chapter reviews the history, diagnosis and therapy of infertility. For complete coverage of this and all related areas of Endocrinology, please visit our FREE on-line web-textbook, www.endotext.org.

HISTORY

Sara was perhaps the earliest documented infertile woman.  At age 90, after many years of marriage to Abraham, Sara’s first born was conceived only after God’s intervention (The Bible, Genesis 17:17-21:2).  Fertility gods pepper hieroglyphic inscriptions on Egyptian temples.  The ancient Egyptian god Seth was god of thunder and storms as well as the desert.  Though married to Nephthys, Seth never fathered children, hence his association with the barren desert and infertility.  In ancient Rome, infertility was an acceptable reason for a man to request divorce.  Even English royalty was pressured to bear their kings’ heirs to the thrones, pressure so daunting that it often resulted in pseudocyesis (1,2).

Modern gynecologists wrote about infertility in the mid-20th century as a descriptive state induced by a certain personality type of the woman (3,4).  As more data become known of the physical causes of infertility, the depression and anxiety thought to be a cause of infertility evolved into an effect of the disease.

The next significant advance in the diagnosis of infertility was laparoscopy.  Laparoscopy enabled tubal factor infertility and endometriosis to be diagnosed in women without major abdominal surgery. It would be much later when surgical intervention was actually introduced through the laparoscope, but, nonetheless, diagnosis became more aggressive.

The first therapeutic breakthrough was probably the introduction of clomiphene citrate in the 1960s for ovulation induction in anovulatory women (5).  This was followed by gonadotropin injections.

Most recently, the birth of Louise Brown in July of 1978, the world’s first child conceived after in vitro fertilization, altered the approach to therapy once again. Because of its technologic glamour, IVF was highlighted by the media in the last quarter of the 20th century as the Mecca of infertility therapy and the prime example of interfering with the intimacy of reproduction.  The biologic fidelity of gametes from the married couple resulting in the wife’s gestation and delivery of their child was broken.  Gestational parent, gamete donors, and surrogate mothers became the topic of heated ethical and legal battles.

DEMOGRAPHICS 

In the United States approximately 16.6% of women ages 15-44 are infertile, and 11.9% (7.3 million) women have received fertility services (6).  Infertility is not considered a disease by most third party payers, and medical diagnosis and treatment usually requires the couple to provide payment. Because infertility is not a life-threatening disease, choice of therapy often rests with the couple, which is frequently a choice of cost-consideration rather than cost-effectiveness.  Couples frequently request therapy which is the least costly even though the efficacy may be low. The most important role of the physician in these cases is education and barring therapy whose risk-benefit ratio is too low for safety.

Thus, the evaluation and therapy of infertile couples is often dictated by the amount of funds available.  This review, however, discusses the disease in an academic manner without emphasis on practicality.  Studies available to evaluate the science rather than the art of medicine will be emphasized in order to provide readers with factual information, but this should not be interpreted as minimizing the practical and sensitive side so important in the clinical practice of medicine.  Rather, armed with as much information as possible, each physician may be best able to tailor the evolution and therapy to the needs and resources of the individual.

EVALUATION

The evaluation of infertility assesses each component of reproductive physiology to identify an abnormality: the cervix, the uterus, the endometrium, the semen, the ovarian function, the fallopian tubes and the peritoneum.  Of course, each evaluation begins with a history and physical.  Often, historical evidence directs and streamlines subsequent laboratory evaluation, frequently eschewing other tests before beginning therapy.  It follows, that each abnormality found should be treated to maximize fertility.

AGE EFFECTS

Infertility increases with age.  The effect of the female’s age is more dramatic and earlier than that of the male, but an age effect is seen with advancing male age also.

Figure 1:Graph of Births over Time as a Function of Maternal Age (7)

Figure 1:Graph of Births over Time as a Function of Maternal Age (7)

Fertility peaks when the woman is in her late teens and early twenties. It begins to decline at age thirty and drops more rapidly after age 35 years (8). It plummets after age 40 and pregnancy after age 45 is rare (7,9).  This phenomenon is also reflected in the live birth rate and has been remarkably stable over time and geography as seen in figure 1.  Miscarriages are also more frequent as maternal age rises (10).  Although one may suggest that this is because of the well-known increase in aneuploid conceptions with maternal age (11), euploid pregnancies are also lost with higher frequency as the mother ages.

The effect of maternal age on fertility is theoretically an effect of a larger proportion of abnormal embryos with increasing maternal age.  Data from in vitro fertilization in which normal appearing embryos were examined with fluorescent in situ hybridization (FISH), revealed 39% abnormal embryos from women who are greater or equal to 40 years of age compared to 5% from women who are 20 to 34 years of age (12). A more recent study utilizing comprehensive chromosomal screening through comparative genomic hybridization (CGH) illustrated a 51.3% aneuploidy rate in embryos from infertile women aged 30 – 43 years. Of these women, those with age 40 and older produced an average of 57% aneuploid embryos (13).

Frequently unaddressed, is the effect of the male’s age.  Sperm count does drop with age, although the individual variation is wide as is the male’s sexual function. The decreasing fertility of the male roughly lags behind that of the female by 10 years.  Significant infertility is seen after age 55 years, although this data is difficult to come by, because of the more impressive effect of maternal age.  In order to gain firm figures, men at various ages older than 45, married to women younger than 30 would have to be surveyed for intercourse frequency and conception.  The only data available are those from the Mormon genealogy registers (see Figure 2)(14).

Figure 2:Data from Mormon Registries: Fertility as a Function of Maternal and Paternal Age (13)

Figure 2:Data from Mormon Registries: Fertility as a Function of Maternal and Paternal Age (13)

Thus, men older than 55 married to women older than 35 may have a synergistic age effect on their fertility, but one which is difficult to quantify.

In vitro fertilization outcomes echo earlier trends between female age and pregnancy. While increasing female age leads to a decrease in live birth when using autologous oocytes, donor-oocyte-IVF cycle outcomes appear unaffected by increasing recipient age (Figure 3)(15).

Figure 3: Live birth rates among IVF cycles performed in the United States in 2013 (15).

Figure 3: Live birth rates among IVF cycles performed in the United States in 2013 (15).

CERVICAL FACTOR

The nulliparous cervix is approximately 4 cm in length.  Its columnar epithelium is pierced by the ducts of mucous secreting glands which spew forth their contents as a protective barrier preventing bacteria from entering the upper reproductive tract and as a welcome channel to lead sperm into the upper tract. Cervical mucus is comprised of hyaluronic acid micelles which are affected by the hormonal milieu exposed to the glands.  Late in the follicular phase, as estrogen rises, the micelles align in a parallel arrangement forming channels to guide the sperm.  Under the microscope this can be seen as the classic "fern" pattern of dried cervical mucus (Figure 4).  The pH is alkaline and nourishing to the sperm. Indeed, sperm can live in normal cervical mucus for as long as 4 days.  The abundant mucus frequent oozes from the cervical os into the vagina to lure the sperm from the ejaculate, while protecting them from the acidity of the vagina.  At mid-cycle, just prior to and after ovulation the rising progesterone increases the salt content of the mucus, breaking the micelle channels and thickening the consistency of the mucus.  The "fern" pattern is no longer seen; the mucus thickens and becomes hostile to sperm and bacteria alike.

Figure 3:The classic fern pattern of dried cervical mucous.

History

Women with a history of cervical infections, surgery or cryotherapy may have damage to the cervical glands and lack mucus.  This may result in the inability of the sperm to survive the harsh vaginal acidity and not make the assent to the uterus.  However, a considerable amount of cervix must be removed or damaged for a true "cervical" factor to cause infertility.

Infections rarely result in infertility.  Although gonorrhea and chlamydia have often been accused, it is difficult to prove that either of these actually destroys cervical glands and are more likely to result in tubal factor infertility (16).  Acute infection may alter cervical pH, killing sperm, although this is not well documented.

Cervical conization which removes the cervical glands is the most likely cause of cervical factor infertility.  Cryotherapy and laser vaporization may destroy the lower canal, but glands above the point of metaplasia usually provide enough mucus to retain fertility (17).

Physical Examination

The normal squamocolumar junction with clear cervical mucus almost always rules out a cervical factor.  When the cervix is scarred with a narrow external os and almost flush with the vagina, the cervical glands are frequently absent.

Diagnosis

This diagnosis of cervical factor is based heavily of the history of cervical damage by surgery or infection.  Nonetheless, the classic diagnosis of cervical factor infertility has been the post coital test (aka Sims Hauser test). The postcoital test was proposed to determine the adequacy of sperm and the receptivity of cervical mucus, however it has been the subject of debate over the last 10 years.  In a randomized 24 month study female patients with abnormal post-coital tests were compared to those with normal tests, and importantly there was no difference in pregnancy rates between the two groups (18).  In a literature review assessing use of the post coital test, the sensitivity of the test ranged from 0.09 to 0.71, specificity from 0.62 to 1.00, predictive value of abnormal from 0.56 to 1.00, and predictive value of normal from 0.25 to 0.75 (19). In light of problems of poor validity, lack of standard methodology, lack of a uniform definition of normal, and unknown reproducibility, the postcoital test in longer considered of significant value in infertility assessments. Without a history of cervical damage by infection or surgery, the diagnosis of cervical factor is largely circumstantial.

Treatment

A true cervical factor is best treated with intrauterine insemination as described below.  Briefly, washed sperm is injected into the uterus at the time of ovulation.  Because the sperm survive a limited time, it is probably best to time ovulation with urinary LH to narrow the insemination-ovulation interval.

In addition, prior cervical surgery may predispose to an incompetent cervix during pregnancy (20).  Thus, the patient should be closely monitored for painless dilation of the cervix during pregnancy and evaluated for cervical cerclage.

ENDOMETRIAL FACTOR

A hostile endometrial environment will impede embryonic implantation as shown by the placement of an intrauterine device into the endometrial cavity for contraception.  This hostile effect is postulated to be reproduced by endometrial polyps and submucous fibroids.   In addition, hormonal, immune and biochemical factors have been postulated to result in a hostile endometrial environment.

Poor progesterone effect resulting in delayed maturation of the endometrial lining, commonly referred to as the luteal phase defect, has been considered as hostile to implantation.  Indeed, luteal phase defect has been demonstrated in a case of trisomy 16, suggesting that the defect is a secondary phenomenon of an primary defect, namely, genetic abnormality of an oocytes and its follicular apparatus resulting in abnormally low hormonal stimulation and finally, poor endometrial maturation (21).  Teleologically, this would prevent pregnancy with abnormal embryos.  Theoretically, this mechanism may go astray during genetically normal cycles and result in failed implantation, but has never been proved to be a cause of infertility, per se.

Other factors, which have been implicated in the development of a hostile environment for implantation, are autoimmune factors such as lupus anticoagulant and Integrin IIIβ (22).  It has been shown that antiphospholipid antibodies are not a factor in implantation, but the concentrations of Integrin III β are yet to be determined (23).

History

Patients with endometrial polyps and submucous fibroids may have premenstrual spotting or heavy menstrual bleeding.  A luteal phase defect is not detected in any medical history.

Physical Examination

Endometrial factors cannot be diagnosed by physical examination.

Diagnosis

Endometrial polyps and submucous fibroids are detected by either hysterosalpingography, a saline sonohysterogram, or hysteroscopy (Figure 5).  A dilation and curettage (D&C) may be performed and histology of the curettings will confirm the diagnosis.  Submucous fibroids may be felt as an irregularity while curetting the endometrial cavity.

Figure 5 :Hysteroscopic view of an endometrial polyp.

Abnormal autoimmune factors do not cause infertility and need not be diagnosed.  Integrin IIIβ is still a research tool and diagnosis of an abnormal concentration should be left to research protocols.

The luteal phase defect is not a documented cause of infertility (24-26). The high prevalence of out-of-phase endometrial biopsies in fertile women makes histological dating of the endometrium of no use in the routine evaluation of infertility (26). Because its role in spontaneous abortion is not known, some physicians prefer to evaluate the endometrium in infertile patients.  The diagnosis is made by performing an endometrial biopsy in the luteal phase.  A histological dating lagging more than 2 days behind the actual postovulatory date diagnoses the defect.

Treatment

Endometrial polyps may be removed by operative hysteroscopy or uterine curettage.  Submucous fibroids may be removed through hysteroscopic surgery.  Depending upon the depth of myometrial invasion, laparoscopy or laparotomy may be necessary.  Pritts et al performed a meta analysis of women undergoing hysteroscopic myomectomy and found a modest improvement in pregnancy outcome (relative risk of 1.72 with a confidence interval of 1.13-2.58)(27).

As mentioned, other causes of endometrial hostility have not been proven to be related to infertility and, thus, treatment is not warranted. 

UTERINE FACTOR

In addition to the endometrium and cervix, the shape and competency of the uterine fundus must be considered.  Anomalies of mullerian fusion and fibroids have been suggested to result in the inability of implantation or growth of the pregnancy.  It is difficult to postulate how abnormalities of the uterus can cause infertility except through abnormal blood flow leading to poor implantation and a subsequent abnormality involving placentation.  Indeed, evidence that does exist relates infertility to large fibroids suggesting tubal factor infertility.  Mullerian fusion abnormalities resulting in a uterine septum or bicornuate uterus are largely associated with recurrent miscarriage rather than the ability to conceive.

History

Historical clues of an abnormal uterus are rare.  Occasionally, patients with incomplete mullerian fusion may have dysmenorrhea, but the symptom itself is so nonspecific, that the physician is not likely to alter either diagnosis or therapeutic course.

Physical Examination

A bimanual pelvic examination will reveal large fibroids.  The bicornuate uterus is typically unable to be palpated.  A compete uterus didelphys will be revealed by a vaginal and cervical examination.

 Diagnosis

Uterine fibroids may be diagnosed by ultrasound (Figure 6).  Their imposition on the endometrial cavity may be illustrated by hysterosalpingogram and confirmed by hysteroscopy.  Ultrasound is less valuable in diagnosing mullerian anomalies, but hysterosalpingogram will identify the abnormal uterus (Figures 7,8,9) .  MRI is often helpful in identifying both fibroids and the type of mullerian anomaly found on hysterosalpingogram (Figure 10).

Figure 6: Transverse sonographic image of the uterus demonstrating overall enlargement and multiple leiomyomata.

Figure 7: Hysterosalpingogram of a normal uterus. Note the normal appearing intrauterine cavity with bilateral tubal spillage of contrast

Figure 8: Hysterosalpingogram of a T-shaped uterus secondary to in utero DES exposure.

Figure 9: Hysterosalpingogram of a uterine anomaly. With HSG, a septum versus a bicornuate uterus cannot be distinguished..

Figure 10: Transverse MRI image demonstrating multiple leiomyomata..

The septate uterus can be differentiated from the bicornuate uterus by laparoscopy and MRI.  Both laparoscopy and hysteroscopy will identify fibroids and their location in the uterus and pelvis.  If fibroids interfere with tubal pick-up of oocytes, mechanical infertility will result.  Similarly, fibroids impinging on the endometrial cavity may interfere with implantation and placentation.

Treatment

Uterine fibroids may be treated by myomectomy.  Depending on the location of the fibroid, hysteroscopic or laparoscopic removal may be possible.  Otherwise laparotomy is necessary. Treatment of the fibroid with a GnRH agonist will shrink the fibroid, but as soon as the agonist is discontinued and estrogen activity resumes, the fibroid will return to at least its original size (28).

Myomectomy is indicated when the myomas result in bleeding severe enough to cause anemia, when pressure is placed on the on the bladder to cause urinary tract infections, or when pressure on the bowel results in constipation.  It is less clear whether myomectomy improves fertility and, in fact, may even result in pelvic adhesions and lead to tubal factor infertility (29).  Myomas which impinge or are present within the endometrial cavity may likely cause spontaneous abortion, but evidence proving this causation is difficult to find.  Even harder to prove is the efficacy of myomectomy resulting in an increase in term pregnancy.

TUBAL FACTOR

The mechanical ability of the sperm to be conducted to the site of the oocyte or the fertilized oocyte to be propelled back into the uterus, demands patent fallopian tubes.  Clearly, the tube must be open to allow fertility.  Also important is the ability of the tube to freely move across the surface of the ovary in order to sweep the oocyte into the tube.  If the tube is not able to pick up the oocyte from the surface of the ovary, no pregnancy will result.

History

Patients with tubal factor infertility often have a history of a pelvic infection, endometriosis, or previous abdominal or pelvic surgery.  Frequently, however, patients are unable to clearly identify a source of their adhesions. For example, a patient who experienced a chlamydial infection may have attributed the lower abdominal pain, fever and cramping to a gastrointestinal viral infection and often cannot recollect the time of infection.

Adhesions may be asymptomatic or may result in pelvic pain.  Normal movement of the bowel and ovaries across the visceral surfaces of the abdomen may be impeded by pelvic adhesions and results in these organs pulling on the abdominal wall. This may result in both pelvic and abdominal pain.

Physical Examination

Tubal disease significant enough to result in hydrosalpinges may result in adnexal masses.  Similarly adhesions, which adhere the ovaries to uterus, may often be interpreted as a uterus with posterior fibroids on physical examination.

Diagnosis

Tubal adhesions or hydrosalpinges are suggested when contrast pools are seen on a  hysterosalpingogram (Figure 11), but definitive diagnosis requires visualization at laparoscopy. Tubal occlusion may be diagnosed by hysterosalpingography or by laparoscopy.  Most recently, fluid extravasation into the pelvis after flushing of an intrauterine cannula may be seen by an experienced ultrasonographer (30).  However, this test is less reliable than the former and is dependent on the skill of the sonographer, patient’s factors such as weight and air in the bowel and resolution capabilities of the ultrasound machine.

Figure 11: Hysterosalpingogram demonstrating hydrosalpinges. Note the marked dilation of the tubes and the pooling of contrast..

Treatment

In vitro fertilization results in the highest chances of pregnancy for the couple whose infertility is the result of pelvic adhesions or blocked Fallopian tubes.  Surgical correction may increase the chances of fertility, however, unless the adhesions are filmy and their extent is limited, surgical lysis incurs a high likelihood of adhesion recurrence.

ENDOMETRIOSIS

Endometriosis is the abnormal development of endometrial glands and stroma outside of the uterus.  It is associated with dysmenorrhea, dyspareunia, and infertility.  The American Society of Reproductive Medicine (ASRM) developed a revised staging scheme in order to standardize communication between physicians regarding their patients, between investigators for research protocols and to follow effects of therapy (31).

The mechanism of action whereby endometriosis causes infertility is complex.  Severe endometriosis with adhesions and adherent pelvic organs results in mechanical infertility.  It is difficult to assign a mechanism to minimal and moderate endometriosis.  Increased peritoneal fluid, increase peritoneal prostaglandin concentration, and interference with normal ovarian folliculogenesis have all been postulated (32). However, no direct evidence exists between mild and minimal endometriosis and resultant infertility (33).  Indeed, some have postulated endometriosis is a result rather than a cause of infertility.

History

Patients with endometriosis experience a spectrum of symptoms ranging from incapacitating dysmenorrhea and severe dyspareunia to none at all.  Although 30% of infertile patients have endometriosis (34), it is unknown how many patients who have endometriosis are infertile.  Classically, patients have dysmenorrhea which begins during the menses and over the years extends to the prior luteal phase.

Cyclical hemoptysis and hematochezia may occur in patients with endometriosis implants in the lung; however, very few of patients with endometriosis have these remote implants.

Physical Examination

Endometriosis may be occasionally palpated on pelvic exam if there are implants on the uterosacral ligaments or the posterior surface of the uterus; however, physical examination is usually not helpful in the diagnosis of endometriosis.

Diagnosis

Endometriosis is diagnosed by visual inspection at the time of laparoscopy or laparotomy or if endometriomas are visualized on pelvic ultrasound.  Endometrial implants over the pelvic organs may be biopsied for histological confirmation.  Endometrial glands and stroma are seen microscopically.  The American Society of Reproductive Medicine recommends using a standard staging system to document the severity of each patient’s disease.  The system incorporates superficial and deep lesions, adhesions, and the location and size of each lesion. Standardized staging allows communication between physicians and evaluation of treatment efficacy.

Treatment

Endometriosis causing pain may be treated with surgical or medical therapy.  Surgical extirpation of lesions and adhesions is successful in alleviating pain for endometriosis but is less effective in increasing fertility caused by endometriosis (35). Preventing menstruation with the use of continuous oral contraceptive or continuous progestin therapy may also relieve pain.  Medical menopause induced by GNRH analogues has also been effective in reducing the implants of endometriosis.  However, once normal cycles and hormones resume, the implants return.  Medical therapy does not have much effect on the adhesions associated with endometriosis (36,37).  Similarly, endometriomas of the ovary are decreased in size by medical therapy but are rarely completely treated.  In vitro fertilization is more effective than either surgery or medicine in increasing fertility caused by endometriosis (32).

OVULATORY FUNCTION

Pregnancy is direct evidence of ovulation.  Patients who do not ovulate cannot conceive without assisted reproductive technology.  Ovulatory function requires the integration of many normally functioning systems.  Normal thyroid function, normal insulin action, normal adrenal function and perhaps normal cerebral function are all required for ovulation.  When these systems are disrupted, follicular function is altered and ovulation becomes disordered.  This results in a series of endocrine events, which leads to elevated estrone production, elevated androgen production and altered insulin action.  These events, in turn, prevent ovulation.  This cycle of disordered events has become known as polycystic ovarian syndrome.  This syndrome is discussed in detail in Chapter 6.

History

Normal, regular menstrual cycles usually reflect ovulatory cycles.  Indeed, 95% of regular menstrual cycles are ovulatory (38).  If cycles are irregular, the patient is most likely not ovulating, obviously a cause of infertility.  Patients who do not ovulate and have polycystic ovarian disease may also have other associated findings of the disorder including insulin resistance, androgen excess, acanthosis nigricans and obesity.

Physical Examination

Anovulation alone is not associated with any physical findings.  Patients will have abundant, thin cervical mucus most of the time.  This is the result of continuous, unopposed estrogen.  In those whose have elevated androgens, hirsutism may be present.  Acanthosis nigricans may also be found at the neckline, in the axilla, and underneath the breasts.

Diagnosis

Anovulation is diagnosed by a serum progesterone measurement less than 4 ng/ml drawn at least 4 days prior to a menstrual bleed.  Proliferative endometrium on an endometrial biopsy performed during the week prior to a menstrual cycle is also diagnostic of anovulation.  Finally, a basal body temperature chart revealing no biphasic increase in temperature is diagnostic of anovulation in 80% of cases; 20% of women with a monophasic basal body temperature will actually ovulate (39,40).

Treatment

Ovulation may be stimulated by administering clomiphene citrate, letrozole, or gonadotropins. Clomiphene citrate is a serum estrogen receptor modulator, which increases pituitary secretion of LH and FSH.  When given to anovulatory women, the gonadotropins stimulate follicular development.  The follicle continues to develop normally and feeds back to signal the pituitary that the oocyte is mature and an LH surge is needed.  The so-called "ovarian clock" is restored and ovulation may be timed by monitoring urinary LH concentrations.  Clomiphene successfully induces ovulation in 85% of women, although pregnancy rates are lower than 65% (41,42).  When clomiphene is unsuccessful, gonadotropins may directly stimulate the ovarian follicle to develop and chorionic gonadotropin may be administered to simulate the LH surge and result in ovulation.

Patients with polycystic ovarian disease have often been treated with insulin sensitizing agents, particularly metformin (43,44).  Most recently however, a multicenter randomized placebo-controlled trial failed to show any superiority of metformin over clomid, and combining clomid with metformin was no better than clomid alone (45) Metformin use in PCOS should be restricted to women with glucose intolerance and the routine use of this drug in ovulation induction is not recommended (46,47).

Letrozole, an aromatase inhibitor, has also been used to induce ovulation in anovulatory women. The medication decreases peripheral aromatization of testosterone to estrogen leading to an increase in FSH secretion from the pituitary gland and resultant follicular maturation (48). The Reproductive Medicine Network performed a double-blind, multi-center study comparing pregnancy outcomes in women with PCOS using either clomiphene citrate or letrozole. Patients using letrozole had a higher cumulative live birth rate than those who used clomiphene citrate (27.5% vs. 19.1%, P=0.007) and a lower multiple pregnancy rate (P=0.03) (49).

Gonadotropins, FSH or a combination of FSH and LH can also be used to induce ovulation in anovulatory women. These medications are generally injected subcutaneously and lead to follicular growth. Recruitment of more than one dominant follicle is not uncommon during a gonadotropin stimulation, so patients are followed closely with ultrasound monitoring and serum estradiol levels. The treatment modality is associated with an increased risk of high-order-multiple pregnancy when compared to natural conception, clomiphene citrate, and letrozole.

CHRONIC DISEASE

History

A thorough menstrual history should be done with all infertile patients to detect chronic diseases such as severe anemia, autoimmune diseases, liver disease and renal disease.  Debilitating disease and chronic malnutrition may also result in infertility (50).  Chronic disease most likely interferes with ovulation by central mechanisms.  Treatment of the disease rarely restores ovulation because such treatment is usually palliative.

Physical Examination

The exam findings may be subtle, and include clubbing of nailbeds, poor skin turgor, subcutaneous adiposity, conjunctival pallor, thinning of hair, jaundice, cushingoid features, recent weight loss, poor skin turgor, delayed capillary refill, gum bleeds, and cheilosis, Fatigue, exercise intolerance, dyspnea on exertion, peripheral edema, cardiac murmurs are consistent with underlying cardiac disease. Splenomegaly, dilated abdominal wall collateral vasculature, and ascites or signs of anemia may represent longstanding gastrointestinal disease.  Pulmonary disease is manifested by a chronic cough, diminished breath sounds, wheezing, dyspnea or simply fatigue.

Diagnosis

In most instances, the diagnosis will already be established.  Physical exam findings may indicate the patient’s overall health, and nonspecific studies may be helpful such as: CBC with peripheral smear, urine dipstick for proteinuria, hematuria, pyuria and microscopic evaluation (epithelial cells, WBC casts, etc.), liver functions, and serum chemistries to include electrolytes, creatinine, BUN, magnesium, phosphate, uric acid, and protein.  A sedimentation rate is a nonspecific test of inflammation, and cardiac status may be initially assessed with an ECG, CXR, echocardiography, and stress test.   Clearly, the diagnostic tests must be tailored to the individual.

Treatment

Treatment of infertility associated with chronic disease requires addressing the underlying health problem and optimizing the patient’s present state of health. This usually requires a multi-specialty effort, specific to the patient’s health needs.  Special consideration should be taken to assess if a relative or absolute contraindication to pregnancy exists, as the pregnancy may well exacerbate an already tenuous medical condition.  Maternal surrogacy is an option in patients who are unable to tolerate the physical stresses of pregnancy, such as patients with severe hypertension, significant structural cardiac anomalies, advanced multiple sclerosis, to name a few.  If premature ovarian failure has occurred, either secondary to chronic disease, or to treatments such as chemotherapy or radiotherapy, donated oocytes are an option.

MALE FACTOR

Extensive discussion of male factor infertility is within Section: Endocrinology of the Male edited by Robert MacLachlan,M.D (www.endotext.org).  A recent study performed by the Reproductive Medicine Network correlated semen parameters to fertility.  The "new norms" by which fertility can now be defined are: a sperm density greater than 15 million/ml, motility greater than 32%, and a strict morphology greater than 4% (51).  Abnormal semen parameters have a host of causes resulting from an absent vas deferens secondary to cystic fibrosis heterozygosity to a varicocele.  Nonetheless, when the semen parameters are low, the number of competent sperm available to fertilize an oocyte is decreased and the monthly fecundity is lowered. It is more difficult to explain why male factor infertility occurs in the presence of normal semen parameters.  Some have suggested that abnormal capacitation, decreased motility, abnormal morphology, chemical composition, or antibody presence compromise fertilization.  These factors remain unproved.

History

Male factor infertility usually presents when the gynecologist orders a semen analysis in the initial infertility evaluation.  Rarely does it present as symptoms in the male. The male partner may have a history of undescended testicles or testicular pain due to a varicocele, but this is usually elicited after the semen analysis is abnormal.

Physical Examination

Males with a varicocele will often have a palpable vessel in the scrotum.  Otherwise no physical findings are abnormal in most infertile males.

Diagnosis

The diagnosis of male factor infertility begins with a semen analysis.  Abnormal parameters include: sperm concentration of less than 15 x 10(6) per milliliter, less than 32% of sperm motility, with less than 4% with normal morphologic features (51). Tests such as the sperm penetration assay, antisperm antibodies, free oxygen radicals have been implicated as resulting in infertility, but the documentation is unconfirmed.  Sonographic evaluation of the scrotum will confirm the presence of a varicocele.

Treatment

If the infertility is due to a mechanical issue, such as retrograde ejaculation, either pharamacologic or surgical correction of the bladder neck is feasible. In patients with neurological damage from spinal cord or pelvic trauma, multiple sclerosis, or retroperitoneal surgery, rectal electroejaculation or surgical sperm recovery may be utilized.  If the male partner is azoospermic due to hypogonadotropic hypogonadism, the patient is treated with gonadotropin therapy.  Obstructive azoospermia  may be corrected surgically or sperm recovery techniques such as TESE (testicular sperm extraction)(Figure 12) or MESA (microsurgical epididymal sperm aspiration)(Figure13) may be used. Since the wide acceptance of ICSI (intracytoplasmic sperm injection)(Figure14), an oocyte is mechanically  fertilized  with a single sperm, a method which ameliorates all but the most severe male factor infertility. Clomiphene citrate is no better than placebo for increasing a low sperm count.  Recently, sperm motility has been treated with carnitine supplements, but randomized trails are pending.  (For a full discussion on male factor infertility, see Endocrinology of the Male edited by Robert McLachlan, M.D.at www.endotext.org).

Figure 12: Testicular sperm extraction (TESE)

Figure 13: Microsurgical epididymal sperm aspiration (MESA)

Figure 14: Intracytoplasmic sperm injection (ICSI)

UNEXPLAINED INFERTILITY

Approximately 15% of patients with infertility have an unidentifiable cause.  Thus, unexplained infertility is a failure of both the couple to conceive and the physician to explain why.  Perhaps even more psychologically difficult to accept than sterility, unexplained infertility poses impressive stress to the couple.  Despite doing all they can correctly, the couple is unable to conceive and has no answer to their problem even after enduring expensive, painful testing.  Their only recourse is empiric therapy.

History

Couples with unexplained infertility have routine intercourse, have regular menstrual cycles and normal infertility testing results.

Physical Examination

Inherent to the diagnosis, physical examination reveals no abnormalities.

Diagnosis

The diagnosis of unexplained infertility is made in couples who have intercourse regularly or well-timed intercourse and fail to conceive despite regular ovulatory cycles, a normal semen analysis, normal laparoscopy, and normal hysterosalpingography.

Treatment

Couples with unexplained infertility may conceive without therapy but it may take from 3 to 7 years (52).  To decrease the waiting time, empiric therapy is instituted.  Empiric therapy usually begins with ovulation induction with oral agents or gonadotropins combined with intrauterine insemination.  This combined therapy increases the cyclic fecundity over either alone in couples with unexplained infertility (53, 54). After three or four unsuccessful cycles or alternatively, in vitro fertilization may be offered.

ASSISTED REPRODUCTIVE TECHNOLOGY

The birth of Louise Brown in 1978, the world’s first "test tube baby", revolutionized therapy for infertile patients.  No longer were women without fallopian tubes unable to conceive and gestate.  For the first time, medicine was able to view the first few human embryonic divisions and reproductive medical scientists learned better how to induce ovulation and handle gametes.  Numerous "spin-off" technologies arose in the subsequent two decades:  controlled ovarian hyperstimulation (COH-IUI), intracytoplasmic sperm injection (ICSI) and oocyte donation allowed pregnancy in couples without gametes.  This section briefly describes the reproductive technologies available to infertile patients and referred to in treatment sections above in the chronological order of their introduction into the therapeutic armamentarium.

In Vitro Fertilization (IVF)

In Vitro Fertilization (IVF) was first successfully performed by Patrick Steptoe, MD and Robert Edwards, PhD in a woman with tubal factor infertility.  The woman timed her ovulation and underwent laparotomy for oocyte retrieval.  In the ensuing years, spontaneous cycles were replaced by first clomiphene citrate ovulation induction and then stimulation with gonadotropins.  Ovulation stimulation allowed the retrieval of more than one mature oocyte, thus increasing the statistical chance of embryonic implantation during one cycle.  The early pregnancy rates were approximately 8% per cycle and cancellation prior to oocyte retrieval was high.  The introduction of GnRH analog allowed down regulation prior to ovarian stimulation and thwarted the numbers of cycles cancelled due to spontaneous premature LH surges. GnRH analog therapy was most likely the reason that IVF pregnancy rates jumped to about 15% per cycle in the late 1980s.

By the time IVF in the United States was introduced by the Jones in 1983, oocytes retrieval was primarily by laparoscopy. In 1983, Gleicher et al reported the first transvaginal ultrasound-directed oocyte aspiration, and currently, most aspirations are performed in this manner (55).

Indications

Initially, IVF was indicated in patients with tubal factor infertility.  The relatively high success rates have allowed extension to couples with endometriosis, drug-resistant polycystic ovarian disease, and unexplained infertility.  Additionally, IVF can be used with donor oocytes to treat women with age-related ovarian dysfunction, ovarian failure or surgically removed ovaries.  Combined with ICSI (see below) IVF can also be used to treat couples with sperm disorders and immunologic infertility.

Procedure:

IVF begins with ovulation induction. Although it may be performed in natural cycles, removal of the oocytes requires intensive hormonal monitoring and the availably of the medical team too often to be practical.  In addition, pregnancy rates are lower with natural cycle IVF.

A variety of ovulation induction regimens are available. Many programs in the United States administer ovarian down regulation with a GnRH agonist in the luteal phase prior to the cycle of stimulation.  Oral contraceptives are often added prior to down-regulation.  After spontaneous ovarian activity is suppressed, folliculogenesis is stimulated with gonadotropins.  The regimen used is particular to each program, but those that have been tested in clinical trials all seem to result in similar pregnancy and delivery rates.  Choices are made based on cost, availability, route of administration, and familiarity of the physician with the regimen.

GnRH antagonists are also used to thwart spontaneous LH surges during ovulation induction.  Thus far, they have not proven superior to the GnRH agonists in pregnancy outcome and are more expensive on a mg per mg basis (56) but offer the advantage of fewer total injections to the patient. Another proposed advantage of GnRH antagonist cycles is a decreased risk of moderate and severe Ovarian Hyperstimulation Syndrome (OHSS) when combining this protocol with a GnRH-agonist induction of final oocyte maturation (57-59).

During ovulation induction, the ovaries are monitored for follicular growth by frequent transvaginal ultrasound examinations and serum estradiol concentrations.  When clinical parameters suggest the presence of mature oocytes, human chorionic gonadotropin (hCG) is administered to mimic the LH surge and allow further progression of the oocytes through meiosis.  Some IVF programs now use a GnRH agonist to trigger ovulation, as it appears that the incidence of moderate and severe ovarian hyperstimulation syndrome (OHSS) is reduced in high-risk patients when an agonist is used. Approximately 35 hours later, the patient undergoes a follicular aspiration.  In the United States, most aspirations are performed transvaginally with ultrasound direction.  Conscious sedation is typically used.  Regional analgesia is not usually employed because the concentration of the local anesthesia in the follicular fluid has been shown to decrease the pH and affect the fertilization of the oocyte (60). Concomitant identification of the oocyte by nearby laboratory technicians informs the physician to proceed with aspiration of each sequential follicle (Figures 15,16).

Figure 15: Human oocyte immediately following retrieval.

Figure 16: Human oocyte after the surrounding cumulus cells have been removed.

Between 4 and 6 hours after aspiration, the oocytes are mixed with 15,000-30,000 motile, previously prepared sperm.  Human fertilization occurs in the next 18 hours and the first two cell divisions usually occur in the subsequent 24 hours (61).  Embryos are then transferred into the uterine cavity typically three or five days later.  The American Society of Reproductive Medicine (ASRM) recommends determining the number of embryos to be transferred by patient criteria, with favorable prognosis patients generally receiving no more than 1-2 embryos, and poor prognosis patients of advanced maternal age receiving no more than 5 embryos (ASRM Practice Committee Report: Guidelines on the Number of Embryos to Transfer, revised September, 2012).  These guidelines were proposed to decrease the incidence of multiple pregnancies, yet still facilitate a favorable pregnancy rate.

The embryos are transferred through the vagina into the uterus.  Approximately 2 weeks later a pregnancy test is performed.  Because aspiration removes granulosa cells which would otherwise produce the progesterone necessary for endometrial development, many programs support the luteal phase with exogenous progesterone supplementation.  This has never been shown to be necessary or effective, but is of low risk (62).  If given, supplementation is continued for approximately 4 weeks after the positive pregnancy test.

Contraindications: 

IVF is contraindicated in women in whom pregnancy is contraindicated.

Outcome:

Federal law requires that the outcome of all IVF cycles be reported to the national database kept by the CDC and ASRM/SART (www.cdc.gov/nccdphp/drh/art99/index99.htmwebsite and www.sart.org).

For the 87,089 IVF cycles performed nationally in 2013, there was a 40.1% live birth rate per initiated cycle for women younger than 35 years, 31.4% for women between ages 35 and 37, 21.2% for women between 38 and 40, and 11.2% for women over 40 years (SART www.sart.org).

Gamete Intrafallopian Transfer (GIFT)

Gamete Intrafallopian Transfer (GIFT) is a procedure developed in 1983 by Dr. Ricardo Asch in San Antonio (63). The procedure begins in a therapeutic manner similar to IVF, however, when the oocytes are aspirated, they are mixed with sperm and immediately replaced in the patient"s fallopian tubes.  GIFT requires less laboratory services and expertise than does IVF, but does require the patient to incur the risks of general anesthesia and laparoscopy.  With increasing training and availability of IVF laboratory personnel, GIFT is rarely utilized.

Indications:

GIFT requires at least one normal, patent fallopian tube.  Patients with unexplained infertility are those who benefit most by this procedure. Additionally, because embryos are not formed in the laboratory, some religious groups may find GIFT more acceptable than IVF.

Procedure: 

GIFT begins with ovulation induction in a manner similar as described above for IVF (see IVF).  When the patient is ready for oocyte aspiration, the aspiration may be performed either transvaginally with ultrasound guidance, or transabdominally under laparoscopic guidance.  The oocytes are immediately combined with previously prepared sperm and allowed to incubate for 10 minutes to allow sperm binding to the zona pellucida.  Approximately 25,000 motile sperm and two oocytes are placed in each Fallopian tube through a catheter placed in the tube under laparoscopic vision.  Luteal support with exogenous progesterone may be given. Two weeks after aspiration a pregnancy test is performed.

Contraindications: 

Patients with abnormal Fallopian tubes are not eligible for GIFT treatment.  Any contraindication to pregnancy is a contraindication to GIFT.

Outcome:

Federal law requires that the outcome of all GIFT cycles be reported to the national database kept by the CDC and ASRM/SART (www.cdc.gov/nccdphp/drh/art99/index99.htmwebsite).
Overall, GIFT was performed with less than 1% of the aspirations in the United States in 2011, and because of the small number of cases, clinic outcome data specific for GIFT is unavailable.  In general, the outcome of GIFT is thought to be comparable to that of ovulation induction with gonadotropins and intrauterine insemination.

Controlled Ovarian Hyperstimulation and Intrauterine Insemination (COH-IUI)

Controlled Ovarian Hyperstimulation and Intrauterine Insemination (COH-IUI) was first introduced in 1983 by Dodson et al. as a direct spin-off of GIFT (64).  These clinical investigators reasoned that if the cyclic fecundity rate may be increase by placing processed sperm into the normal fallopian tube with 4 oocytes, perhaps the physical placement can be replaced by the patient’s natural physiology. Their first series of 85 patients were stimulated with gonadotropins to develop 4 mature follicles and the normal fallopian tubes were entrusted with oocyte pick-up.  After administering hCG, processed sperm were delivered into the intrauterine cavity and allowed to be propelled naturally to the site of the oocyte.  Previous studies revealed that this transport occurred within two minutes.  This therapy was applied to patients with unexplained infertility so rapidly that it resulted in a demand on the supply of urinary gonadotropins so large that a backlog of drugs in the United States occurred.

Indications: 

Unexplained infertility was the initial indication for COH-IUI.  This was later extended to include some patients with pelvic adhesions (but patent tubes) and also patients with male factor infertility.

Procedure: 

Beginning early in the menstrual cycle, gonadotropins are administered daily to patients.  After approximately 5 days of therapy, ultrasound folliculograms and serum estradiol measurements are performed every 1 to 2 days to determine the dose and frequency of further gonadotropin administration.  When three to four mature follicles are detected, human chorionic gonadotropin (hCG) is administered to trigger ovulation.  Between 24 and 48 hours after hCG administration, an intrauterine insemination (IUI) is performed.

The sperm is prepared for IUI in a manner similar to that first described for IVF.  After liquification, the ejaculate is diluted with a buffered media and centrifuged.  The sperm is thus separated from the seminal plasma when the supernatant is discarded and the sperm-rich pellet is re-suspended in media.  Processing thereafter varies with the laboratory and sperm characteristics.  The sperm suspension may be layered in a sephadex column (Percoll), recentrifuged with media, or allowed to "swim-up" into an overlying layer of media.  These procedures are intended to remove non-motile sperm, debris, bacteria, and white cells before intrauterine insemination.  They also remove 60-80% of the motile sperm.  No technique has proved superior for pregnancy outcome.

Contraindications:

Blocked fallopian tubes are a contraindication to COH-IUI because the tubes are required to pick up the oocytes from the ovary and facilitate the mechanical apposition of sperm and oocyte.

Outcome:

Pregnancy rates and pregnancy outcome after COH-IUI depend upon diagnosis.  Few randomized studies are available to assess accurate outcomes after this therapy because it is often attempted in patients who would otherwise be treated with IVF.  For example, a patient with pelvic adhesions who cannot afford IVF may opt for a cycle of COH-IUI in hope that more ovulations may increase the chance of an oocyte being geographically close to the fimbrial opening and afford tubal pick-up.  The more ovulated oocytes, the better statistical chance of an oocyte reaching the tubal opening.  Nonetheless, patients such as this would be less likely to conceive that a patient with normal tube-egg pick-up.

Strict criteria for unexplained and male factor infertility were applied in the randomized trial conducted by the Reproductive Medicine Network (65).  This study revealed that patients with truly unexplained infertility had a 33% pregnancy/cycle of COH-IUI, higher than couples treated with either COH or IUI alone, and higher than controls.

The multiple pregnancy rate was 13.4% overall and the spontaneous abortion rate was 19% overall (22.3% in the COH treated groups).  Interestingly, this study treated couples with more than one motile sperm and no other infertility factors.  In those couples with sperm counts less than 0.2-21.8 x 10-6, couples were found to have and higher pregnancy rate when treated with COH-IUI than either COH or IUI alone, and higher than controls.

Not all couples with unexplained infertility are optimal candidates for COH-IUI. Women with an elevated serum AMH or high basal antral follicle count may experience production of multiple dominant follicles, leading to a higher risk of high-order-multiple pregnancy and/or OHSS. These negative outcomes were noted to be more prevalent in the above study with 6 cases of OHSS, 3 sets of quadruplets, and 4 sets of triplets among the 186 total pregnancies in the COH-IUI group (65). Many argue the relatively low reported pregnancy rates in COH-IUI compared with risks do not justify its use after failure of CC-IUI since IVF success rates have improved greatly over the last decade (66).

OOCYTE AND EMBRYO DONATION

Oocytes used in IVF are not required to be from the female partner of the infertile couple.  Indeed, these procedures allowed women to utilize donor gametes as their male counterparts have been doing for over a century.  A fertile woman who agrees to donate her oocytes anonymously or to a known couple, undergoes ovulation induction, and the retrieved oocytes are fertilized.  Of course, the sperm may also be from a sperm donor.  After embryonic development, the embryos are replaced into the female partner of the infertile couple.  She is treated with exogenous hormones to synchronize her cycle with the donor.

Similarly, a couple can use embryos donated from another person or couple to become pregnant. The hormonal preparation of the female partner’s uterus is similar to an oocyte-donation cycle. In the US, each state has a unique set of laws governing use of donated embryos and oocytes, so practitioners must stay updated on changes in reproductive laws in their state. Legal restrictions and variable costs associated with embryo and oocyte donation has led to reproductive “tourism” where couples travel to other states or countries for reproductive treatment.

Indications:    

Oocytes or embryos obtained from a donor is indicated for patients with medical or surgical menopause or genetic disorders that may impact offspring.  Oocyte donors must be physically and mentally healthy and without major genetic diseases in the family, have attained the state’s age of legal majority, and preferably be within 21-34 years of age. The donor must be screened for HIV, hepatitis B surface antigen, hepatitis C antibody, syphilis, chlamydia, and gonorrhea (American Society of Reproductive Medicine Practice Committee Report: Recommendations for gamete and embryo donation: 2012.)

Procedure:   

Both oocyte donors and recipients are carefully selected, counseled and screened before being accepted into the program.  Donors may be known to the patient, a volunteer that altruistically donates to an anonymous recipient (usually reimbursed for missed work and effort), or an individual that is undergoing IVF and donates her spare oocytes.  Stimulation and transfer are performed as with routine IVF.

Contraindications:  

Any contraindication to pregnancy, spontaneous or induced ovulation would preclude either donation of oocytes or use in an IVF cycle.

Outcome:  

For the 8,921 fresh treatment donor-oocyte cycles initiated in 2013, the reported delivery rate was 49.6% per initiated cycle (www.SART.org).  Neither recipient age nor diagnosis plays a substantial role in the success of oocyte donation (67).

INTRACYTOPLASMIC SPERM INJECTION (ICSI)

Advancements in IVF promulgated its extension to couples with male factor infertility since in vitro fertilization required fewer moving sperm than in vivo fertilization, even after IUI.  Furthermore, in the early 1990’s the zona pellucida was incised (zona slitting) or treated with hyaluronic acid (zona drilling) to facilitate sperm transgression across it in cases of male factor infertility. Another technique injected sperm under the zona pellucida (SZI).  Indeed, inadvertent penetration of the cytoplasm and injection of a sperm during a SZI procedure in resulted in a pregnancy and sired the best treatment of male factor infertility since donor sperm:  Intracytoplasmic Sperm Injection (ICSI) (68).

Indications:

ICSI is indicated for male factor infertility in which the count, motility, or strict morphology is low.  The definite parameters will depend upon each program; in general, a sperm density <5 x 106, motility <20% and strict morphology <5% are indications.  In addition, patients with antisperm antibodies may be best treated with ICSI. For 2013, 67% of reported IVF cycles in the United States utilized ICSI as the means for fertilization (www.SART.org).
ICSI also enabled men without sperm in their ejaculate to sire a pregnancy.  Sperm aspirated from the epididymis (MESA) and from the testicle (TESE) may be used for injection.  It is important, however, to realize that men with azoospermia may have a genetic defect which may possibly be inherited and result in infertility in subsequent male offspring.  Similarly, men with at least one vas deferens congenitally absent may be a carrier of cystic fibrosis.  His wife should be screened for cystic fibrosis mutations to assure that she, too, is not a carrier, lest they have a child affected with the disease.

ICSI has been proposed in non-male-factor infertility, but current literature does not support routine use (69). A randomized, multi-center trial showed similar clinical pregnancy rates with conventional insemination and ICSI in non-male-factor patients (33% vs. 26%) and a higher rate of failed fertilization (5%) in the conventional insemination group. Based on these data, the number needed to treat (NNT) to prevent one failed fertilization with ICSI is 33 (70-71).

Procedure:

Patients treated with ICSI undergo the same procedures as with IVF except that 5 hours after aspiration, one sperm is injected into each oocyte.  The sperm is pretreated to remove it from the seminal plasma and placed in poly vinyl propylpyrrhidol (PVPP) which slows its movement and allows a single sperm to be aspirated into a glass micropipette. The tail of the sperm is frequently broken off to prevent migration from the cytoplasm after injection.

Contraindications:

Contraindications to ICSI are those of IVF.

Outcome:  

ICSI requires IVF and Federal law requires that the outcome of all IVF cycles be reported to the national database kept by the CDC and ASRM/SART.

PREIMPLANTATION GENETIC TESTING

Preimplantation genetic testing encompasses procedures involving the removal of polar bodies from oocytes,blastomeres from cleavage-stage embryos, and trophectoderm cells from day 5-6 embryos to test for mutations in gene sequence or chromosome number prior to embryo transfer. The term "preimplantation genetic diagnosis" (PGD) is used when one or both parents carry a specific gene mutation or chromosomal rearrangement and testing is performed to determine whether that genetic abnormality has been transmitted to the oocyte or embryo. The term "preimplantation genetic screening" (PGS) applies when the genetic parents are presumed to be chromosomally normal and their embryos are screened for
abnormalities in chromosome number.

In 1990, the first established pregnancies using this procedure were reported (72). In two couples known to be at risk of transmitting adrenoleukodystrophy and X-linked mental retardation, two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six to eight cells stage, and sexing by DNA amplification of a Y chromosome-specific repeated sequence. Both women were confirmed as carrying normal female twins.

Indication:

PGD is indicated for couples at risk for transmitting a specific genetic disease or abnormality to their offspring. Historically evidence did not currently support the routine use of PGS (aneuploidy screening) to increase live birth rates in women of advanced maternal age, with multiple miscarriages or multiple IVF failures (73), but emerging research with newer PGS techniques has shown a modest improvement in implantation rates. A meta-analysis of 11 studies illustrated a higher sustained implantation rate (IR) with pooled relative risk (RR) of 1.39 (95% CI 1.21 – 1.60) among the randomized trials and RR of 1.75 (95% CI 1.15 – 1.45) among observational studies (74). PGS technologies continue to evolve and improve, making elective single embryo transfer of the embryo with highest implantation potential more attainable.

Procedure:

Following oocyte recovery with IVF (see IVF) material from an oocyte or embryo is
extracted by creating a perforation in the zona pellucid via a laser, acid Tyrode’s
solution, or a sharpened glass needle. The polar body, blastomere(s) or trophectoderm cells are then extracted using a small suction pipette or by gently compressing the oocyte or embryo to
extrude material through the opening. To identify specific gene mutations, PGD employs techniques involving the polymerase chain reaction (PCR) to amplify a segment of the genome that contains the specific gene of interest. Fluorescence in situ hybridization (FISH) is a technique that uses DNA probes labeled with distinctly colored fluorochromes. These probes bind to specific DNA sequences unique to each chromosome and detect missing or excess chromosomal material. Where FISH limits the number of chromosomes able to be tested at once, newer technologies have combined DNA microarrays and comparative genomic hybridization (CGH) to screen more genetic loci in PGS. More recent PGS platforms utilize single-nucleotide polymorphism (SNP) microarray, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) to screen day 5-6 embryos for genetic abnormalities. In order to utilize newer PGS technologies, IVF centers must have experience in trophectoderm biopsy, ability to grow embryos to the blastocyst stage, and a successful cryopreservation program.

Contraindications:

Contraindications to PGD/PGS are those of IVF.

Outcome:

The estimated risk of transferring an affected embryo mistakenly identified as normal by traditional single-gene PGD is approximately 2% for recessive disorders and 11% for dominant disorders (75). Prenatal diagnostic testing to confirm the results of PGD is encouraged strongly because
the methods used for PGD have technical limitations that include the possibility for a false negative result.

NGS involves embryonic DNA being divided into 100-200 base pair sequences and comparing these sequences to a reference genome, and both whole chromosome and segmental chromosome imbalances can be identified (76). However, with this technology, mosaic DNA will labelled “abnormal,” and many argue embryos with some mosaicism may actually be viable in vivo. NGS outcomes are being followed closely as the technology evolves.

FERTILITY PRESERVATION

An increasing number of women are delaying reproduction until their late 30s, 40s and even into their fifth decade of life.  Many women understandably have an interest in preserving their fertility, and are seeking the option to cryopreserve their oocytes for later use. Oocyte cryopreservation however, is still considered an experimental procedure and data related to clinical outcomes are limited.

According to the American Society for Reproductive Medicine, the only established methods of fertility preservation for women are oocyte and embryo cryopreservation (77,78). Cryopreservation of embryos (as opposed to oocytes) is well established, with the first successful pregnancy from frozen human embryos reported in 1983. Women in committed relationships may elect to cryopreserve embryos with their partners’ sperm, and single women may elect to cryopreserve embryos utilizing donor sperm. Oocyte cryopreservation has become more prevalent in the last decade for fertility preservation in women facing cancer treatment as well as those planning to electively defer childbearing. The American Society for Reproductive Medicine (ASRM) now views elective oocyte cryopreservation as a viable, non-experimental treatment option for women (78). Currently in the US, employers such as Apple and Facebook offer to help offset costs associated with oocyte cryopreservation in attempt to help career-oriented women preserve fertility. Thawed oocytes appear to have similar fertilization and implantation rates as fresh oocytes (79) due to improved cell survival from newer vitrification techniques, and there does not appear to be an increase in congenital anomalies in babies born after this process (80).

In the past, retransplantation of cryopreserved ovarian tissue has been proposed as a mechanism for fertility preservation in cancer patients. With only a small number of live births in the United States to date, this methodology will not be discussed further (for a concise review, refer to citation 77 and 81).

Indications: 

Fertility preservation is an option for women with cancer or other illnesses requiring treatment that may compromise their fertility. Fertility preservation is also an elective option for women who are interested in childbearing at a later time, but patients need to be informed of the potential benefits, limitations and risks of this developing technology.

Procedure:

Procedures for oocyte and embryo cryopreservation begin in a manner similar as described for IVF (see IVF). The oocytes are cryopreserved immediately upon
harvest, or oocytes are fertilized and the embryos are cryopreserved typically on day three, five or six of culture.

Contraindication:

Fertility preservation procedures are contraindicated in women in whom exposure to fertility medications or oocyte retrieval is contraindicated.

Outcome:

In the absence of clinic-specific out comes, there is an overall 2% live birth rate per oocyte thawed via traditional slow freeze techniques and 4% live birth rate per oocyte thawed via vitrification (82,83). For cryopreserved embryos transferred in 2013, the live birth rate was 44.4% for women under age 35, 40.6% for women ages 35-37, 36.1% for women ages 38-40, 31.6% for women 41 to 42 years (www.SART.org).

Oocyte Nuclear Transfer:

Research continues in ART among women who are carriers for mitochondrial disorders, a diverse group of maternally-derived mutations resulting in progressive and life-threatening diseases. Animal studies replacing the cytoplasm and mitochondrial DNA (mtDNA) from an affected oocyte with that of a healthy donor oocyte have shown promise. The FDA in the US currently does not support human clinical trials to confirm or refute animal studies due to unknown implications for the offspring (www.fda.gov).

Summary:

Advances in technology have dramatically altered the treatment of infertile couples and have improved pregnancy and live birth outcomes.  However, the methodical approach beginning with a detailed medical history and physical examination is a mainstay.  Avoiding empirical tests and treatments will afford couples the most economical and successful therapy for this emotionally devastating disease.

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Central Causes of Amenorrhea

ABSTRACT

Normal ovulatory menstrual cycles reflect a coordination of an intact hypothalamic-pituitary-ovarian unit. This process involves neuroendocrine messaging between the hypothalamus, pituitary, and gonads. Common central causes of amenorrhea include functional hypothalamic anovulation, exercise-associated anovulation, pharmacologic-induced anovulation, psychiatric associated disorders such as anorexia nervosa and bulimia, and genetic-developmental defects of the gonadotropin releasing hormone (GnRH) neuronal unit. While brain radiation, trauma or a congenital deficiency of gonadotropins can certainly result in amenorrhea, many more common subtle factors can also affect this pathway. Neuroendocrine regulation of ovulation involves the interaction between neurons secreting GABA, kisspeptin, and neurokinin which modulate the function of GnRH neurons. There is also a link between the reproductive axis, nutritional-energy balance status, and dysregulation of two key peptides, leptin and ghrelin, that occurs when there are extreme, nutritionally stressful conditions such as with anorexia nervosa, extreme exercise, or starvation. In the evaluation of patients with central amenorrhea, the history and physical are crucial to determine the laboratory evaluation and if brain imaging is required. Understanding the patient’s goals will allow an individualized treatment regimen, as it will likely differ if the patient desires children imminently. For complete analysis of this and all areas of Endocrinology, please visit our FREE web-textbook, www.endotext.org.

Introduction

The hypothalamic-pituitary-ovarian axis must be fully synchronized for normal ovulation and successful reproduction. Factors that modulate hypothalamic gonadotropin hormone releasing hormone (GnRH) include pubertal maturation, alterations in energy balance, body composition and metabolism, and stress and emotional changes. Disruption of normal menstrual cycles and normal ovulation are often associated with a variety of life style factors such as excessive exercise, nutritional deprivation, and psychological stress. In the vast majority of cases, this is associated with normal neuroanatomic findings. In a small subset, neuroendocrine abnormalities such as isolated gonadotropin deficiency (Kallmann syndrome), head trauma, radiation effects, Sheehan syndrome, and pituitary apoplexy are identified (see Table 1). Regardless of the etiology, the final common pathway is a change in the normal pattern of episodic secretion by the GnRH pulse generator resulting in disruption of ovulation and amenorrhea. These types of disorders will be discussed in this chapter.

Table 1. Classification of Anovulation Associated with the CNS Hypothalamic-Pituitary System

Functional Hypothalamic Anovulation
  • Exercise-related factors
  • Nutritional factors
  • Psychogenic or stress factors
Physiologic Anovulation
  • Prepubertal phase
  • Postpartum phase
  • Breastfeeding phase
Pharmacologic-Associated Anovulation
  • Opiate agonist
  • Dopaminergic agonist
Psychiatric-Associated Disorders
  • Pseudocyesis
  • Anorexia nervosa
  • Bulimia
Organic Defects of the Hypothalamic-Pituitary Unit
  • Kallmann syndrome
  • Isolated gonadotropin deficiency
  • Pituitary tumors
  • Sheehan syndrome
  • Pituitary apoplexy/aneurysm
  • Empty sella syndrome
  • Inappropriate prolactin secretion
  • Infection (human immunodeficiency virus, tuberculosis)
  • Head trauma
  • Post-radiation effects

NEUROENDOCRINE CONTROL OF GNRH SECRETION DURING THE REPRODUCTIVE CYCLE

Prior to the existence of laboratory techniques to assess gonadotropin secretion, physicians relied on clinical judgement to arrive at the diagnosis of central amenorrhea (1). The advent of the radioimmunoassay in the late 1960s paved the way for a laboratory diagnosis of this disease. The secretion of endogenous GnRH is difficult to assess in the human because this decapeptide is rapidly metabolized within 2 to 4 minutes in the peripheral circulation. Thus, it is not possible to directly assess GnRH secretion and the majority of clinical investigations utilize frequent measurements of LH concentrations as the surrogate marker for hypothalamic GnRH secretion.   In women with regular menstrual cycles, clinical studies have demonstrated a characteristic pulsatile secretion of LH at a frequency of 90-120 minutes during the follicular phase and a frequency of 180-240 minutes during the luteal phase (2). Small alterations in LH pulsatile frequency and or amplitude can result in a range of disorders including luteal phase defects, oligo-ovulation and anovulation. Thus, most studies have examined for changes in LH pulsatile frequency and amplitude as the major endpoint in studies to investigate functional hypothalamic amenorrhea.

Puberty

Normal female pubertal development requires an elaborate orchestration of the hypothalamic-pituitary-gonadal axis. Prepubertal girls have low LH, FSH, and estradiol. Prepubertal girls respond with a rise in gonadotropin secretion (predominantly FSH) in response to exogenous GnRH administration (3). This response indicates that the hypothalamic GnRH pulse generator is less active during this period of development. At pubertal onset there is a distinct diurnal variation as the gonadotropins rise (4). Although the exact triggers at the time of puberty are poorly understood, there are three distinct changes that are observed in the hypothalamic-pituitary unit (5).

  1. A nocturnal sleep-related augmentation of pulsatile LH secretion begins as a result of the increase in GnRH pulsatility.
  2. The sensitivity set point of the hypothalamus to estradiol and testosterone decreases, thereby resulting in an increased basal gonadotropin secretion.
  3. In the female, a positive feedback capability develops, and critical levels of estradiol can trigger a large release of GnRH, and subsequently LH, leading to ovulation.

Central axis control of gonadotropins

The effect of estradiol on the CNS is generally dichotomous. While E2 sensitizes the pituitary gonadotrophs to GnRH by inducing GnRH receptor expression, it also suppresses the release of GnRH from the hypothalamus (6). This "negative feedback" is the dominant effect of estradiol for the vast majority of the ovulatory cycle. The negative control of gonadotropin release is augmented by inhibin secreted from ovarian granulosa cells. Unlike other endocrine systems, continued exposure to higher levels of estradiol (>250 pg/mL) for prolonged duration (>48hours) produced by the dominant ovarian follicle results in a sudden release of a GnRH-LH surge termed "positive feedback" that triggers ovulation at midcycle (7).

Neuroendocrine regulation of ovulation

During embryogenesis in primates, the GnRH cells migrate from the nasal placode into the bilateral arcuate nuclei in the mid-hypothalamus. The neurons of the arcuate nucleus that target the GnRH cells synapse with incoming axons from extrahypothalamic brain areas such as the amygdala, hippocampus, and cortex that target the GnRH cells. Conceptually, these interneuronal connections release excitatory (E) or inhibitory (I) neurotransmitters. Variations in the synaptic balance of these neurotransmitters exist during the ovulatory cycle. During negative feedback, the E/I ratio is low and during positive feedback the E/I ratio is high (8). Gamma amino isobutyric acid (GABA) neurotransmitters are primarily associated with inhibitory GnRH activity, while kisspeptin and neurokinin are associated with excitatory GnRH activity (9). Estradiol effects on GnRH pulsatility appears to be mediated by altering the E/I ratio through synaptogenesis and synaptolysis of neuronal pathways targeting GnRH secretion (10). During the bulk of the cycle (negative feedback) the effect of E2 is synaptogenic and induces neurotransmitters with a net inhibition of GnRH secretion. In contrast, during the pre-ovulatory phase (positive feedback), estradiol triggers synaptolysis, thereby causing the ovulatory surge.

Kisspeptins are G protein-coupled receptor ligands originally identified as human metastasis suppressor gene products that have the ability to suppress melanoma and breast cancer metastasis. These peptides have recently been found to play an important role in initiating the secretion of GnRH at puberty (11). Kisspeptin, encoded by the KISS1 gene is located on the long arm of chromosome 1 (1q32) and is a peptide consisting of 145 amino acids. In the brain, the gene is transcribed within the hippocampal dentate gyrus and activates the G protein-coupled receptor GPR54.

In 2003 the discovery of the necessary role kisspeptin has in puberty led to the understanding of the neuroendocrine effect on human reproduction (12). The GnRH neurons of primates, rodents and sheep are found in close apposition with kisspeptin neurons. GnRH neurons express the kisspeptin receptor, and when kisspeptin is incubated with hypothalamic explants, it stimulates the release of GnRH. This effect is not observed in kisspeptin receptor knockout mice. While GnRH neurons show an increase in firing rate in vitro following kisspeptin treatment, this effect is attenuated by the application of a kisspeptin receptor antagonist. The discovery of inactivating mutations in the kisspeptin/GPR54 further ascertained its role in human reproduction. In humans, inactivating GPR54 mutations are associated with normosmic hypogonadotropic hypogonadism while activation of GPR54 signaling is associated with precocious puberty. Central and peripheral administration of kisspeptin stimulates the hypothalamic-pituitary-gonadal axis while pre-administration of a GnRH antagonist abolishes this effect. Collectively, these observations strongly suggest that kisspeptin can be a primary mediator for activation of GnRH neurons.

All of the current studies initiating puberty with exogenous kisspeptin were performed on monkeys and rodents; however, one study demonstrated return of reproductive hormone production in women with hypothalamic amenorrhea who received twice-weekly kisspeptin injections for 8 weeks (13). Another potential clinical application for kisspeptin is to use it as a trigger for the LH surge during ovulation induction for in vitro fertilization (IVF). It has been shown to effectively induce oocyte maturation, and may be a more physiologic mechanism for inducing ovulation than hCG, which is currently used for ovulation induction. There is speculation that a kisspeptin trigger may also decrease the risk for ovarian hyperstimulation syndrome; however, studies comparing kisspeptin and a GnRH agonist need to be completed prior to recommending the use of kisspeptin (14). Further studies are ongoing to further establish the role of kisspeptin in restoring reproductive function in certain conditions.

Modulation of GnRH Secretion by Opioidergic, Dopaminergic, and Excitatory Amino Acids

Animal studies have shown that other neurotransmitter systems such as dopamine, norepinephrine, and serotonin can regulate GnRH or LH secretion. These studies suggest that activation of the noradrenergic system is associated with increased release of GnRH whereas dopaminergic or serotonergic activation can either inhibit or stimulate GnRH release (15-21). These observations can explain in part the CNS-associated disruption of normal menstrual cycles in patients who take phenothiazine (dopamine receptor antagonists), stimulants, antidepressants, and sedatives on a chronic basis.

Excitatory amino acids such as glutamate and aspartate have been shown to be localized to the arcuate nucleus in the media basal hypothalamus adjacent to GnRH neurons and have been implicated in a regulatory role for GnRH secretion primarily during pubertal maturation (22). These two amino acids appear to activate GnRH secretion during puberty in monkeys.

Endogenous opiates peptides such as endorphins, enkephalins, and dynorphins appear to play largely an inhibitory role in GnRH and LH secretion. The same functional neuronal network that secretes kisspeptin also co-secrete dynorphin, and collectively it is known as the kisspeptin-neurokinin B-dynorphin (KNDY) pathway (23). KNDy neurons in the infundibular/arcuate nucleus have an effect on GnRH by influencing both the GnRH cell body and the neurosecretory terminals. Given the KNDy cells express both neurokinin B receptors and the kappa opioid peptide receptor (dynorphin receptor), it is suspected that the stimulatory role of neurokinin B and the inhibitory role of dynorphin work together to cause a pulsatile release of kisspeptin, which results in a GnRH pulsatile release (24). In patients with hypothalamic amenorrhea, blockade of endogenous opiate receptors with the receptor antagonists such as naloxone or naltrexone (dynorphin antagonists) will induce an increase an increase in pulsatile release of GnRH and LH (25). Long term treatment of hypothalamic amenorrhea patients with naltrexone can result in return of normal menstrual cycles in some individuals (26). These findings indirectly suggest that endogenous opiate activity is suppressing GnRH secretion. The use of an inhibitory neurotransmitter may be beneficial in patients who require a suppression of GnRH or LH secretion. For example, patients with PCOS generally have a dysregulation of gonadotropin secretion which likely contributes to irregular ovulation may benefit from exogenous regulation of the opiate peptides (27).

LINKAGE BETWEEN NUTRITIONAL STATUS AND HYPOTHALAMIC AMENORRHEA

For many years, clinicians have sought to identify the functional link between nutritional status and reproduction. Recent identification of neuropeptides that alter feeding behaviors has provided a physiological mechanism to explain the shutdown of the H-P-O axis for individuals who experience significant changes in nutritional status (i.e. starvation and obesity). Two key peptides, leptin and ghrelin have been identified and appear to regulate feeding behavior which may mediate the dysregulation of the reproductive axis under extreme, nutritionally stressful conditions.

Leptin is a 16 Kd, 167 amino acid polypeptide that was first isolated in 1994. This peptide is a product of the ob gene. Based on phylogenetic studies, leptin is conserved and has been identified in amphibians, rat, sheep, and human (28). Leptin is primarily produced by the adipocytes but has also been shown to be synthesized in other tissues such as skeletal muscle, heart, stomach, and the placental-fetal unit. Leptin is a member of the cytokine family and is classified as an anorexigenic peptide. Other peptides in this class include pro-opiomelanocortin and the cocaine and amphetamine-regulated transcription peptides. Leptin’s action is opposed by orexigenic peptides such as neuropeptide Y and the agouti-related peptide.

Leptin is known to circulate in two forms: as a free form and as a bound form complexed to soluble leptin-binding proteins or to circulating leptin receptors. Physiological studies demonstrate that leptin is secreted in a pulsatile manner with a diurnal rhythm. Current commercial assays for leptin appear to measure total leptin in the serum compartment. Transport of leptin into the brain has been described as unidirectional through the blood brain barrier into brain tissue.

For patients with HA, most studies report a decrease in total circulating leptin with a loss of the normal diurnal rhythm (29). This lower leptin level is a common characteristic of several energy-deficient conditions and is associated with a decrease of the LH pulse frequency. There have now been six receptor isoforms described for leptin. These receptors have been localized to the brain, ventral hypothalamus, lung, kidney, pancreas, adrenals, ovaries, skeletal muscle, and hematopoietic stem cells. Within the brain, the arcuate nucleus appears to have the greatest concentrations of leptin receptors (as well as GnRH receptors).

Binding of leptin to its receptor causes functional dimers to form and activates the JAK/STAT3 intracellular signaling pathways. The effects of leptin on GnRH release may be mediated through kisspeptin. The leptin receptor (Ob-Rb) is not expressed by GnRH neurons of the hypothalamus. However Ob-Rb mRNA is found in 40% of Kiss1 mRNA-expressing cells of the arcuate nucleus (30). The expression of Kiss1 mRNA in the mutated leptin receptors is reduced in comparison with wild-type mice. Furthermore, the expression of Kiss1 mRNA has been shown to be influenced by nutritional status. In pre-pubertal rats, which have been food deprived for 72 h, the hypothalamic expression of Kiss1 mRNA is markedly reduced (31).

In contrast to leptin, ghrelin, another energy-balance peptide, was found to be elevated in individuals with HA. Ghrelin is a 28-amino acid orexigenic peptide that was first isolated in the stomach and is a potent stimulus to GH release. In the fasting state, ghrelin serves as the hunger signal from the periphery to the CNS, acting on the hypothalamic arcuate nucleus, a region which is known to control food intake. Based on these relationships, it is tempting to rationalize that leptin and ghrelin simply serve as natural on/off switches for regulation of feeding behavior.

Functional Hypothalamic Amenorrhea

A practical definition of functional hypothalamic amenorrhea (HA) is the absence of menstrual cycles for more than 6 months without evidence of anatomic or organic abnormalities (32). Three main types of functional hypothalamic amenorrhea have been recognized, associated with stress, weight loss, or exercise (33). Other more serious organic disorder can mimic HA such as isolated gonadotropin deficiency (see table 1).   Thus, this diagnosis should be made after exclusion of other causes.
HA is associated with increased cortisol secretion that reflects increased activity of the hypothalamic-pituitary-adrenal (HPA) axis (34). There appears to be an increased secretion of a "stress response complex" of hormones such as CRH, ACTH, cortisol, PRL, oxytocin, vasopressin, norepinephrine, and epinephrine (TABLE 2). CRH has been shown to directly inhibit GnRH secretion in rats, monkeys, and humans at the hypothalamic level in in vitro and in vivo experimental models. This inhibition can be negated by administration of a CRH receptor antagonist or by naloxone, an opiate receptor antagonist. Taken together, these observations suggest that the inhibitory effect of CRH is mediated in part by increased opioidergic activity.

The increased secretion of ACTH at the pituitary level may also suppress pituitary response to GnRH. In addition, increased cortisol levels may also dampen pituitary response to GnRH. It must be emphasized that an acute stress response will be unlikely to alter ovulatory function since the H-P-O axis is quite resilient. On the other hand, it appears that chronic environmental stressors lead to long term activation of the HPA axis which in turn can induce ovulatory dysfunction at either the hypothalamic or the pituitary level.

Modification of the stress response can restore normal HPO function. The validation for this comes from evidence of complete resumption of normal ovulatory function in women with HA treated with cognitive behavioral therapy (CBT) (35). Treated women exhibited increased LH pulsatility and decreased cortisol levels. A similar study at the same institution found that CBT resulted in an increase in leptin and TSH levels, and improved the neuroendocrine and metabolic components of HA (36). This provides evidence that stress-reducing behavioral changes may be successful in restoring normal ovulation and metabolic function in women with HA.

A constellation of other neuroendocrine alterations occur in HA. There is a suppression of the hypothalamic-pituitary-thyroid axis with a resultant decrease in circulating thyroid hormones (37). There also may be a decrease in prolactin secretion as well as alterations in other neurohormones such as melatonin (38). There may be difficulties in recognizing the stressors that have elicited the stress response in FHA since the stressors may not be objective or quantifiable (39). Human stressors often reflect attitudes of self or society. Subjective stressors such as these may not be as easy to quantitate as stressors with objective measures such as food deprivation or outright violence.

Evaluation

The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists have advocated for menstrual status to be considered a “vital sign” at routine clinical visits, given the importance of estrogen for bone and other tissues (40). Many individuals with this disorder will provide a history of normal menarche and regular menstrual cycles between 26 and 35 days in length. These women typically are intelligent, high-achievers, who are usually thin or of normal body weight. A detailed interview may identify a stressful event or emotional crisis (divorce, relation breakup, death of a friend or relative) preceding the amenorrhea. Other interpersonal and environmental stressors may also be present such as academic pressures, job stresses, or psychosexual problems. A careful review of the patient's current lifestyle including exercise intensity, dietary choices, and the use of sedatives or hypnotics may be helpful in characterizing the psychogenic stress components. The patient may have features of the female athlete triad, which consist of amenorrhea, insufficient caloric intake with or without an eating disorder, and low bone density or osteoporosis.

The physical examination should focus on identifying galactorrhea, thyroid dysfunction, and evidence of hyperandrogenemia (i.e., acne, hirsutism). The pelvic examination should be normal except for a thinned vaginal mucosa or absent cervical mucus which are characteristics of hypoestrogenism. Despite these findings, these patients do not usually experience hot flushes.

(Table 3, 4)

Laboratory evaluation should include FSH, prolactin (PRL), and TSH. Most of the other pituitary hormones should be in the normal range (Table 4). In many patients, the progestin challenge test (medroxyprogesterone acetate 10 mg for 7 days) will demonstrate an absence of withdrawal uterine bleeding or vaginal spotting. This test is a bioassay for the absence of estrogen priming of the endometrium and reflects the chronically low circulating levels of estradiol.

The basic defect in women with functional hypothalamic amenorrhea is the failure of the hypothalamus to increase GnRH output in the presence of severe hypoestrogenism. Most investigators believe that there is a slowing of the GnRH pulse generator as reflected by a decrease in peripheral pulsatile LH secretion in these women. The pattern of LH secretion may vary. During the early onset, LH pulse frequency and amplitude may be normal, and in more severe cases, regression to a prepubertal pattern may occur. During recovery from HA, sleep- associated increases may be observed (see Figure 1).

The pituitary gland is fully functional and capable of synthesizing and release of LH and FSH. However, responses to exogenous GnRH in these individuals may vary depending on the endogenous GnRH priming of the pituitary gland. LH and FSH responses to exogenous GnRH may be absent, normal, or supernormal. In these patients, after a period of priming with intravenous pulsatile GnRH (1-2 mg/90 minutes), normal levels of LH and FSH can be restored and responses to exogenous GnRH become normal. Taken together, these observations suggest that endogenous GnRH secretion is deficient and, gonadotropin secretion and ovarian function can be normalized with physiologic replacement of exogenous, pulsatile GnRH.

Magnetic resonance imaging (MRI) of the brain is not routinely needed in patients with

presumed hypothalamic amenorrhea (41). However, it is indicated in patients who have a history of severe or persistent headaches, persistent vomiting that is not self-induced, central hypothyroidism, hyperprolactinemia or galactorrhea, or a change in thirst, urination, or vision.

Figure 1

CLINICAL MANAGEMENT OF FUNCTIONAL HYPOTHALAMIC AMENORRHEA

The clinical evaluation of HA should focus on a carefully conducted history and physical examination that reviews life style variables and interpersonal relationships. Since this is a diagnosis of exclusion, significant organic diseases must be excluded (Table 1). In many patients, spontaneous recovery of menstrual function will take place following accommodation to environmental stressors or after modification of life style. Psychological counseling may also be appropriate for these individuals. Because of the functional nature of HA, an individualized and expectant management should be considered the initial approach. In those who remain amenorrheic, periodic evaluation of menstrual status every 4 to 6 months is prudent.

In the infertile patient with this diagnosis who fails to resume normal cycles, a trial of low dose clomiphene citrate (25-50 mg for 5 days) is appropriate. In these patients, higher doses of clomiphene may suppress the H-P axis due to the weak estrogenic properties of clomiphene in an already hypoestrogenic environment. For clomiphene failures, use of human menopausal gonadotropins as an alternative method is highly successful. If available, pulsatile administration of GnRH 5 mg/90 minutes intravenously using a modified insulin pump will successfully induce ovulation after a 13 to 14 day treatment period (42). This latter approach is associated with ovulation rates of greater than 90% with generation of a single dominant ovarian follicle and much lower rates of ovarian hyperstimulation. In these patients, corpus luteum support can be maintained with either GnRH or human chorionic gonadotropin 1500 units intramuscularly every three days for 4 doses.

For the woman who remains amenorrheic for more than 1 year, the long term risks of hypoestrogenism including reduced bone mineral density and osteoporosis become factors. In young women with persistent hypoestrogenism, the bone mass can decrease at a rate of 2-5% per year for the first three to five years. Dual energy X-ray absorptiometry (DEXA) is often necessary to convince patients to begin estrogen therapy. The minimal dose of estrogen necessary to conserve bone has been established in menopausal women. At least 0.3 mg of conjugated estrogen, 1 mg of micronized estradiol, or 0.025 mg of transdermal estrogen is necessary to protect against bone loss. Use of a progestin on a cyclic basis such as medroxyprogesterone acetate for 10 to 12 days each month is necessary to ensure regularly shedding of endometrium and prevent endometrial hyperplasia. Calcium and vitamin D supplements may also be taken; however, the primary therapy requires treatment of the underlying process. The use of bisphosphonates in reproductive-aged women should be carefully considered.

As previously discussed, Leptin plays a critical role in regulation of kisspeptin and in HA. A possible future treatment for HA may include leptin replacement as it has been shown to restore pulsatility of gonadotropin-releasing hormone and ovulation as well as full development of secondary sexual characteristics in females with hypogonadotropic hypogonadism (43). However, the effect of leptin therapy on bone health is unknown. Similarly possible future treatments options may include exogenous kisspeptin.

Table 2. Associated Neuroendocrine Abnormalities in Hypothalamic Amenorrhea.

Increased daytime cortisol secretion

Increased amplitude and duration of nocturnal melatonin secretion

Increased nocturnal secretion of GH

Elevated CRH levels in cerebral spinal fluid

Blunted elevation of PRL, ACTH, and cortisol during the noon meal

Table 3. Common Features of Women with Psychogenic Hypothalamic Amenorrhea

Single marital status

Obsessive-compulsive habits

History of significant stressful life events

History of sexual abuse

History of prior irregular menstrual cycles

Normal or thin habitus

Tendency to use sedatives or hypnotic drugs

Involved in professional occupations

High intelligence

Table 4. Expected Serum Hormonal Parameters in Functional Hypothalamic Amenorrhea

Hormone Expected Values
LH

 

 

 Normal or low

 
FSH Normal or low
PRL Normal or low
TSH Normal
GH Normal or low
Estradiol Normal or low
ACTH Normal
Cortisol Daytime elevation with diurnal variation
Testosterone Normal or low
T3 Normal
T4 Normal
Figure 1. Examples of LH pulsatile secretion at various stages of the hypothalamic-pituitary activation. GnRH-LH secretion can progress from an apulsatile stage; to a high amplitude infrequent pulse stage; to a sleep-entrained pulse stage; and to regular every ninety minute pulse (1).

Figure 1. Examples of LH pulsatile secretion at various stages of the hypothalamic-pituitary activation. GnRH-LH secretion can progress from an apulsatile stage; to a high amplitude infrequent pulse stage; to a sleep-entrained pulse stage; and to regular every ninety minute pulse (1).

BULIMIA AND ANOREXIA NERVOSA

Severe eating disorders such as bulimia and anorexia nervosa are also associated with disruption of reproductive function. Bulimia is characterized by alternating episodes of consumption of large amounts of food over a short time (binge eating) followed by periods of self-induced vomiting, excessive use of laxatives or diuretics and food restriction. The incidence of bulimia is estimated to be 4.5 to 18 percent among high school and college students (44). Bulimia usually begins between the ages of 17 to 25 years.

Anorexia nervosa is a severe eating disorder characterized by extreme weight loss (greater than 25% of ideal body weight), body-image disturbance, and an intense fear of becoming obese (45). Anorexia patients are usually between the age of 12 yrs and the mid-thirties and have a bimodal age of onset at 13-14 years and 17-18 years. There is a 90 to 95 % female predominance with the majority of patients coming from Caucasian, middle-class or upper-middle class families. The overall incidence of anorexia ranges from 0.64 per 100,000 to 1.12 per 100,000.   The mortality associated with anorexia has been reported to be as high as 9 percent usually secondary to cardiac arrhythmia which is precipitated by electrolyte abnormalities and/or diminished heart muscle mass. Suicide has also been more common (2 to 5%) in patients with anorexia nervosa. These statistics are sobering making it extremely important for the clinician to recognize early signs of this disorder so that appropriate intervention and treatment can be initiated.

The clinical features of bulimia and anorexia are listed in Tables 5 and 6. Due to the marked reduction in caloric intake in anorexia, basal metabolism is lowered by decreased conversion of thyroxine to triiodothyroxine to maintain homeostasis. Thyroxine is converted via an alternative pathway to reverse triiodothyroxine, a relatively inactive isoform (Table 7). This protective functional mechanism is also commonly seen in severely ill patients and during prolonged starvation. Anorexics also suffer from defects in thermoregulation and are hypothermic. Because secretion of vasopressin is impaired, anorexics also have partial diabetes insipidus and are unable to concentrate their urine.

Anorectic and bulimic patients have hyperactivation of the HPA axis (46). Studies show a persistent hypersecretion of cortisol throughout the day with an increase in 24 hour free cortisol secretion. Despite the increased cortisol production, the manifestations of hypercortisolism are not present due to a decrease in cellular glucocorticoid receptors (47). The reduced number of these receptors may also provide an explanation for the incomplete suppression of the pituitary-adrenal axis by dexamethasone. Pituitary CRH responses are also blunted in bulimics and anorectics. As with other stress response syndromes, anorexics have evidence for increased central opioid activity with reported increases in cerebrospinal fluid levels of b-endorphins (48). Hypolepitinemia is commonly seen in women with anorexia. In these patients the low levels of leptin are representative of a chronic energy deficiency (49).

Like functional hypothalamic amenorrhea, anorexics have a prepubertal pattern of LH secretion presumably due to marked decrease in GnRH secretion. With weight gain, anorexics can display transitional patterns of LH secretion and may have normal or supranormal responses to GnRH (Figure 1). In some patients, despite return to normal body weight, up to 50% remain anovulatory.

Table 5. Common Features of Bulimia

 

Irregular menstrual cycles

Dental enamel erosion

Acute irritation of esophageal mucosa

Esophageal or gastric rupture

Hypokalemia

Aspiration pneumonia

Ipecac poisoning

Table 6. Common Features of Anorexia Nervosa

Preoccupation with handling of food

Bulimic behavior

Calorie counting

Distortion of body self-image

Hyperactivity

Obsessive-compulsive personality

Increased incidence of past sexual abuse

 

Amenorrhea

Constipation

Coarse, dry skin

Soft, lanugo-type hair

 

Hypothermia with defective thermoregulation

Mild bradycardia

Cardiac arrhythmia

Hypotension

 

Hypokalemia secondary to diuretic or laxative abuse

Low bone mass

Increased serum beta-carotene levels

Anemia

Leukopenia

Elevated hepatic enzymes

Table 7 Neuroendocrine Abnormalities Associated with Anorexia Nervosa

 Diminished GnRH-LH pulsatile frequency and amplitude

Low blood LH and FSH levels

Impaired ACTH response to CRH stimulation testing

Resistance to dexamethasone suppression

Increased ACTH levels

Increased 24 hour urinary free cortisol levels

Low prolactin levels

Low TSH levels

High reverse T3 and low T3 levels

Elevated GH levels

Decreased IGF-1 levels

Diabetes insipidus

 The success rates for treatment of anorexia nervosa and bulimia remain low. Therapeutic approaches include behavior modification, group therapy, and individual psychotherapy. Generally, a team approach consisting of a psychiatrist and a general medicine specialist with expertise in eating disorders is desirable. Because of the high mortality rate and the significant morbidity associated with anorexia, it is important to obtain psychiatric consultation and follow-up in all patients with eating disorders. For patients who fail to resume menstrual function even after restoration of body weight, estrogen replacement therapy is indicated.

EXERCISE-INDUCED HYPOTHALAMIC AMENORRHEA

With the increased participation of women in all types of recreational and competitive athletic activities, health issues related to exercised-induced amenorrhea have become common. Depending on the type of sport and competition level, the incidence of amenorrhea varies from 5 to 25%. The incidence of menstrual irregularities appears to be greatest in sports that favor a low body weight physique such as ballet dancers (6-43%) and middle and long distance runners (24-26%). The incidence appears to be less frequent in bicyclers (12%) and swimmers (12%) (50).

In those athletes with menstrual irregularity, LH pulsatility is altered and can range from a decreased frequency to a transitional pattern (Figure 1). It is well known that acute exercise leads to hyperactivation of the HPA axis. Is it the stress of exercise or the low energy availability that alters LH pulsatility in exercising women? This key question has been answered in part by controlled studies in which women undergo dietary caloric restriction imposed in the face of increasing exercise demands. It would appear that LH pulsatility is not disrupted by the stress of exercise but rather LH pulsatility is disrupted because of reduced energy availability (51).

It is important to emphasize for so many of these athletes that the “female athlete triad” amenorrhea, osteoporosis, and eating disorders coexist. Management of these patients should emphasize measurement of bone density, counseling about diets, weight changes, trying to keep weight near normal levels, and calcium intakes. Women with exercise-related amenorrhea tend to have impaired lipid and metabolic profiles including an elevated serum total cholesterol, LDL cholesterol, and triglycerides compared with healthy women (52). One goal of therapy should be to decrease the level of exercise, improve the diet, and achieve weight gain. For others, exercise may not induce amenorrhea but may be associated with longer menstrual cycles, luteal phase defects, and intermenstrual spotting. These reproductive defects may be reversible with a decrease in exercise level or intensity. Alternatively, clomiphene given at doses between 25-100 mg for 5 days may increase GnRH pulsatility sufficiently to correct this defect. If amenorrhea persists for over 6 months, it may be prudent to begin estrogen replacement therapy or oral contraceptive pills. Long term health risks in these individuals are reproductive dysfunction and skeletal abnormalities.

OTHER CAUSES OF HYPOGONADOTROPINISM

Isolated Gonadotropin Deficiency (IGD)

This disorder is characterized by a decrease or absence of endogenous GnRH secretion resulting in very low to undetectable LH and FSH levels. Individuals with this disorder have incomplete development of secondary sexual characteristics, primary amenorrhea, eunuchoid features, and in some cases a decreased sense of smell or anosmia (Kallmann syndrome) (53). This disorder can have an autosomal dominant inheritance pattern. IGD has been linked to variance in over 15 genes. Common genetic variants include KAL1, FGFR1, PROKR2, PROK2, CHD7, FGF8, KISS1, KISS1R, TAC3, TACR3, GNRHR, and GNRH1 (54). The underlying defect is due to a failure of GnRH neurons to form completely in the olfactory placode or to migrate from the olfactory bulb to the media basal hypothalamus during early embryo development. For individuals with anosmia or hypo-osmia, there is evidence of hypoplasia of the olfactory bulbs on magnetic resonance scan (55).

Baseline levels of LH and FSH may be in the prepubertal or normal range. However, levels of other pituitary hormones such as TSH, GH, PRL, and ACTH are normal. Due to the failure to increase gonadal sex steroid secretion during puberty, secondary sex characteristics fail to develop and closure of the epiphyseal plates of the long bones is delayed resulting in a eunuchoid habitus where the arm span is greater than the height.

In many cases individuals are started on birth control pills without the diagnosis being made, such that there is partial or complete development of secondary sexual characteristics. In the untreated patient, breast development is usually Tanner stage I or II while pubic hair development will be Tanner IV or V. Treatment of IGD will require estrogen therapy to induce progressive pubertal maturation (100 ng/kg/day of ethinyl estradiol). Patients should be monitored at 2 or 3 month intervals to determine the rate of skeletal growth and development. The addition of a progestin such as medroxyprogesterone acetate 10 mg/day for 12 days can then be used to shed the endometrium. Progesterone therapy should only be started after optimal breast development is achieved. Once sexual maturation is completed, the estrogen dose can be gradually increased and maintained on 2 mg of micronized estradiol or 0.625 mg to 1.25 mg of conjugated estrogens with 12 days of progestin each month. When fertility is desired, ovulation induction can be carried out with either human menopausal gonadotropins or pulsatile GnRH administration.

Postpartum Pituitary Necrosis (Sheehan Syndrome)

Postpartum pituitary necrosis is usually preceded by a history of severe obstetrical hemorrhage with hypotension, circulatory collapse, and shock. After fluid resuscitation of the patient, this condition may be manifested by clinical evidence of partial or panhypopituitarism. Simmonds was the first to describe this clinical syndrome although the most complete description has been attributed to Sheehan (56). This condition constitutes an endocrine emergency that can be life-threatening.

The pathophysiology of this process is not entirely clear. With pregnancy, there is an increase in blood supply to the pituitary bed and the pituitary gland enlarges. During the period of profound hypotension, Sheehan postulated that occlusive spasm of the arteries that supply the pituitary and stalk occurs. This leads to venous stasis and thrombosis of the pituitary portal vessels causing a variable degree of pituitary ischemia and cell death. Many patients initially present with a failure to have breast engorgement and lactation due to a deficiency in PRL secretion. These women may also have other anterior pituitary deficiencies. The posterior pituitary is usually spared because it is less dependent on the portal blood supply. In some patients, the absence of ACTH secretion leads to inadequate cortisol secretion resulting in postural hypotension, nausea, vomiting, and lethargy. Hypothyroidism may be noted later in this scenario. However, in the majority of women, there is a diagnostic delay due to the vague symptoms. Recovery of pituitary function has been reported in a few cases.

The extent of pituitary deficiencies can be characterized by provocative testing with a combined intravenous injection of the hypothalamic releasing factors GnRH, TRH, GHRH, and CRH (57). Appropriate replacement therapy can be instituted once the pituitary reserve is defined. For the patient who presents with hypotension, immediate administration of glucocorticoids is required (cortisone acetate 100 mg, IM). Once the patient stabilizes, a maintenance dose of cortisone acetate 20-25 mg/day or prednisone 5 mg/day can be given. With increased stressful conditions such as an infection, a doubling or tripling of daily doses should be used. For hypothyroid patients, thyroxine replacement should be replaced gradually beginning at 50 mg/day and increased at 50 mg increments at 1 week intervals until full replacement doses are reached (0.1 to 0.2 mg/day). Patients should be instructed to wear a medical alert bracelet. Estrogen replacement therapy will be required for persistent amenorrheic patients. If fertility is desired, ovulation induction with exogenous gonadotropins is required.

Post-traumatic Hypopituitarism

This condition can arise following severe head trauma as a result of a sudden deceleration of the head and occult damage to the pituitary stalk or hypothalamus during a traffic accident (58). Trauma may also be associated with a basal skull fracture or an episode of unconsciousness. The risk for post-traumatic hypopituitarism is generally higher after a severe brain injury requiring neurosurgical interventions than for a mild or moderate injury (59). These individuals will often manifest a delay from injury to presentation of up to 10 years post-injury, possibly due to ongoing atrophy of the sellar and perisellar structures. The symptoms range from partial to complete panhypopituitarism. These symptoms can include amenorrhea, galactorrhea, hypogonadism, loss of axillary and pubic hair, anorexia, and weight loss. For these patients, evaluation of the pituitary-adrenal axis is most important because hypocortisolism can be potentially life-threatening. The diagnostic evaluation and management is similar to that described for Sheehan syndrome.

Pituitary Apoplexy

This medical emergency is characterized by an acute infarction of the pituitary gland. Patients will complain of a sudden onset of a severe retro-orbital headache and visual disturbances which may be accompanied by lethargy or loss of consciousness (60). These symptoms may mimic other neurological emergencies such as hypertensive encephalopathy, cavernous sinus thrombosis, ruptured aneurysm, or basilar artery occlusion. CT or MRI imaging may indicate hemorrhagic changes in the pituitary sella region. Patients with pituitary tumors are at higher risk for this complication. For some patients, neurosurgical consultation and emergency decompression may become necessary. Provocative testing as describe for Sheehan syndrome should be performed to evaluate for multiple pituitary deficits. Appropriate replacement of target tissue hormones should be instituted based on testing.

Post radiation-Induced Hypopituitarism

Exposure to therapeutic radiation sources for treatment of midline CNS tumors can place patients at increased risk for delayed development of hypopituitarism (61). In general, sensitivity to radiation is greatest for somatrotropes and gonadotropes, followed by corticotropes and thyrotropes. The onset of pituitary deficiencies may be insidious but can occur within 1 year of radiotherapy. Periodic assessment of hypothalamic-pituitary function should be performed for an indefinite period of time and appropriate replacement hormone therapy should be instituted as these deficiencies develop.

SUMMARY

In this chapter, we have reviewed a variety of disorders that can disrupt the normal menstrual cycle. Whether the disorder is linked to central organic lesions, changes in life style, or functional, stress components, all appear to have a common pathway(s) resulting in alterations in the secretion of the GnRH pulse generator.

 

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  38. Lam KSL, Tse VKC, Wang C, Yeung RTT, Ma JTC, Ho, JHC. Early effects of cranial irradiation on hypothalamic-pituitary function. J Clin Endocrinol Metab 1987;64:418-424.

Clinical Management of Male Infertility

ABSTRACT

This chapter provides a comprehensive overview of the clinical, "hands-on" approach to the diagnosis and treatment of male infertility. It is a resource of evidence-based information for those who are practicing in this fascinating field as reproductive endocrinologists, fertility specialists, urologists, gynecologists and general practitioners. This chapter has been revised with a particular focus on current challenges in the management of severe male infertility. The chapter gives an overview of the genetic and chromosomal contribution to abnormal sperm production and covers the latest advances in the investigation of the topical DNA fragmentation and newest sperm selections strategies. Therapeutic interventions are presented in detail, mainly in the areas of sperm retrieval, varicocele repair and intra-cytoplasmic sperm injection, including potential complications and extreme challenges as complete failed fertilization. Various approaches to fertility preservation are described, including advances in prepubertal fertility preservation. The current chapter is a resource for those who wish to familiarize themselves with multiple aspects of donor sperm usage. For complete coverage of this and all related areas of Endocrinology, please visit our FREE web-book, www.endotext.org.

NATURE AND CAUSES OF MALE INFERTILITY

Definitions

Infertility is the inability to produce a pregnancy or failure to do so within a reasonable period of trying, usually 6 to 12 months. Sterility is a total inability to produce a pregnancy, and this may be reversible or irreversible. Subfertility is infertility without an absolute barrier to reproduction that would cause sterility, such as azoospermia. Hypogonadism is a nonspecific term for decreased testicular or ovarian function that could include a disorder of gamete production or function or a disorder of sex hormone production or action. Usually male hypogonadism indicates testicular failure associated with androgen deficiency. Primary hypogonadism results from disorders that affect the gonads directly, and secondary hypogonadism results from defective pituitary gonadotropin secretion.

Incidence and Distribution

Of couples planning a pregnancy up to 50% conceive in the first cycle and in the remainder, the percentage who conceive in each successive month declines as the proportion of subfertile couples left continuing to try increases. Approximately 85% conceive a first pregnancy by 6 to 12 months.[1-3] The 6- to 12-month period used to define infertility means that it afflicts approximately 15% of couples.[4] Infertility is thus common and the male contribution is substantial.[5] Infertility results from female disorders (anovulation, tubal obstruction, or other pathology) in approximately 30%, a male disorder in 30%, and disorders in both partners in 30%. No abnormalities are found in approximately 10%. Because male and female factors frequently coexist, both partners of the infertile couple are investigated and managed together.[6]

Controversial reports were published with regard to declining sperm count in some regions of the world, highlighting the potential adverse environment effect on infertility [7-10]. Other reports could not show this effect [11-13].

ETIOLOGY AND CLASSIFICATION OF INFERTILITY

At present, the precise cause cannot be determined in most men investigated for infertility.[6, 14] Relationships between testicular damage, semen quality, and fertility are not strong.[15-17] Even genetic disorders may have marked phenotypic variation. For example, with microdeletions in the long arm of the Y chromosome, testicular histology may show Sertoli-cell-only syndrome, germ cell arrest, or hypospermatogenesis.[18, 19] There are many suggestions for the causes of the common forms of male infertility, but new genetic causes are being found with the use of more efficient DNA sequencing technologies and large clinical databases (table 4). There are many other suggested etiological or contributory factors for which data is lacking or circumstantial including minor hormonal disturbances, dietary deficiencies of vitamins and minerals, disturbed scrotal thermoregulation with or without varicocele and accessory sex gland inflammation.[20] Currently the testicular dysgenesis syndrome and reactive oxygen species (ROS) damage are topical (see chapter 12 by Giwercman and Giwercman) [21, 22]. The concept of the testicular dysgenesis syndrome arose from concerns that toxins in the environment, acting in concert with genetic predisposition, are affecting testicular development. It encompasses hormonal inhibition (“endocrine disruption”) of the proliferation of Sertoli cell precursors, Leydig cells and germ cells during fetal life via adverse environmental, dietary, lifestyle or other influences affecting the mother and resulting in increased risks of cryptorchidism, hypospadias, primary spermatogenic defects, and testicular cancer.[21] Despite the attractively unifying nature of the TDS hypothesis for these disease association, the actual disease mechanism remain unknown and it remains unproven. ROS are believed to be causal in the relationship between abnormal spermatozoa in semen, DNA strand breaks in sperm heads and markers of apoptosis in sperm.[22] The importance of these pathogenetic mechanisms in male infertility remains to be determined.[23, 24]

A classification of causes of male infertility based on the effectiveness of treatment is shown in Table 1. In this classification, effective treatment means medical intervention known or proved by clinical trial to improve the chances of the man producing a conception by coitus or artificial insemination and does not include the use of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) to bypass the impairment.

Table 1. Classification Of Male Infertility By Effectiveness Of Medical Intervention To Improve Natural Conception Rate
TYPE OF INFERTILITY  FREQUENCY (%)
Untreatable sterility           12%
   Primary seminiferous tubule failure 12%
Treatable conditions           18%
   Sperm autoimmunity 7%
   Obstructive azoospermia 10%
   Gonadotropin deficiency 0.5%
   Disorders of sexual function 0.5%
   Reversible toxin effects 0.02%
Untreatable subfertility           70%
Oligospermia 35%
   Asthenospermia and teratozoospermia 30%
   Normospermia with functional defects 5%

CLINICAL EVALUATION

Patients with irreversible sterility can be separated from those with potentially treatable conditions or subfertility usually by standard clinical evaluation (Table 2) and some simple investigations (Table 3).

 

Table 2 Clinical Evaluation of the Infertile Man

History:

  • illness or injury affecting testes,
  • pubertal development
  • sexual performance
  • fertility exposures
  • occupation, habits

Physical examination:

  • general
  • virilization
  • gynecomastia
  • body proportions and BMI
    • scrotal examination
      testicular size
      epididymides
      vasa
      varicocele

Table 3 Basic Investigation of the Infertile Man

Semen analysis:

Semen volume, sperm concentration, motility, morphology, and sperm antibodies

Hormone measurements:

Luteinizing hormone, follicle-stimulating hormone, prolactin, testosterone, sex hormone–binding globulin

Imaging:

Scrotal ultrasound, transrectal ultrasound of prostate and seminal vesicles, magnetic resonance imaging of pituitary

Testis biopsy:

Needle aspiration: cells or tissue, open

History

It cannot be overemphasized that both members of the couple need to be involved in the assessment and discussion of the results. The emotional reaction of the couple to the diagnosis of infertility may interfere with clinical evaluation and management. Intimate information may not be disclosed while the couple is embarrassed, hostile, or confused. Previous sexually transmissible infections or pregnancy may be concealed from the partner.

Nature and Duration of Infertility

Previous pregnancies and time taken to conceive each pregnancy and duration of infertility are important prognostic factors. The couple may be aware of an infertility-related problem, such as previous undescended testes or orchitis. Some who present with a short duration of infertility may be unaware of the normal human pregnancy rates. The plan for investigation depends on the possibility of finding remediable abnormalities and on the age of the female partner.

Family History

The family history should be considered but may not be known because infertility is often not discussed openly.[25, 26] Increasing numbers of chromosomal and genetic causes for male infertility are being discovered (Table 4).[27-29] Some of these cause sterility and are recessive disorders or de novo mutations. Others may only affect fertility slightly so that there may be no family history of infertility. The most important include Kallmann syndrome, myotonic dystrophy, androgen receptor defects, gonadotropin and gonadotropin receptor defects, cystic fibrosis and bilateral congenital absence of the vasa, and chromosomal rearrangements / aneuploides and Yq microdeletions.[18, 19, 27, 30-37] However, collectively they explain less than 10% of male infertility.

There are many pediatric syndromes that involve hypogonadism or undescended testes in association with ambiguous genitalia, multiple malformations, obesity, or mental retardation, but patients with these generally do not present for management of infertility. Other genetic diseases may be associated with infertility, for example, congenital adrenal hyperplasia, hemoglobinopathy, Huntington’s disease, polycystic kidneys, and mitochondrial disorders.[27, 38-41] Predispositions to some conditions may also have a genetic basis such as the anatomic variant of the tunica vaginalis which predisposes to testicular torsion, the association of Young’s syndrome with mercury poisoning in infancy and the familial aspects of sperm autoimmunity. Men with sperm autoimmunity have increased frequencies of both family histories of organ-specific autoimmune diseases and autoantibodies to thyroid and gastric parietal cells in their serum.[42] Furthermore, brothers of men with poor semen analysis results are more often infertile than expected.[25, 26] Thus, it is postulated that genetic causes or predispositions will be found for most male infertility. However, genetic factors remain unclear for the common types or associations of male infertility: idiopathic oligospermia, asthenospermia, teratospermia and undescended testes.

Table 4. Genetic And Chromosomal Defects In Infertile Men: Known Or Suspected
Function  Defect  Phenotype
(Approximate frequency)
Hormonal regulation KALIG 1

Prokineticin-2

FGFR1

 Kallmann syndrome, isolated gonadotropin
GnRH receptor deficiency (1/10,000)
DAX 1 Adrenal hypoplasia congenita (rare)
Steroidogenic enzymes Congenital adrenal hyperplasia (rare)
Haemochromatosis iron deposition in gonadotroph: (1/1000)
FSHβ

T allele of rs10835638

 oligospermia (rare)
FSH receptor oligospermia (rare)
Androgen receptor

 

CAG/CGG polymorphism

 oligospermia (1/20,000)

asthenozoospermia(?)
Spermatogenesis XXY and variants Klinefelter syndrome (1/800)
XYY oligospermia (1/5000)
Translocations oligospermia (1/3000)
Yq microdeletions (AZF regions, CDY) Sertoli cell only, oligospermia (1/500)
ETV5 gene variants Sertoli cell-only syndrome
TSPY gene (short arm of Y chromosome – Yp) oligospermia
DMPK CTG ext. myotonic dystrophy (1/8000)
INSL3 undescended testes (?)
Sex hormone binding globulin (SHBG) gene Oligoasthenoteratozoospermia?
Estrogen receptors (ESR1, ESR2) undescended testes (?)
MTHFR oligospermia
USP26 de-ubiquitinating enzyme family Spermatogenic failure?
TAF7L Spermatogenic failure?
PRM1-PRM2 (protamines responsible for chromatin compaction)
TNP1-TNP2 (transition nuclear protein)
Meiosis Translocations germ cell arrest (rare)
?CREM germ cell arrest (?)
SYCP3 Azoospermia
DAZL (T54A) germ cell arrest (?)
Spermiogenesis Fibrous sheath or dysplasia (1/50,000)
DNAI1,DNAI2 DNAH5, DNAH11, DNAAF2 (dynein arm)

RSPH4A, RSPH9 (radial spoke)

TXNDC3 (thioredoxin-nucleoside diphosphate kinase)

 immotile cilia (1/50,000)
? absent acrosomes (rare)
? decapitate sperm (rare)
protamine II teratozoospermia(?)
LDH-x asthenozoospermia(?)
Genital tract CFTR BCAV (1/2000)
? Other obstructions (rare)
? Necrozoospermia (rare)
Sperm-oocyte interaction ZPBP1 Disordered zona pellucida induced acrosome reaction (1/4000)
? Defective sperm-zona binding with normal sperm morphology (rare)

Coital Adequacy and Timing

Information on impotence and ejaculatory disturbances is important because intravaginal deposition of semen near the time of ovulation is crucial for fertility. Infrequent coitus is common in couples seen for infertility. Low libido may result from androgen deficiency, general illness, or a psychological reaction to the infertility. 

Childhood and Pubertal Development

Treatment in childhood for penile or scrotal disorders (e.g. hypospadias, epispadias, urethral valves, undescended testes, inguinal hernia, hydroceles) could be relevant (see chapter by Hutson). Sexual maturation may be delayed and incomplete with primary or secondary hypogonadism. There may have been associated growth problems that required treatment. Early puberty and growth resulting in short stature suggest congenital adrenal hyperplasia.[38, 39]General Health: Any illness, acute or chronic, can impair sperm production in a nonspecific manner.[43] Acute critical illness, such as severe trauma, surgery, myocardial infarction, burns, liver failure, intoxication, and starvation, is often accompanied by suppression of gonadotropin secretion and secondary hypogonadism. In contrast, a primary testicular disorder with elevated gonadotropin levels may occur with chronic illnesses. Increased peripheral conversion of androgens to estrogens may produce some features of feminization such as gynecomastia. The association of hypogonadism and feminization with chronic liver disease is well known. Similar primary, secondary or mixed hypogonadism may occur with other chronic illnesses such as chronic anemia, chronic renal failure, rheumatoid arthritis, chronic spinal cord injury, thyroid diseases, Cushing’s syndrome, obesity, human immunodeficiency virus (HIV) infection, and neoplasia. Sex hormone–binding globulin levels are increased with some conditions such as cirrhosis and thyrotoxicosis but suppressed with others such as obesity, hypercortisolism, and hypothyroidism.[43] Numerous drugs have side effects on the reproductive system.[43] Heroin addiction and intrathecal narcotic infusions to control chronic pain suppress luteinizing hormone secretion.[44] Fever can cause transient declines of a few months’ duration.[43, 45, 46] Diabetes mellitus may be associated with impotence in early uncontrolled stages, ejaculatory disorders with autonomic neuropathy, sperm autoimmunity and ROS damaged sperm.[47] Men with renal disease may have infertility of multifactorial origin, including testicular failure from chronic illness, cytotoxic drug exposure, zinc deficiency, and damage to the vasa or penile blood supply during kidney transplantation. However, as with cirrhosis, provided that metabolic decompensation is not severe, semen quality often is adequate for fertility.[43] Epididymal obstruction associated with chronic sinopulmonary disease (Young’s syndrome) was diagnosed frequently in Australia and the United Kingdom in the past yet is rare elsewhere.[48] Some cases of Young’s syndrome may have been caused by mercury poisoning in childhood from calomel-containing teething powders.[49] These were withdrawn from the market in the mid-1950s when it was found that they caused pink disease, and Young’s syndrome is seen rarely. Fetal rubella may result in defective development of the vasa. Bronchiectasis and sinusitis are common in men with immotile sperm from cilial defects.[50] Situs inversus may also be present.

Testicular Symptoms

Previously undescended testes are common in men being investigated for infertility.[14, 51, 52] Undescended testes may be associated with other congenital malformations and disorders of testicular hormone production or action during fetal development, such as Kallmann syndrome, insulin-like factor 3 receptor mutations, androgen receptor mutations or defects of androgen metabolism, and diethylstilbestrol exposure in utero. In Western countries, this condition is usually treated in early childhood, but whether early surgery reduces the severity of the subsequent spermatogenic disorder is unclear.

A randomized controlled trial of orchiopexy for unilateral palpable maldescended testis at 9 months versus 3 years of age showed that surgery at 9 months was followed by significant growth of the testis up to age 4 years but there was no change in testis size in those treated at age 3 [53]. This has led to clinical guidelines for treatment of maldescended testes that recommend orchiopexy for congenital forms between 6 and 12 months of age and as soon as possible for those discovered later and for acquired maldescent. Hopefully this will reduce the frequency of subsequent testicular tumours and spermatogenic defects. A testicular dystrophy may cause both the failure of descent and defective sperm production in adult life despite early surgery. It is difficult to explain otherwise how men with unilateral undescended testes are so frequent in the infertile population. Bilateral undescended testes carry a poorer outlook for fertility than unilateral undescended testes. Infertility after bilateral orchiopexy is approximately six times more common than in the general population and occurs in approximately half of the men, whereas after unilateral orchiopexy, infertility is increased by a factor of two and affects approximately 10%.[51] There may be associated malformation of the epididymides.[54] Rarely the testes may atrophy after surgery because of interference with the blood supply or coincidental torsion.

Episodes of severe testicular pain and swelling may result from torsion, orchitis, or epididymo-orchitis and may be followed by loss or atrophy of the testis. Post-inflammatory atrophy is particularly frequent with mumps orchitis but rare with other illnesses such as glandular fever and brucellosis.[55] Epididymo-orchitis of bacterial origin is commonly associated with urethritis or urinary tract infections and may follow straining with heavy lifting. Sexually transmitted diseases are important, particularly if there was associated epididymal pain or swelling. Some patients have post-gonococcal obstructions in the tails of the epididymides without clear or admitted histories of epididymitis.

Failure of development and a decrease in size of one or both testes are important symptoms of spermatogenic defects. Torsion of the testes may cause atrophy. The vasa may be damaged during hernia repairs and kidney transplantation. Testicular biopsy may inadvertently damage the epididymis, especially if retroversion of the testis is not recognized and the biopsy is performed without taking the testis out of the tunica. Similarly, surgery for torsion, hydroceles, or epididymal cysts may result in the obstruction of the epididymis. Hematomas in the scrotum and infarction of the testes may follow interference with the vascular supply of the testes. Rarely, autoimmune orchitis results from testicular injury or inflammation. Testicular tumors and carcinoma in situ occur with increased frequency in infertile men, even without a history of undescended testes.[21, 56]

Iatrogenic Infertility

Vasectomy and Sertoli-cell-only syndrome caused by cytotoxic chemotherapy and radiation therapy for malignant tumors of the testes, leukemia, lymphoma, and serious autoimmune diseases are the most common forms of medically induced infertility.[57, 58] Although some treatment regimens only suppress spermatogenesis temporarily, recovery of fertility is unpredictable. Alkylating agents, such as cyclophosphamide and busulfan, destroy spermatogonia.[58] Antimetabolites may be used to treat psoriasis, rheumatoid arthritis, or xenograft rejection and can have transient adverse effects on spermatogenesis.[43, 59] Treatment with sulfasalazine for inflammatory bowel disease or arthritis causes a reversible impairment of semen quality.[43] Cessation of sulfasalazine often results in a marked improvement in semen quality over several months. Many other drugs have real or potential adverse effects on spermatogenesis or sexual performance, including androgens, anabolic agents, estrogens, glucocorticoids, cimetidine, spironolactone, antibacterials (especially nitrofurantoin), antihypertensive drugs, and psychotropic agents. However, in practice these are not common causes of infertility.[43]

Anti-spermatogenic Factors

Occupational and environmental exposures may affect reproduction.[60, 61] Exposure to heat from frequent sauna baths, vehicle driving, furnaces, and perhaps working outdoors in summer may cause a decline in spermatogenesis. Impaired testicular heat exchange from obesity and varicoceles may accentuate the effect. Exposure to chemicals in the workplace or elsewhere, particularly nematocides; organophosphates; estrogens; benzene; and welding, zinc, lead, cadmium, and mercury fumes, may have anti-spermatogenic effects. Various social drugs, including tobacco, alcohol, marijuana, and narcotics, are potentially anti-spermatogenic, but these usually require heavy use for an adverse effect.[43, 62, 63] Some addicts have other organ damage, such as cirrhosis, which may further impair testicular function.[43]

Physical Examination

A general physical examination is performed (see Table 2) and specific abnormalities are sought in particular circumstances, for example, of the respiratory system with suspected genital tract obstructions or immotile sperm, the prostate for ejaculatory duct obstruction or prostatitis, the endocrine system for hypopituitarism or other defects associated with testicular failure, the nervous system for autonomic neuropathy with coital disorders, optic field defects with pituitary tumors, and hyposmia with Kallmann syndrome.

Virilization

Hair distribution varies markedly between men. The loss or reduced growth of facial, pubic, axillary, and body hair is an important feature of androgen deficiency but is often unrecognized by patients. Men may note a reduced frequency of the need to shave. The stages of genital and pubic hair development can be recorded according to the method of Tanner. Eunuchoidal proportions (arm span 6 cm greater than height or pubis-to-floor measurement greater than 6 cm longer than one half the height) result from delayed fusion of the epiphyses and are a sign of delayed or incomplete puberty in whites or Asians but can be found with normal testicular function.

Gynecomastia

Gynecomastia of mild degree is common in men with testicular failure of any cause.[43] Marked gynecomastia may be associated with Klinefelter’s syndrome, cirrhosis, androgen receptor defects, estrogen-producing tumors, or anabolic steroid and human chorionic gonadotropin abuse. Galactorrhea is rare in men and usually but not always associated with hyperprolactinemia.[64]

External Genitalia

Examination of the penis for the position of the meatus, phimosis, urethral strictures, and Peyronie’s disease is important because these may influence the adequacy or completeness of ejaculation. Inadequate penile size appears to be an exceptionally rare cause of infertility.[65]

Examination of the scrotal contents is critical in the evaluation of male infertility. A general approach to the examination is outlined in Figure 172-1. This examination should be performed with the man lying and standing.The body of the testes, the head, the body, and the tail of the epididymis and vas are palpated on both sides as shown. Sometimes it is difficult to examine the scrotum thoroughly because of ticklishness or because the scrotum is very tight. Testes may retract into the superficial inguinal region, especially if small. Testes not present in the scrotum may be palpable in the subcutaneous tissue in the groin or, occasionally, in the inguinal canal. Palpable remnants of the vas and epididymis in the scrotum suggest the testis has atrophied completely—the vanishing testis.[66]

Orchiometry

The volume of the testis is determined by comparison with an orchiometer (normal: 15–35 mL).[67] In the absence of varicoceles, the right and left testes are approximately equal in size. Testicular volume is related to body size and number of sperm per ejaculate. As seminiferous tubules occupy more than 90% of the volume of the testes, impairment of spermatogenesis is commonly associated with reduced testicular size. Testicular atrophy suggests severe impairment of spermatogenesis.

Figure 1. . Prader orchiometer for measuring testis volume.

Figure 1. . Prader orchiometer for measuring testis volume.

Testicular Abnormalities

Pain on palpation or excessive tenderness suggests inflammation. Loss of normal testicular sensation may occur with chronic inflammations, neuropathy, or neoplasia. Reduced consistency or softness of the testes is a feature of reduced spermatogenesis. Abnormalities of shape and hard lumps suggest tumors or scars.

Epididymal Abnormalities

Palpable abnormalities include congenital absence of the vas or other failures of development, enlargements of the heads or nodules in the tails of the epididymides with obstruction, spermatoceles, and other cysts and tumors. In men with very small testes (<5 mL), small epididymides suggest severe androgen deficiency, and normal-size epididymides suggest postpubertal testicular atrophy or a severe seminiferous epithelial disorder, such as Klinefelter’s syndrome.

Vasal Abnormalities

Abnormalities of the vas include absence, nodules and gaps with vasectomy, and thickening or beading of the vas with severe post-inflammatory scarring as from tuberculosis.

Miscellaneous Abnormalities

Incidental scrotal findings include scars from surgery, scrotal dermatitis, and pubic fat pads around the genitals in extreme obesity. Inguinal hernias and lipomas and encysted hydroceles of the cord are palpated above and behind the epididymis. Cysts “hydatids” of the appendix testis or epididymis are typically anterior to the head of the epididymis. Spermatoceles and cysts of the paradidymis are in the head or body of the epididymis. Retroversion of the testes, where the vas and epididymis are anterior rather than posterior to the testes is common. Hydroceles of mild degree are common. A tense hydrocele may hide a testicular tumor. Unilateral absence of the vas may be associated with ipsilateral agenesis of the kidney and ureter on the same side. Many of these anomalies have little relationship with infertility.

Checking for Varicocele

With the man standing up, the scrotum can be inspected for swelling of the pampiniform plexus and a cough or Valsalva impulse seen or palpated by holding the spermatic cords between the thumb and index finger of each hand and elevating the testes toward the external inguinal ring. This maneuver reduces the risk of confusing contractions of the cremaster muscles with venous impulses. Varicocele size is graded: cough impulse without palpable enlargement of the spermatic cord (grade 1), palpable enlargement (grade 2), and visible enlargement (grade 3). Although predominantly a left-sided condition, varicoceles may occasionally be on the right side.

The accuracy and reproducibility of clinical examination, even for structures as accessible as those in the scrotum, may not be high. Varicoceles may vary in size from day to day. Even absence of the vasa may be overlooked. With practice, orchiometry can be repeated to within one orchiometer size.

SEMEN ANALYSIS AND OTHER INVESTIGATIONS

Investigations are outlined in Table 3.

Semen Analysis

The most important laboratory investigation in male infertility is semen analysis. The variables assessed and the methods are in the World Health Organization’s laboratory manual.[68] Automation of semen analysis is in progress and should be used in most specialized laboratories soon.[17]

It is crucial that the laboratory is experienced in the performance of semen analyses, and participates in quality assurance activities.[69] There should be a room nearby for the collection of semen. Semen may be obtained by masturbation or coitus using a special nontoxic condom. Ordinary latex contraceptive condoms are unsatisfactory because the rubber usually immobilizes the sperm.[70] If these methods of collection are not possible, postcoital examination of midcycle cervical mucus may give some information about the likelihood of adequate semen quality if many motile sperm are found. In contrast, a negative postcoital test on its own is of little diagnostic value because conception can occur in the same cycle.[71]

The man should be provided with a wide-mouth, sterile, and nontoxic collection jar and written instructions about collection and delivery to the laboratory. A period of abstinence from ejaculation from 2 to 5 (preferably 2) days, delivery of the sample to the laboratory within 1 hour of collection, and avoidance of exposure to lubricants or extremes of temperature are specified.

Because of the variability of results, several semen analyses at intervals of 2 or more weeks are necessary in a man with an abnormality in the first test. Even with complete collection of samples, there is variability caused by counting error, other technical errors, and differences in the ejaculate from day to day (Fig. 2).[68, 72] These large variations need to be remembered when interpreting results of semen analysis.

To check for retrograde ejaculation, urine collected immediately after ejaculation is centrifuged and the pellet examined for sperm.

Figure 2. Variability of semen analysis results in a fertile sperm donor. C, sperm concentration; V, semen volume; M, total motility; MI, motility index-product of grade and percentage of sperm with progressive motility graded 0 to 3. (Mallidis C et al: Variation of semen quality in normal men. Int J Androl 14:99-107, 1991. Used by permission Blackwell Scientific Publications.)

Figure 2. Variability of semen analysis results in a fertile sperm donor. C, sperm concentration; V, semen volume; M, total motility; MI, motility index-product of grade and percentage of sperm with progressive motility graded 0 to 3. (Mallidis C et al: Variation of semen quality in normal men. Int J Androl 14:99-107, 1991. Used by permission Blackwell Scientific Publications.)

Assays of semen constituents from the accessory glands and testis are available: zinc and acid phosphatase from the prostate, fructose from the seminal vesicles, neutral α-glucosidase, glycerophosphocholine, and l-carnitine from the epididymis and inhibin B from the Sertoli cells. Prostatic fluid is acid (pH approximately 6.0), but the ejaculate is alkaline because of the admixture with seminal vesicle fluid. Semen biochemistry is of limited usefulness in clinical practice. Some examples are given in Table 5.

Table 5. Common or Characteristic Patterns of Semen Abnormality
Volume (mL) >1.5* Concentration (106/mL) >15*  Motility (%) >40* Normal Morphology (%) >4*  Comment Cause
0.4 0 - - Fructose 1 nmol/L (low)

pH 6.5 (low)

 Congenital absence of vasa
Ejaculatory duct obstruction
Partial retrograde ejaculation
Testicular failure with androgen deficiency
(Spill or incomplete collection)
4.0 0 - - Fructose 15 nmol/L Genital tract obstruction
Primary seminiferous tubule failure
Secondary seminiferous tubule failure with androgen treatment
3.0 100 0 35 Live 70% (Contamination or condom collection)
Immotile cilia
3.0 100 5 35 Live 20% (Contamination or delayed examination)
Necrospermia
Sperm autoimmunity
3.0 100 65 0 Small round heads Total teratospermia: absent acrosomes
3.0 100 25 10 Liquefaction delayed
Sperm aggregation 2+Live 40% Polymorphs 1 x 106/mL		 Idiopathic asthenospermia Sperm autoimmunity
Prostatitis
(Delayed examination)
3.0 4 30 3 Mixed abnormal morphology Oligospermia of specific or nonspecific causes
3.0 <1 - - Motile sperm present Severe oligospermia of specific or nonspecific causes
Primary seminiferous tubule failure
Partial genital tract obstruction
* WHO Reference Value (5%centile for fertile men)

Immunobead Test

Tests for sperm antibodies should be done routinely on all men being evaluated for infertility despite some clinical guidelines suggesting it is not necessary, because no semen analysis pattern is characteristic of sperm autoimmunity.[42, 68] The immunobead test (IBT) with beads binding to more than 50% of motile sperm is regarded as positive, but there is usually more than 70% to 80% immunoglobulin A (IgA) bead binding with clinically significant sperm autoimmunity. Tail tip–only IBT binding is not significant.[73] The mixed antiglobulin reaction test is an alternative to the IBT.[68] The indirect IBT, in which normal donor sperm are exposed to test serum or seminal plasma, can be used when there are too few motile sperm for the direct IBT. An alternative screening method for sperm autoimmunity in men with sperm in the semen would be to perform a sperm-mucus penetration test.[68]

Sperm-mucus penetration tests can be performed by postcoital examination of sperm in cervical mucus collected at mid-cycle or after estrogen treatment (ethinyl estradiol, 50 g twice daily for 4 days) to produce mucus of equivalent quality.[68] In vitro capillary mucus penetration (Kremer) tests are particularly important for evaluating the significance of sperm autoantibodies; failure of sperm to penetrate more than 2 cm in 1 hour indicates severe sperm autoimmunity with a poor prognosis and a high likelihood of failed fertilization with standard IVF.[42, 73]

Sperm Function Tests

A number of tests of sperm function are available to examine the human fertilization process (Fig. 3). These are only performed in specialist laboratories. If simpler approaches or active preparations of zona pellucida (ZP) or sperm receptor proteins become available, they will be widely used to improve the assessment of human sperm. IVF has permitted many conventional and new tests of sperm function to be examined. Groups of sperm variables that are independently significantly related to the proportion of oocytes that fertilize in vitro can be determined by regression analysis.[74] This approach has confirmed the importance of sperm morphology in the ability of sperm to interact with the coverings of the oocyte.

Figure 3. Stages of human fertilization. Spermatozoa swim through the surrounding medium and cumulus mass (not shown) and bind to the surface of the zona pellucida. The acrosome reaction is stimulated by zona proteins and the acrosome reacted sperm penetrates the zona, enters the perivitelline space and binds to the oolemma via the equatorial segment. Oocyte processes surround the sperm head and it enters the ooplasm and decondenses. Infertility could result from defects of any of these processes. For example, abnormal sperm particularly with defective head morphology bind poorly to the zona. (From Baker, H.W.G., Male Infertility. Chapter 141 In Endocrinology, 6th edition, Jameson J. L. and DeGroot L.J. (Chief Eds.), Saunders Elsevier Philadelphia PA. pp 2556-2579, 2010)

Figure 3. Stages of human fertilization. Spermatozoa swim through the surrounding medium and cumulus mass (not shown) and bind to the surface of the zona pellucida. The acrosome reaction is stimulated by zona proteins and the acrosome reacted sperm penetrates the zona, enters the perivitelline space and binds to the oolemma via the equatorial segment. Oocyte processes surround the sperm head and it enters the ooplasm and decondenses. Infertility could result from defects of any of these processes. For example, abnormal sperm particularly with defective head morphology bind poorly to the zona. (From Baker, H.W.G., Male Infertility. Chapter 141 In Endocrinology, 6th edition, Jameson J. L. and DeGroot L.J. (Chief Eds.), Saunders Elsevier Philadelphia PA. pp 2556-2579, 2010)

Human Sperm–Zona Pellucida Binding Ratio Test

Because the number of sperm bound to the ZP is strongly related to the fertilization rate, human sperm–ZP interaction tests have been developed using oocytes that failed to fertilize in vitro.[74] These oocytes can be used either fresh or after storage in concentrated salt solutions. Because the ZP binding capacity is variable, control (fertile donor) and test sperm are labeled with different fluorochromes (fluorescein and rhodamine). After incubation with equal numbers of control and test sperm, the oocytes are aspirated through a wide bore pipette to dislodge loosely adherent sperm and the numbers of sperm tightly bound to the ZP are counted with a fluorescence microscope. Results are expressed as a ratio of the number of test and control sperm bound to the ZP of four oocytes. An alternative method is to cut the zona and expose one half to test and the other to control sperm (Hemizona assay).[75]

Human Sperm–Zona Pellucida Penetration Test

It is difficult to determine the number of sperm penetrating the ZP when many sperm are bound to the surface. The sperm bound to the surface of the ZP can be sheared off by repeatedly aspirating the oocyte with a pipette with an internal diameter less than the diameter of the oocyte (120 m). The sperm penetrating the ZP or perivitelline space can then be counted easily, and the results of this test are the most predictive of fertilization rates with standard IVF.[74]

Zona Pellucida–Induced Acrosome Reaction Test

Sperm dislodged from the ZP can be stained with a fluorescein-labeled lectin such as pisum sativum agglutinin or an antibody specific for the acrosomal contents to determine the proportion that are acrosome reacted. This test is useful for diagnosis of disordered ZP-induced acrosome reaction.[74]

Human Sperm–Oolemma Binding Ratio Test

Sperm-oolemma binding has been studied in a similar way to the sperm-ZP binding test, using oocytes that have had the ZP removed. [74]

Interpretation of Semen Analysis Results

Table 5 shows various patterns of abnormality of semen quality and their common causes. It is always important to consider whether the result is spurious. Repeated tests are necessary to establish an average and to determine the variability within an individual man (see Fig. 2).

Variations in Semen Volume and Appearance

Low semen volume suggests incomplete collection, short duration of abstinence from ejaculation before the test, absence or obstruction of the seminal vesicles, or androgen deficiency. High semen volume (>8 mL) may be seen in association with oligospermia but is of little practical significance. Hemospermia is usually the result of minor bleeding from the urethra, but serious conditions, such as genital tract tumors, must be excluded. Other discoloration of the semen may indicate inflammation of accessory sex organs. The semen may be yellow with jaundice or salazopyrine administration. Defects of liquefaction and viscosity are relatively common and presumably result from malfunction of the accessory sex organs. Although these may cause problems with semen analysis and preparation of sperm for assisted reproductive technology (ART), they are probably of little relevance to fertility. Sperm agglutination is common with sperm autoimmunity but can also occur for other reasons.

Azoospermia

The total absence of sperm from the semen needs to be confirmed in repeated tests with vigorous centrifugation of the semen and careful examination of the pellet.[68] Cooper and others have shown that sperm maybe found in apparently azoospermic samples using more sensitive sperm-counting methods (e.g. fluorescence microscopy).[76] Rarely, an illness or difficulty with collection will cause transient azoospermia; however, this can also occur for unexplained reasons. With severe spermatogenic disorders and some obstructions, sperm may be present in the semen intermittently. If any live sperm can be found, these can be cryopreserved for intracytoplasmic sperm injection (ICSI).

Oligospermia

Sperm concentrations of less than 15 million/mL or preferably total number (concentration X volume) less than 39 million are classified as oligospermic.[68] This figure represents the 5th percentile derived from analysis of semen analyses preformed using WHO methods in about 1900 healthy volunteers whose partners had a time-to-pregnancy of ≤12 months and is used in the 5th edition of the WHO semen analysis manual .[77] There is a correlation between sperm concentration and other aspects of semen quality. Both motility and morphology are usually poor with oligospermia.

Asthenospermia

Asthenospermia is defined as less than 40% sperm motility or less than 32% with rapid progressive motility.[68] Spurious asthenospermia caused by exposure of sperm to rubber (particularly condoms), spermicides, extremes of temperature, or long delays between collection and examination, should be excluded. Low sperm motility is a frequent accompaniment of oligospermia, and is often also associated with a mixed picture of morphologic defects suggesting defective spermiogenesis.

Specific ultrastructural defects of the sperm can be evaluated by electron microscopy when there is zero sperm motility or extreme asthenospermia (less than 5% motile sperm).[50, 78] Absent dynein arms, other axonemal defects, mitochondrial abnormalities, disorganized fibrous sheath or outer dense fibers, or normal ultrastructure may be found. Standard semen analyses usually show normal sperm concentrations and morphology but there may be tail abnormalities: short, straight, or thick tails, or midpiece defects. Viability tests help to distinguish this group of patients from those with necrospermia.[79] Patients with structural defects in the sperm may be able to be treated by ICSI. Asthenospermia may also be associated with sperm autoimmunity. The causes of other motility defects of moderate degree are unidentified.

Absolute asthenospermia (the condition in which only immotile spermatozoa can be retrieved) is reported at a frequency of 1 in 5000 men and it implies a very poor fertility prognosis. Selection methods currently used to choose a viable sperm for ICSI in the case of absolute asthenospermia are based on chemical substances added to the sample before the procedure or biophysical approaches accomplished during the procedure. [80]

Immotile sperm treatment with pentoxifylline, a nonselective inhibitor of phosphodiesterase has been investigated and proved to be an efficient approach. [81, 82]

Theophylline, a nonselective inhibitor of phosphodiesterase with an increased half-life compared to pentoxifylline was also investigated, recently, [83] in a prospective study on thawed testicular sperm selected for ICSI. Theophylline-treated thawed sperm compared

with untreated sperm showed a significant improvement in searching time, increased fertilization and blastulation rates and higher implantation and clinical pregnancy rates. In a recent case report [84], theophylline has been demonstrated to be an efficient agent for stimulating immotile spermatozoa also in a patient with retrograde ejaculation and total sperm asthenospermia, and a healthy live birth was reported.

Other sperm selection methods as the sperm tail flexibility test (STFT) [85], the hypo-osmotic swelling test (HOS)[86, 87] and laser shot system [88] were reported to be useful.

Necrospermia

It is important to distinguish necrospermia from other types of severe asthenospermia because some patients with necrospermia produce pregnancies despite the low sperm motility.[40, 79, 89, 90] Necrospermia is characterized by usually less than 20% to 30% total motility, less than 5% progressive motility, and a viability test less than 30% to 40%, indicating a high proportion of dead sperm. Other causes of severe asthenospermia such as sperm autoimmunity and collection problems must be excluded. Necrospermia may fluctuate in severity, particularly with changes in coital frequency.[79, 90] Characteristic of necrospermia is an improvement of sperm motility with increased frequency of ejaculation. The condition may be caused by defective storage of sperm in the tails of the epididymides or stasis in the genital tract, and it also occurs with chronic spinal cord injury and with adult polycystic kidney disease associated with cysts in the region of the ejaculatory ducts.[40, 89] There are ultrastructural features of degeneration in the ejaculated sperm but normal structure of late spermatids in testicular biopsies.[79, 89] Treatment with antibiotics may have a beneficial effect, but this is not proved. The couple should have intercourse once or twice every day for 3 to 4 days up to the time of ovulation.

Teratospermia

Teratospermia is a reduced percentage (<4%) of sperm with normal morphology assessed by light microscopy.[68] It is important to distinguish mixed abnormalities of sperm morphology from those in which all or the majority of sperm show a single uniform defect, such as spherical heads with absence of the acrosomes (globospermia) and pinhead sperm. Pinhead sperm result when the centrioles from which the sperm tails develop are not correctly aligned opposite the developing acrosome. On spermiation, the sperm heads are disconnected from the tails and absorbed during epididymal transit so that there are only sperm tails in the ejaculate, the cytoplasmic droplet on the midpiece giving the pinhead appearance.[91] Both these conditions cause sterility but are extremely rare.

In general, human spermatozoa are very variable in appearance and the microscopic assessment of sperm morphology is highly subjective and difficult to standardize between laboratories. Only a small proportion (<25%) of the motile sperm from fertile men are capable of binding the ZP in vitro, and this zona binding capacity is closely related to the morphology of the sperm head.[92] The morphometric characteristics of the sperm that bind to the ZP may be useful as a standard for sperm morphology.[17, 93] Various histological assessments of morphology have been used. The simplest is to record as normal only those sperm that have no shape defects in head, midpiece or tail, regions.[68] In the strict morphology approach, although size measurements are set, the sperm are assessed by eye and those marginally abnormal are assigned abnormal. Automated methods involving image analysis by computer have been developed that could overcome the between-laboratory variability and greatly improve the predictive value of semen analysis for natural conception.[17, 93]

Before the introduction of ICSI, the percentage of sperm with normal morphology assessed by strict criteria after washing the sperm and adjusting the concentration to 80 million/mL, provided one of the most useful predictors of fertilization rates with standard IVF. There was a progressive reduction in oocytes fertilized from 60% to 20% as abnormal morphology increased from less than 70% to more than 95%.[94] Patients with high proportions of sperm with abnormal morphology are now treated by ICSI because of the risk of failure of fertilization with standard IVF. ICSI results are independent of sperm morphology.

Sperm chromatin and abnormal DNA assays

A variety of flow cytometric and other assays to measure sperm chromatin integrity, DNA fragmentation, sperm apoptosis or nuclear integrity have been developed. [95, 96] The usefulness of these tests for prediction of fertility remains controversial [97]

Hormone Assessment

It is not necessary to perform hormone measurements routinely. Follicle-stimulating hormone (FSH) levels in patients with azoospermia, normal testicular volume, and normal virilization may help distinguish genital tract obstruction from a spermatogenic disorder. The most useful FSH value for the upper limit in reproductively normal young men is ~8 IU/L.[98, 99] However, some men with primary seminiferous tubule failure have normal FSH levels. Normal FSH is common with germ cell arrest at the primary spermatocyte stage. Rarely, high FSH levels are seen with normal spermatogenesis.[100] Measurement of FSH, luteinizing hormone, and testosterone is useful in men with reduced testicular volume and signs of androgen deficiency, to distinguish primary from secondary hypogonadism. Inhibin B measurement may provide additional information about the state of spermatogenesis.[101] but is rarely used in routine practice.

Prolactin should be measured in men with galactorrhea or androgen deficiency and loss of libido.[102] Other hormone investigations are occasionally required, such as thyroid function tests with hyperprolactinemia, 17-hydroxyprogesterone measurements with congenital adrenal hyperplasia, estradiol with liver disease or tumors, iron studies to exclude hemochromatosis, human chorionic gonadotropin with tumors and estrogen excess, and pituitary function tests for panhypopituitarism.[43]

Chromosome and Genetic Studies

Chromosomal anomalies are 8-10X more common in infertile than fertile populations, and in many cases there are no other phenotypic changes. The prevalence is inversely correlated to the sperm density, being 14% of azoospermia.[103, 104] Accordingly a routine assessment is recommended in men with unexplained infertility and sperm densities < 5-10 million/ml. A karyotypes is performed in men with clinical evidence of primary testicular failure and small testes to confirm a clinical diagnosis of Klinefelter syndrome. Usually the karyotype is 47,XXY, but there may be higher numbers of X chromosomes or a sex-reversal 46,XX karyotype.[105-107] Although most men with Klinefelter syndrome produce no sperm in the semen, some are oligospermic and very rarely fertile.[105] Also, sperm for ICSI may be obtained by testicular biopsy in about 50% of patients.[106, 107] Defective spermatogenesis may occur with 47,XYY, but the clinical picture is much less uniform than it is for Klinefelter syndrome. The extra Y chromosome is deleted early in gametogenesis because the sperm, embryos, and children generally have normal karyotypes. However, an increased rate of sex chromosomal and autosomal aneuploidy has been noted in studies of sperm from XXY and XYY men.[107, 108] Some Y chromosome abnormalities, such as an isochromosome of two short arms, are associated with absences of spermatogenesis.

Infertile men have much higher rate of aneuploidies compared to fertile men and most of them have no other phenotypic features. An increased frequency of autosomal abnormalities is found with defective spermatogenesis, particularly balanced autosomal translocations (reciprocal and Robertsonian), which may be transmitted in unbalanced form to their offspring.[109] As part of their infertility investigation, it is imperative to screen severely oligospermic men as the result may affect treatment outcome.

Cystic fibrosis gene studies are important for evaluation of patients with congenital absence of the vas and their partners.[110] If the woman has a cystic fibrosis gene mutation, preimplantation genetic diagnosis of their embryos can be offered. Microdeletions in the long arm of the Y chromosome (AZF regions) have been found in 3% to 15% of men with severe primary spermatogenic disorders.[18, 19, 27, 36, 37] Sons of men with these microdeletions have the same microdeletions.[111]

Another gene involved in spermatogenesis (histones replacement) and different from the AZF regions Yq deletions is CDY. ([112] TSPY gene is located on the short arm of Y chromosome and may regulate the timing of spermatogenesis by signaling spermatogonia to enter meiosis. [29]

The sex hormone-binding globulin (SHBG) gene has been studied for possible role in spermatogenesis.[113] Other genes that have been investigated for a possible involvement in fertility are DAZL (sperm count) MTHFR and the estrogen receptors genes (ESR1 and ESR2). Polymorphisms of the promoter region of the estrogen receptor gene have been shown to be related to sperm production. Men with higher numbers of TA repeats have lower sperm counts. [29]

Androgen receptor defects have also been found in some men with unexplained primary spermatogenic disorders. Mutations in the gene impairing androgen receptor activity produce androgen insensitivity, which has a variable phenotypic expression from testicular feminization to otherwise normal males with gynecomastia or hypospermatogenesis and oligospermia.[33] Increases in the number of CAG repeats in exon 1 over approximately 40 are associated with Kennedy disease (progressive spinobulbar atrophy) and men with this condition may be infertile.

The field of epigenetic errors has also been studied for its possible contribution to male infertility. [114] (Table 4)

Other specific genetic tests and family studies may be indicated on clinical grounds (see Table 4).

At present, it is reasonable to screen all infertile men with otherwise unexplained primary spermatogenic defects with average sperm concentrations less than 5-15 million/mL by karyotype and Yq microdeletion testing.[115] All patients should be counseled about the possibility of transmitting known and unknown genetic defects.

Testicular Biopsy

Testicular biopsies are necessary to assess spermatogenesis in men with presumed genital tract obstruction. A significant proportion of men with azoospermia, normal testicular size, and normal FSH are found to have severe spermatogenic disorders.[14] Some severe spermatogenic defects may be incomplete, and because ICSI can be performed if sperm can be obtained from the testes, diagnostic testicular biopsies should be offered to men with severe primary spermatogenic tubule disorders with persistent azoospermia. If any elongated spermatids can be found, it should be possible to perform ICSI. However, if no elongated spermatids are seen in the diagnostic biopsies it still may be possible to find spermatids by more extensive sampling of testicular tissue with open biopsies (see later).

It is most important that tissue for histology is removed from the testes with minimal damage and placed in a suitable fixative, such as Bouin’s or Steive’s solution. Standard formalin fixatives destroy the cytoarchitecture.

Testis biopsies may be performed under local or general anesthesia. Needle biopsy many obtain only isolated cells but these may be sufficient for diagnosis based on cytology or for flow cytometry. The technique shown in Fig. 4 usually provides sufficient material for a histologic diagnosis of the state of the seminiferous epithelium despite some deformation artifacts.[116] It is also useful for obtaining testicular sperm for ICSI.[117] Complications are rare and include minor bleeding in the skin and testis, and rarely hematoma or reactions to the local anesthetic. Failure to obtain tissue occurs particularly with fibrosed or small (<5mL) testes

In the presence of azoospermia, an open testicular biopsy might not just be a therapeutic approach in the way of sperm retrieval, but also the only way of excluding early testicular germ cell neoplasia or even an overt testicular cancer.

Infertile men are more likely to develop testicular cancer compared to men with normal fertility.[118] Testicular intra-tubular germ cell neoplasia of the unclassified type (ITGCNU) also called carcinoma in situ (see below) is the precursor of testicular germ cell tumors in which the neoplastic cells are confined within the seminiferous tubules. This makes it a non-invasive stage of the disease. It can be found in testicular tissue adjacent to germ cell tumours in more than 90 percent of adult cases. [119, 120]

The incidence of ITGCNU in men undergoing fertility evaluation ranges between 0.4 - 1.1 percent.[121, 122] As ITGCNU is asymptomatic, patients remain undiagnosed until an overt tumour can be identified usually by palpation. A sample from a sperm retrieval open biopsy should be sent to histopathology routinely.

Figure 4. (A) Fine-needle tissue aspiration biopsy of the testis. Local anesthetic is injected around the vas to block testicular sensation. (B) Fine-needle tissue aspiration biopsy of the testis A 21 gauge butterfly needle is inserted into the testis. An assistant applies suction to the needle tubing via a 10mL syringe and the operator makes thrusting movements of the needle into the substance of the testis. (C) Fine-needle tissue aspiration biopsy of the testis while maintaining the suction the needle is removed carefully and any seminiferous tubules protruding from the needle are grasped with fine forceps to avoid them falling back into the puncture hole. With this technique seminiferous tubule sections are sucked into the needle and these are expelled into some culture medium. Portions can be sent for histology and the remainder used for extraction of sperm in the IVF laboratory by stripping the seminiferous tissue out of the connective tissue membrane of the seminiferous tubule.

Figure 4. (A) Fine-needle tissue aspiration biopsy of the testis. Local anesthetic is injected around the vas to block testicular sensation. (B) Fine-needle tissue aspiration biopsy of the testis A 21 gauge butterfly needle is inserted into the testis. An assistant applies suction to the needle tubing via a 10mL syringe and the operator makes thrusting movements of the needle into the substance of the testis. (C) Fine-needle tissue aspiration biopsy of the testis while maintaining the suction the needle is removed carefully and any seminiferous tubules protruding from the needle are grasped with fine forceps to avoid them falling back into the puncture hole. With this technique seminiferous tubule sections are sucked into the needle and these are expelled into some culture medium. Portions can be sent for histology and the remainder used for extraction of sperm in the IVF laboratory by stripping the seminiferous tissue out of the connective tissue membrane of the seminiferous tubule.

For clinical purposes, testicular histology is classified as follows: normal or hypospermatogenesis (all the cellular elements of spermatogenesis are present but in reduced numbers), germ cell arrest (the earlier cellular elements of spermatogenesis are present but at a certain stage, the process stops, most often at the primary spermatocytes), Sertoli-cell-only syndrome or germ cell aplasia (the tubules contain Sertoli cells but no germ cells), hyalinization (the cellular elements have disappeared, leaving only thickened seminiferous tubule walls as in Klinefelter syndrome), and immature testis (no gonadotropin stimulation, prepubertal appearance).[123] Examples are shown in Figure 5. Other classifications such as partial or incomplete maturation arrest and partial germ cell aplasia cause confusion in the literature and should not be used.[124]

Figure 5A. Testicular histology from fine-needle aspiration samples. Normal.

Figure 5A. Testicular histology from fine-needle aspiration samples. Normal.

Figure 5B. Testicular histology from fine-needle aspiration samples. Left mild hypospermatogenesis with elongated spermatids with poor head morphology: right normal.

Figure 5B. Testicular histology from fine-needle aspiration samples. Left mild hypospermatogenesis with elongated spermatids with poor head morphology: right normal.

Figure 5C. Testicular histology from fine-needle aspiration samples. Mild-moderate hypospermatogenesis.

Figure 5C. Testicular histology from fine-needle aspiration samples. Mild-moderate hypospermatogenesis.

Figure 5D. Testicular histology from fine-needle aspiration samples. Moderate-severe hypospermatogenesis.

Figure 5D. Testicular histology from fine-needle aspiration samples. Moderate-severe hypospermatogenesis.

Figure 5E. Testicular histology from fine-needle aspiration samples. Germ cell arrest at the primary spermatocyte stage.

Figure 5E. Testicular histology from fine-needle aspiration samples. Germ cell arrest at the primary spermatocyte stage.

Figure 5F. Testicular histology from fine-needle aspiration samples. Sertoli cell-only syndrome: low and high power.

Figure 5F. Testicular histology from fine-needle aspiration samples. Sertoli cell-only syndrome: low and high power.

Figure 5G. Testicular histology from fine-needle aspiration samples. Germ cell arrest at the spermatogonial stage from gonadotropin deficiency. Upper panel atrophic Leydig cell stained brown with anti-testosterone antibody.

Figure 5G. Testicular histology from fine-needle aspiration samples. Germ cell arrest at the spermatogonial stage from gonadotropin deficiency. Upper panel atrophic Leydig cell stained brown with anti-testosterone antibody.

Figure 5H. Testicular histology from fine-needle aspiration samples. Carcinoma in situ, only transformed spermatogonia and Sertoli cells present.

Figure 5H. Testicular histology from fine-needle aspiration samples. Carcinoma in situ, only transformed spermatogonia and Sertoli cells present.

Other Investigations

Ultrasonography is useful to check for tumors in the testes, particularly when the testes are difficult to palpate because of a tense hydrocele.[125] Other abnormalities may also be found (Figs 6-8). It can also be used to measure testicular size and confirm the presence and nature of cysts or other abnormalities in the scrotum. Some argue scrotal ultrasound should be performed routinely in infertile men to measure testicular volume, assessing the texture and exclude impalpable malignant tumors in the testes.[126] However, some clinical guidelines do not support this. [127] Doppler blood flow assessment is valuable in assessing a painful swollen testis for torsion or inflammation and for evaluating varicoceles. Other tests of a varicocele, including thermography, technetium scans, and venography may be performed but, as pointed out later, the value of treating varicoceles to improve fertility is uncertain. Rectal ultrasound may demonstrate cysts in the prostate, enlarged seminal vesicles, or dilated ejaculatory ducts associated with distal genital tract obstructions (Figure 8).[128] Clinical suspicion of the presence of a pituitary tumor should be followed up by appropriate radiology. Abdominal imaging is necessary to check the position of impalpable testes.

FIGURE 6. Ultrasounds of testes with tumours. A Longitudinal, testis with seminoma in a man presenting with infertility, severe oligospermia and a large hard right testis; B Low power histological section of radical orchiectomy specimen with active tumour stained blue, and necrotic centre and seminiferous tubules stained red; C, Londitudinal and D transverse of impalpable 1cm diameter seminoma in the upper pole of the right testis in a man presenting with severe oligospermia and normal sized testes.

FIGURE 6. Ultrasounds of testes with tumours. A Longitudinal, testis with seminoma in a man presenting with infertility, severe oligospermia and a large hard right testis; B Low power histological section of radical orchiectomy specimen with active tumour stained blue, and necrotic centre and seminiferous tubules stained red; C, Londitudinal and D transverse of impalpable 1cm diameter seminoma in the upper pole of the right testis in a man presenting with severe oligospermia and normal sized testes.

FIGURE 7. Miscellaneous findings on scrotal ultrasound. A,B multiple epididymal and intratesticular cysts in a man with von Hippel Lindau syndrome; C Simple hydrocele; D Multiloculated hydrocele; E Simple cysts in head of the epididymis; F Ectasia of the rete testis; G Hypoechoic area in periphery of testis of uncertain significance; H Microlithiasis in a severely atrophic testis; I Transverse blood vessel; J Intratesticular varicocele; K Blood flow in intratesticular varicocele.

FIGURE 7. Miscellaneous findings on scrotal ultrasound. A,B multiple epididymal and intratesticular cysts in a man with von Hippel Lindau syndrome; C Simple hydrocele; D Multiloculated hydrocele; E Simple cysts in head of the epididymis; F Ectasia of the rete testis; G Hypoechoic area in periphery of testis of uncertain significance; H Microlithiasis in a severely atrophic testis; I Transverse blood vessel; J Intratesticular varicocele; K Blood flow in intratesticular varicocele.

FIGURE 8 Ultrasound of undescended testes. A - In neck of scrotum at external inguinal ring with small tumour; B - In inguinal canal; Incidental prostatic cyst in a healthy man: C - transverse, D - sagittal; Ejaculatory duct obstruction: E - prostate transverse - dilated ejaculatory duct; F - transverse, dilated seminal vesicle; G - sagittal, dilated duct and seminal vesicle.

FIGURE 8 Ultrasound of undescended testes. A - In neck of scrotum at external inguinal ring with small tumour; B - In inguinal canal; Incidental prostatic cyst in a healthy man: C - transverse, D - sagittal; Ejaculatory duct obstruction: E - prostate transverse - dilated ejaculatory duct; F - transverse, dilated seminal vesicle; G - sagittal, dilated duct and seminal vesicle.

MANAGEMENT OF SPECIFIC CONDITIONS

This section addresses the management of sperm autoimmunity, male genital tract obstructions, coital disorders, genital tract inflammation, and varicocele. Treatment of gonadotropin deficiency and androgen replacement therapy are covered in Chapters by Hayes and Pitteloud, and Handelsman.

Sperm Autoimmunity

Sperm autoimmunity is present in 6% to 10% of infertile men.[42] They have sperm coated with antibodies to the extent that sperm function is impaired, particularly sperm - mucus penetration and sperm zona-pellucida binding, resulting in severe infertility. Natural pregnancy rates without treatment are very low and fertilisation rates with standard IVF are low or zero. Other men have positive IBT, sometimes with only to tail tip binding and normal or only marginally impaired mucus penetration.[73] These low-level sperm autoantibodies are irrelevant to the infertility and other causes of the couple’s infertility should be sought. Sperm autoimmunity can be treated by glucocorticoids in immunosuppressive doses. Antibody levels fall and semen quality improves in about 50% of patients and about 25% produce natural conceptions during a 4-6 month course of treatment.[42] There are significant risks of severe side effects particularly aseptic necrosis of bone. The superior results of ICSI make this treatment obsolete and only useful in exceptional circumstances.

Genital Tract Obstruction

Clinical Characteristics

Most men with genital tract obstruction have azoospermia, normal testicular size, normal virilization, and normal serum FSH levels. However, some have combined obstruction and spermatogenic disorders or partial obstructions and severe oligospermia. There may be a history of an event that caused the obstruction, such as epididymitis with gonorrhea or associated respiratory disease. Because a few men with normal spermatogenesis have elevated FSH levels and some spermatogenesis may occur in association with a severe spermatogenic disorder, all patients should be offered further investigation. The presence of sperm antibodies in blood serum by indirect IBT indicates sperm are being formed but is an adverse prognostic factor for natural conception after surgery. With bilateral congenital absence of the vasa or ejaculatory duct obstruction, semen volume, pH, and fructose levels are low. The semen also does not have its characteristic smell and does not form a gel after ejaculation because it contains only prostatic and urethral fluid. Rectal ultrasound shows absent or atrophic seminal vesicles with bilateral congenital absence of the vasa but with ejaculatory duct obstruction the seminal vesicles and ejaculatory ducts are dilated and the cause of the obstruction may be obvious such as a cyst of the prostatic utricle.[128] Testicular biopsy is usually normal but there may be some reduction in spermatogenesis either as a coincidence or as a result of the obstruction, particularly after vasectomy.[129]

Pathophysiology

Degeneration of the Wolffian duct structures occurs with cystic fibrosis gene mutations but can be of variable extent. Although most often only the heads of the epididymides are palpable, some men with bilateral congenital absence of the vasa have parts or all of the epididymides and scrotal vasa present with absent or atrophic pelvic vasa and seminal vesicles. Other conditions may cause defective development of the vasa such as congenital rubella. Young’s syndrome which is now rare is not related to cystic fibrosis gene mutations. The pathology shows inspissated material in the head of the epididymis, and there are lipid inclusions in the epithelial cells.[48, 49] As some men with this syndrome have fathered children, the blockage may develop in adulthood.

Post-inflammatory obstructions after gonorrhea typically occur in the tail of the epididymis, whereas nonspecific bacterial inflammation produces more widespread destruction, and tuberculosis usually causes multiple obstructions in the epididymides and vasa or destruction of the prostate and seminal vesicles. Back pressure ‘blowout’ obstructions in the epididymis are frequent after vasectomy. Iatrogenic causes of genital tract obstruction include inadvertent epididymectomy during testicular biopsy, vasal damage during hernia repair or pelvic or lower abdominal surgery such as renal transplantation, and ejaculatory duct obstruction from prostatectomy or complicated bladder catheterization.

Differential Diagnosis

Men with persistent azoospermia, normal testicular size, normal virilization, and normal FSH levels can be assumed to have obstruction until proved otherwise. As many as one third of men with this clinical picture are found to have a serious spermatogenic disorder on testicular biopsy despite the normal serum FSH level.[14] There are rare instances of normal men who show azoospermia on single occasions or over a short period.[6, 20] This “spurious azoospermia” must be excluded before surgery is contemplated. Once diagnosis of obstruction is confirmed, it is necessary to determine the feasibility of surgery. Intratesticular and caput-epididymal obstructions have a poor prognosis, but cauda-epididymal and vasal obstructions can often be treated successfully with surgery.[130] Distal obstructions are important to diagnose because they may be reversed at transurethral endoscopy.[128] It is important to test for sperm in urine after ejaculation in patients with possible ejaculatory duct obstruction as partial retrograde ejaculation can produce the same the semen characteristics as ejaculatory duct obstruction (Table 5). Also large cysts causing ejaculatory duct obstruction can affect the bladder neck and cause retrograde ejaculation.

Treatment

General Management

Patients in whom one or both vasa deferentia are not palpable have congenital unilateral or bilateral absence of the vas deferens. Fifty to eighty-two percent of healthy men with CBAVD harbor detectable mutations in one or both alleles of the CFTR gene . These men should all be tested for CFTR abnormalities. CFTR testing is also indicated in azoospermic patients with normal testis volume, normal serum FSH, and palpable vasa deferentia, who have low semen volume (<1.5 ml). Female partners of carriers should be screened for cystic fibrosis gene mutations and the couple counseled accordingly. Although most consider genetic testing unnecessary, high proportion of men with idiopathic epididymal obstruction were shown to be carriers of CFTR mutations.[131] Preimplantation or prenatal genetic diagnosis may be performed if mutations are found in both partners. The woman should be investigated in detail to ensure her potential fertility before surgery is contemplated in the man. The prognosis of the procedure and the availability of other forms of treatment, including donor insemination, should be discussed with the couple. Sperm retrieval for ICSI, either from the testis or other parts of the genital tract, is an alternative to surgery.[117] ICSI is also used when reconstructive surgery is not possible, the female partner has an infertility problem, or the couple cannot wait 6 to 12 months to have a reasonable attempt at conceiving naturally after surgery. For ICSI, sperm may be obtained by testicular biopsy or percutaneous sperm aspiration from the epididymis under local anesthesia. If a spermatocele is present, usable sperm may be obtained by direct puncture through the scrotal skin. It may be possible to combine vasoepididymostomy with sperm aspiration for cryopreservation or ICSI.

Epididymal and Vasal Surgery

Treatment of male genital tract obstructions is best undertaken by specialist microsurgeons.[130] The testis is exposed and the most proximal (to the testis) level of obstruction determined. The patency of the vas is determined by syringing with saline or by vasography. The vas or epididymal tubule is opened proximal to the obstruction, and, if possible, the presence of motile sperm is demonstrated by microscopy. Microsurgical anastomosis between the ends of the vas or between the vas and the epididymal tubule is then undertaken.

Results

Vasovasostomy and vasoepididymostomy for caudal blocks produce relatively good results, with 50% to 80% of patients having sperm present in the semen. However, less than half of these produce a pregnancy within the first year.[130] There may be continuing obstruction, sperm autoimmunity, or coexisting spermatogenic disorders. The results of vasoepididymostomy for proximal blocks are poor. Although sperm may appear in the semen, pregnancies are extremely uncommon after vasoepididymostomy for caput epididymal blocks. In contrast, the results of ICSI with testicular or epididymal sperm, fresh or after cryopreservation, are similar to those obtained with sperm from semen.[117] 

Gonadotrophin deficiency and suppression

Clinical Characteristics

Most men seeking treatment for infertility associated with gonadotropin deficiency have been treated with androgens, following presentation in adolescence with delayed puberty. The main diagnoses are Kallmann’s syndrome, other isolated gonadotropin deficiencies, combined gonadotropin and growth hormone deficiency and rarely pituitary tumours, trauma or craniopharyngiomas treated in childhood. [30] Occasionally men with previously undiagnosed prepubertal gonadotropin deficiency present with infertility. The clinical features are usually very small testes (<4mL) and severe androgen deficiency. There may be a child like appearance with lack of secondary sex hair development, failure of male pattern scalp hair recession and balding and eunuchoidal proportions. Gonadotropin deficiency may develop after puberty because of tumours, surgery or trauma of the pituitary or hemochromatosis. These men usually note loss of libido and may note reduced beard and body hair growth, low ejaculate volume and decreased testicular size. General lethargy, muscular weakness and hot flushes are also common but non-specific symptoms. Physical examination may show testicular atrophy, reduced secondary sex hair and dry finely wrinkled skin on the face. Gynaecomastia may be present. Features of underlying or associated conditions may be present for example: headache, visual disturbance and hormone excess or deficiency with pituitary tumours, or pigmentation, liver disease or diabetes with haemochromatosis.

Hyperprolactinemia is uncommon in men.[132, 133] It usually presents with loss of libido and impotence, low testosterone levels and variable semen analysis results from azoospermia to relatively normal. Galactorrhea may occur, sometimes with only minimal gynaecomastia. There is usually a pituitary tumour. Hyperprolactinemia associated with a pituitary macroadenoma is rare but important: as well as loss of libido there is usually progressively severe headache and visual field impairment. A number of paediatric syndromes include mental deficiency and gonadotropin deficiency but the patients rarely seek treatment for infertility as adults. Mutations of DAX1 cause adrenal hypoplasia and gonadotropin deficiency.

Gonadotropin suppression may occur in a variety of circumstances. The most common now appears to be the illicit use of anabolic and androgenic steroids or chorionic gonadotropin or opioid for chronic pain. [6, 14] [43, 134]Other hormones and drugs can cause gonadotropin suppression. Rarely men are seen with hormone producing tumours for example adrenal adenomas, Leydig cell tumours or hCG producing teratomas which will suppress gonadotropins, usually there are features of marked hyperestrogenization with progressive gynecomastia. Very rarely men are seen with congenital adrenal hyperplasia with gonadotropin suppression and azoospermia who can be treated successfully by glucocorticoid suppression of ACTH. [38, 39]

Spermatogenesis may occur despite severe androgen deficiency - the so-called fertile eunuch syndrome. This is believed to be due to predominant LH deficiency or partial gonadotropin deficiency. There may be normal sperm concentrations but usually there is low ejaculate volume and sperm motility. The fertile eunuch syndrome commonly occurs with hyperprolactinemia, hemochromatosis, starvation, illness or in athletes in negative energy balance. It is also seen with partial or mild Kallmann syndrome.

Pathophysiology

Commonly gonadotropin deficiency is caused by genetic disorders of gonadotropin releasing hormone production or the GnRH receptor, loss of function of gonadotrophs, or suppression of gonadotropin secretion by extraneous steroids, other drugs or illness. There is usually a combined defect of androgen and gamete production. If the underlying cause cannot be corrected life-long androgen replacement therapy is required. This is usually in the form of testosterone but when fertility is desired, treatment must be changed to gonadotropins. While experimental conditions may be found to indicate that either FSH or LH alone may be able to initiate spermatogenesis in humans, for practical clinical purposes treatment with LH alone (as hCG) is effective for fertile eunuch syndrome and may be effective where spermatogenesis has been stimulated before, either by natural puberty or previous gonadotropin therapy. In other situations both FSH and LH are required (see chapter by Hayes and Pitteloud).

Differential Diagnosis

In men with gonadotropin deficiency it is necessary to determine the cause of the disorder, or if this is not possible to exclude a serious underlying cause such as a pituitary tumour. With Kallmann syndrome there is hyposmia or anosmia from malformations of the rhinencephalon. Other abnormalities may also be present including colour blindness, cleft lip and cerebellar ataxia. [14] [30] Except where the diagnosis is obvious, detailed radiological examination of the pituitary and hypothalamic area is necessary, together with full pituitary function tests to determine if there are other hormone deficiencies.

Treatment

Gonadotropin suppression from administration of steroids or other agents is treated by withdrawal of the agents, and starvation induced gonadotropin suppression by refeeding. Hyperprolactinemia can be treated with bromocriptine or other dopamine agonist. [20] [102]

Gonadotropin deficiency caused by of gonadotrophe destruction or abnormalities of the GnRH receptor require treatment with gonadotropins. [20, 135] Some men with gonadotropin releasing hormone deficits can be treated successfully with pulsatile GnRH administration. For details of the gonadotropin and GnRH treatment, see chapter by Hayes and Pitteloud.

Coital Disorders

Male coital disorders impacting fertility include erectile dysfunction (impotence), failure of ejaculation, and retrograde ejaculation. Many men have problems with sexual performance after first learning about their infertility, but this usually ameliorates with time. Infrequent and poorly timed intercourse may result from incorrect advice, low libido, or the psychological reaction to infertility.[6]

Erectile dysfunction

Erectile dysfunction may be associated with low libido from androgen deficiency with primary or secondary hypogonadism. (This topic is considered in detail in Chapter 8 by Bochinski et al.) Erectile dysfunction related to vascular or neurologic abnormalities (diabetic autonomic neuropathy or pelvic nerve damage) is uncommon in men presenting with infertility.[14] Selective impotence at the time of ovulation may indicate psychological problems and ambivalence about having children.

Failure of Ejaculation

Failure of ejaculation is usual with chronic spinal cord injury and may also be caused by antihypertensive and psychotropic drugs but otherwise is an infrequent cause of infertility in most societies.[89, 136] Healthy men who cannot ejaculate with intercourse may be able to produce semen by masturbation, with a vibrator, or other stimulation.

Retrograde Ejaculation

Retrograde ejaculation occurs when the bladder neck fails to contract at the time of ejaculation so that all or most of the semen passes into the bladder. Usually, there is an obvious cause: prostatic surgery, diabetic neuropathy, pelvic nerve damage, or spinal cord injury. Retrograde ejaculation is diagnosed by the finding of sperm in urine passed after ejaculation.

Differential Diagnosis

Recognition of a coital disorder is crucial and thus all infertile patients must discuss their sexual history in detail. Once recognized, the contribution of organic and psychological factors needs to be evaluated.

General Treatment

An optimistic prognosis can be given provided that live sperm can be obtained. The couple is advised about the various techniques that might be used for collecting the sperm for artificial insemination or other ART. The woman’s potential fertility must be evaluated.

Specific Treatment

A drug, such as an antihypertensive or a tranquilizer, that may be contributing to the sexual disorder should be stopped temporarily or permanently.[43] Erectile dysfunction may respond to sex behavioral therapy, administration of phosphodiesterase 5 inhibitors, intrapenile injections of vasodilators and physical approaches with pumps and rubber occlusion devices to initiate and maintain erections or penile implants, but these are seldom needed in men with infertility. Some men with failure of ejaculation or retrograde ejaculation may be able to ejaculate during intercourse with a full bladder or after the administration of phosphodiesterase 5 inhibitors, imipramine, or cholinergic antihistamines, such as brompheniramine or ephedrine.[136] Others require more powerful stimulation with vibrators or electroejaculation.[89] If these are unsuccessful, sperm may be collected by needle biopsy of the testis.[137]

Use of Collected Semen

If semen can be obtained by masturbation or by wearing nontoxic condoms to collect nocturnal emissions, the couple can be taught to inseminate samples at home. The timing of ovulation can be determined by calendar and either symptoms of ovulation or luteinizing hormone surge detected with a urinary luteinizing hormone dipstick kit. Cryopreservation of samples for AIH or ICSI may also be possible.

Assisted Ejaculation

Ejaculation may be stimulated by applying a vibrator to the underside of the penis near the frenulum of the glans. Vibrators with a 2-mm pitch and frequency of 60 Hz or more are most effective. Men with complete spinal cord injuries below T10 are unlikely to respond and will require electroejaculation. Modern electroejaculation equipment is safe. The probe includes a thermal sensor and proctoscopy is performed before and after the procedure to ensure there are no burns or other damage to the rectum. A balloon catheter in the bladder is used to prevent retrograde ejaculation.[89]

Semen obtained by assisted ejaculation from able-bodied men or in the acute stages of spinal cord injuries is often normal.[89] In contrast, with chronic spinal cord injury, there is frequently low volume, high sperm concentration, and poor motility.[89] As with necrospermia, repeated ejaculation over several days can improve sperm motility. If the semen quality is too poor for AIH or the risks associated with electroejaculation are considered unacceptable, aspiration of sperm from the testis and ICSI produces good results. Assisted ejaculation may cause autonomic hyperreflexia with chronic spinal cord injuries above T6.[89] The resulting uncontrolled hypertension may cause cerebral hemorrhage. Careful monitoring of blood pressure and prophylactic nifedipine treatment usually prevents serious problems. Men without complete sensory deprivation require general anesthesia for electroejaculation.

Retrieval of Sperm with Retrograde Ejaculation

Motile sperm may be obtained from the urine after retrograde ejaculation.[138] Urinary pH is adjusted to above 7 and osmolality to between 200 and 400 mOsm/kg by administration of 80 g of sodium bicarbonate and 2.0 to 2.5 L of water daily for 3 days before the expected time of ovulation. On the day of ovulation, the man ejaculates and passes urine. Sperm are recovered from the urine by centrifugation, washed, and resuspended in an IVF culture medium. The final pellet is resuspended in approximately 0.5 mL of culture medium for insemination. It is also possible to cryopreserve the sample obtained. If this method fails, electroejaculation and catheterization of the bladder could be considered.

Effects of Systemic Illness and Reversible Exposures to Toxins or Drugs

A very large number of exposures to agents in the environment, drugs, and illnesses can adversely affect testicular function, but it is rare to find patients in whom such exposures can be confirmed as contributing to male infertility. However, this should always be considered during clinical evaluation. The most commonly encountered problems clinically are impairment of spermatogenesis by salazopyrine used for treatment of inflammatory bowel disease or arthritis, testosterone administration, anabolic steroid abuse, long-term high-dose opiate use, and febrile illnesses causing transient reduction of spermatogenesis.[43] Workplace exposures may be implicated in some patients, but the association is rarely clear-cut enough to advise change of occupation.[60, 61]

Acute Illnesses

Fever

The adverse effect of acute febrile illness on the semen quality is well known but only occasionally seen.[43, 45, 46] Frequent hot baths or saunas may also have a similar effect. There is a temporary suppression of spermatogenesis that recovers over 3 to 6 months. Whether increased scrotal temperature because of clothing, varicocele, obesity, or environmental temperature contributes to male infertility is controversial.

Critical Conditions

Suppression of gonadotropin secretion can occur with critical illness such as hepatic failure, myocardial infarction, head injury, stroke, respiratory failure, congestive cardiac failure, sepsis, burns, starvation, general anesthesia and severe stress, both psychological and physical.[43] Transient decreases occur after drug or alcohol intoxication, anesthesia, and surgery. The reduction in testosterone is proportional to the severity of some of the critical conditions and may predict the likelihood of recovery. There may also be direct effects on the testes and alterations in sex hormone–binding globulin levels. The shutdown of testicular function may be a useful adaptation to illness or starvation. During recovery from the critical condition, pulsatile secretion of gonadotropins increases in a manner reminiscent of the changes with puberty, and gynecomastia may develop.[43]

Nutritional Aspects

Starvation is associated with gonadotropin suppression. Specific deficiencies of vitamins and minerals such as B12, C, folate, and zinc may affect testicular function, but these are rare in Western countries.[139] Simple obesity may be associated with alterations in the hypothalamic-pituitary-testicular axis and impaired scrotal thermoregulation. The most common changes are increased conversion of androgens to estrogens in peripheral tissues and low sex hormone–binding globulin levels related to insulin resistance. Total testosterone, sex hormone–binding globulin levels and gonadotropin levels may be low and estrogen levels elevated. There are reports of hypogonadotrophic hypogonadism associated with gross obesity and indications that it can be treated with aromatase inhibitors.[140, 141] However, clinical androgen deficiency, estrogen excess, and abnormal semen analysis are not regularly seen in moderately obese men and the cause and effect relationship is not clear. It may be reduced testicular function predisposes to or aggravates obesity.

Obesity

Obesity in men is associated with low serum gonadotropin, total testosterone, and free testosterone concentrations. The low serum total testosterone concentrations is attributed to associated decrease in serum sex hormone binding globulin (SHBG)

Free testosterone concentrations appear to be inversely related to BMI, independent of changes in SHBG [142, 143] Other factors contributing to the hypogonadotropic hypogonadism seen with obesity include an increase in estrogens through aromatization in adipose tissue, insulin resistance, metabolic syndrome, diabetes mellitus, and sleep apnea [144-146]

Sperm quality has been reported to be inversely related to BMI. [147] U shaped relationship with lower sperm numbers was shown with both low and high BMI [148] Reversibility of obesity-associated male infertility with weight loss should be further investigated.[149]

Chronic Illnesses

Impairment of testicular function is common in uncontrolled or poorly controlled chronic diseases.[43] There are usually elevated gonadotropin levels, indicating a primary testicular defect, but impaired gonadotropin secretion, hyperprolactinemia, changes in sex hormone–binding globulin, and increased aromatization of androgens to estrogens may occur. Although this pattern of abnormal testicular function is a common nonspecific response to chronic illness, the mechanism is obscure. There may be symptoms and signs of androgen deficiency and estrogen excess. Hepatic cirrhosis is one of the classic conditions known to have a profound adverse effect on the male reproductive function. Testicular function may recover after liver transplantation. Similar primary hypogonadism may occur with non-cirrhotic liver disease, chronic alcoholism without liver disease, and a variety of chronic diseases without alcoholism: chronic anemias, chronic renal failure, thyroid hyper- or hypofunction, HIV infection, lymphoma, leukemia, advanced metastatic cancers, rheumatoid arthritis, severe cardiac disease, and chronic respiratory disease.

Effects of drugs

Drugs may contribute to male infertility by affecting gonadotropin (e.g., steroids, opiates) or prolactin secretion (psychotropic agents), or spermatogenesis (salazopyrine, alkylating agents) or by reducing sexual performance (psychotropic and antihypertensive drugs). Some drugs may also cause gynecomastia (antiandrogens, estrogens).[43, 134]

There is currently no place for testosterone treatment of infertile men, either continuously for low testosterone levels resulting from primary or secondary testicular failure, or as “testosterone rebound” therapy because testosterone suppresses gonadotropin secretion and reduces spermatogenesis. Abuse of androgens is widespread in people hoping to enhance athletic performance or bodybuilding. Some men are seen for infertility with azoospermia or oligospermia as a result. Others have sexual performance problems after stopping drug use. The patient may conceal the abuse. Normal virilization but low testosterone, low sex hormone–binding globulin, and low, normal, or transiently high gonadotropin levels may be seen. Recovery can take several months sometimes longer than 12 months, particularly after depot anabolic steroids.

Salazopyrine used for bowel disease and arthritis commonly causes spermatogenic defects. Usually, there is poor sperm motility and morphology or oligospermia. The semen may be stained yellow. The antispermatogenic effect is caused by the sulfapyridine in the drug. Stopping the drug results in a recovery of sperm output within a few months provided the patient’s health remains good and he does not have an underlying defect of spermatogenesis.

Other drugs and toxins are claimed to have adverse effects on spermatogenesis such as colchicine and anticonvulsants, and some antihypertensive drugs, calcium-channel blockers, and antiparasitic chemotherapeutic drugs may impair sperm motility, capacitation, or the acrosome reaction.[43, 134]

Smoking

A meta-analysis of 20 observational studies showed that men who smoked cigarettes were more likely to have low sperm counts.[150]

In utero exposure to smoking was studied in 1770 young, healthy, potential military recruits and results showed the possibility of a small effect. [151] Exposure to smoking in utero was associated with mean sperm concentrations which were 20 percent lower when compared with unexposed men. In another study, there were no significant differences in mean sperm concentrations in men whose mothers either smoked or did not smoke during pregnancy. [152] However, men whose mothers had smoked ≥10 cigarettes per day while pregnant were at higher risk of having oligospermia (sperm concentration <20 x 10(6)/mL)

Genital Tract Inflammation

Specific inflammations of the genital tract such as mumps orchitis or gonococcal epididymitis may cause sterility. Nonspecific inflammations in the accessory sex organs are more common in men with infertility than in fertile men.[6, 153-156] Also, male accessory sex organ inflammation and infertility may be more important in some countries than in others.[6] Symptoms include chronic low back pain, intermittent dysuria, discharges from the penis on straining, and discomfort in the pelvic region or testes after ejaculation or prolonged sexual abstinence. The prostate may be enlarged and tender. The semen may show discoloration, variations in volume, increased viscosity, delayed liquefaction, high pH, sperm agglutination, bacteriospermia, and pyospermia. The bacteria in semen are frequently not pathogens but the commensals of the urethra or skin.[153

To have more than 1 million polymorphs per milliliter in semen, as determined by peroxidase reaction or monoclonal antibodies to leukocyte antigens, is considered abnormal.[68] Although inflammatory cells could damage sperm by releasing oxygen free radicals or cytokines, bacteria could impair sperm motility, and inflammation could also cause partial genital tract obstruction, the actual contribution of nonspecific genital tract inflammation to male infertility is contentious.[155, 156] Routine cultures of semen are not warranted except for sperm donors.

General Management

Men with clinical evidence of prostatitis require full urologic assessment.[157] Specific infections with pathogenic agents are treated with appropriate agents. It remains unclear what should be done about the very common issue of asymptomatic pyospermia and nonspecific male accessory gland inflammation. Therapeutic trials generally show no benefit from antibacterial therapy on semen quality.[158, 159] Antibiotics or other agents may be used if it is thought that the pyospermia compromises semen quality or that bacteria might contaminate the IVF culture media. Because the organisms commonly implicated in nonspecific genital tract inflammation include chlamydia, mycoplasma, and various bacteria, broad-spectrum antimicrobial therapy is required if treatment it is to be given. Also, many of the standard drugs do not enter inflamed accessory sex organs. Trimethoprim, erythromycin, doxycycline, and norfloxacin are potentially effective.[158, 159] Increased frequency of ejaculation to facilitate drainage of the accessory glands and stress management may also help.

Varicocele

The mechanism by which varicoceles cause infertility and the effectiveness of treatment in improving semen quality and natural fertility are controversial.[6, 15, 160-162]

Varicoceles are found in approximately 25% of men being examined for infertility. An additional 15% may have a subclinical left varicocele indicated by a faint cough impulse in the spermatic cord or increase in diameter of the veins on ultrasound.[6, 15, 160] Varicoceles are also found in fertile men. Varicoceles are more common in tall men and in men with larger testes.[14] They are less frequent in men with severe testicular atrophy, for example, in Kallmann and Klinefelter syndromes. When there is a moderate to large left varicocele, the left testis is usually smaller than the right testis.

Pathophysiology

Men with varicoceles generally have poorer semen quality than those without varicoceles.[160, 163] Thus, varicoceles can have an adverse effect on testicular function. Various theories have been advanced for the effect, including vascular stasis, back pressure, interference with oxygenation, reflux of renal or adrenal products into the pampiniform plexus, ROS generation and interference with the heat exchange function of the pampiniform plexus.[160] Varicoceles are usually first noticed at puberty and thereafter may increase in size but remain relatively stable in size throughout the man’s lifetime. Symptoms, including swelling and a dragging sensation in the scrotum, are infrequent, and many men with a large varicocele are unaware of its presence. The sudden appearance of a varicocele in an adult should be taken seriously because it may be a feature of a renal carcinoma with extension into the left renal vein.

Differential Diagnosis

The semen quality in men with varicoceles varies from azoospermic to normal. There is no specific pattern of abnormality with varicocele. Testicular histology is also variable, the only common feature being that the defect in spermatogenesis is more severe on the left side than on the right. Varicocele may be an association rather than the cause of a couple’s infertility. Therefore, full evaluation of other aspects of male and of the female partner is necessary.

Treatment

The value of treatment of varicocele for infertility is particularly contentious.[160-162] One view is that treating varicoceles may not improve fertility; therefore, the varicoceles should only be treated for other reasons, such as symptoms.[15, 161] The other extreme is the belief that varicocele is the most important treatable cause of male infertility, therefore all varicoceles should be treated even if small.[162] In the middle are those who would select cases. When there is an absent, obstructed, or atrophic right testis and all sperm in the semen come from the left testis, treatment of the varicocele may produce a reasonable result.[164]

Treatment of the varicocele involves embolization of the incompetent veins or surgery to prevent venous back flow from the abdomen to the pampiniform plexus. Radiographic techniques involve placement of a sclerosant, glue, or coils that promotes clotting in the veins. They have lower morbidity than surgery. A variety of operations can be performed for varicocele. In the past, retroperitoneal ligation and division of the testicular veins with or without preservation of the testicular artery and lymphatics was performed. Inguinal and scrotal microsurgical approaches have lower failure, recurrence, and hydrocele rates. Successful venous occlusion will relieve pain and reduce the size of large varicoceles. Whether semen quality and fertility are improved is not certain.

Results

Because varicoceles are so frequent, treatment of varicocele for infertility became common and several large series were published with claims of high success rates for improving semen and fertility. Floating numerator pregnancy rates averaging 35% (range, 20%–60%) were commonly reported. Regression toward the mean in semen variables, the nature of subfertility, and the need to include time in the denominator of pregnancy rates were ignored.[165] Although there are reports of successful treatment of azoospermic men by varicocelectomy, transient azoospermia may follow a minor illness or occur for unknown reasons, and, thus, such examples do not prove the value of treatment. Most exponents of varicocele treatment regard azoospermia as a bad prognostic sign, especially if the FSH level is elevated.

Follow-Up Studies and Controlled Trials

Follow-up studies of groups of treated and untreated patients with varicoceles suggest pregnancies are as frequent without treatment as with treatment of the varicocele.[15, 161] Attempts have been made to conduct randomized, controlled clinical trials of varicocele treatment. Such trials are difficult because the ideal design with sham operations and blinding, which is so important in controlling for outcomes affected by psychological factors, is not possible. Large trials are also needed. For example, approximately 250 pregnancies are required to have a high chance of finding a 25% increase in pregnancy rate after treatment significant at the 5% level.[20]

So far, the trials have produced conflicting results and meta-analysis does not support varicocelectomy improves fertility.[161] But the trials are generally small and have problems. For example, the World Health Organization set up a multicenter controlled trial of Palomo ligation in men with infertility of more than 1-year duration, abnormal semen analyses, a moderate to large left varicocele, and a potentially fertile female partner. Volunteers were randomized to immediate operation or operation delayed for 12 months to provide an untreated control group. One of the participating centers reported their results separately.[166] There was a substantial effect on pregnancy rate. Two pregnancies occurred in 20 couples during the 1 year of observation without treatment compared with 15 pregnancies in 20 couples in the year after the operation. During the year after the operation in the remaining 18 control patients, there were 8 pregnancies. Semen analysis results also improved after the operation. There were another 248 couples in 12 countries in the trial, and there was a less marked but significant improvement, the life table pregnancy rates at 1 year being 35% for the operated group and 17% for the un-operated group (relative pregnancy rate, 2.7; 95% confidence interval, 1.6–4.4). Semen analysis results also improved over the first year in the operated group. In the control patients having the delayed operation, the life table pregnancy rate at 1 year after the operation was 21%. However, there were possible irregularities of randomization in some centers early in the trial and high dropout rates, and the results were not published in detail.[160] Also, the pregnancy rates in the control group are lower than expected for untreated subfertile men with varicoceles: approximately 30% produce a pregnancy in 12 months.[15, 167]

Thus, although some people remain convinced of the value of treating varicoceles for infertility, it is not easy to demonstrate this unequivocally and the apparent improvements in semen quality and fertility may result from random fluctuations and regression toward the mean. Although better trials are needed, meta-analyses do not support treatment of varicocele for infertility. It is clear that normal fertility is not achieved in a high proportion of patients treated for varicocele. ART is a realistic alternative for most couples who have not conceived after a reasonable time.

GENERAL MANAGEMENT

This section covers aspects of the management of couples with male infertility not amenable to specific treatment (Table 6). A number will conceive during investigation. Others will decide not to continue with medical intervention. Some patients with treatable conditions may choose ICSI instead of treatment or after a treatment has been unsuccessful. However, most couples with male infertility have conditions for which there is no clearly defined and certainly effective treatment. In these cases, it is important to discuss the prognosis for a natural pregnancy occurring, the ineffectiveness of treatments, and the availability of IVF and ICSI, donor insemination, and adoption. The investigation of the female partner should be reviewed and abnormalities treated when possible. Patients should be acquainted with the physiology of the menstrual cycle and symptoms of ovulation to help time sexual intercourse over the fertile phase of the cycle.[168] Good health practices should be promoted, particularly cessation of smoking because it reduces fertility in women. The psychological upheaval experienced by the couple should be discussed and additional help offered if necessary. Specialist infertility counselors and patient support groups are particularly valuable in this area.

Table 6 Current Management of Subfertility

  • Estimate prognosis for natural conception
  • Discuss doubtful value of “empirical therapies”
  • Advise of alternatives: donor insemination, adoption, childlessness
  • Review coital timing
  • Review female partner’s potential fertility
  • Consider artificial reproductive technology: in vitro fertilization/intracytoplasmic sperm injection

Prognosis for Natural Pregnancy

A number of factors in addition to semen quality affect the likelihood of natural pregnancies occurring. [1, 2, 4, 17, 169, 170] Some are obvious, such as female disorders and coital dysfunction. Female age is important because fertility declines after approximately 35 years of age. Duration of infertility is a major factor in most studies: The longer the infertility, the worse the outlook. The prognostic factors found in a study to determine the effect of varicocele surgery were duration of infertility (negative), mean sperm concentration (positive), untreated sperm autoimmunity (negative), ovulatory disorders (negative), occupational group (farmers doing better than other occupations), female age (negative), and previous fertility in the couple (positive).[15] Interestingly, varicocele presence and size were positive prognostic factors even though varicocele surgery was not significant. The pregnancy rate curves for different sperm concentration groups are shown in Figure 9. Subfertile patients seen in the late 1990s had similar natural conception rates.[17] Such factors can be used to advise patients about their chances of producing a natural pregnancy over time. The accuracy of prediction is low because the statistically significant factors only explain a small part of the variability of the pregnancy rates. New studies using automated methods for semen analysis reveal the percentage of sperm with characteristics conforming to morphometrics preferred for binding to the ZP, and the straight line velocity, may have better predictive value.[17] However, other factors currently not assessable, such as gamete transport, may have an important bearing on conception and may explain the occurrence of pregnancies in some couples despite severely abnormal semen analysis results. Patients should not be told natural conception is impossible unless there is an absolute barrier to fertility.

Figure 9. Pregnancy rate curves grouped according to average pretreatment sperm concentration. The number (n)of patients followed each year is shown. The numbers of men and pregnancies in each sperm concentration group are shown in the inset table (From Baker, H.W.G., Male Infertility. Chapter 141 In Endocrinology, 6th edition, Jameson J. L. and DeGroot L.J. (Chief Eds.), Saunders Elsevier Philadelphia PA. pp 2556-2579, 2010)

Figure 9. Pregnancy rate curves grouped according to average pretreatment sperm concentration. The number (n)of patients followed each year is shown. The numbers of men and pregnancies in each sperm concentration group are shown in the inset table (From Baker, H.W.G., Male Infertility. Chapter 141 In Endocrinology, 6th edition, Jameson J. L. and DeGroot L.J. (Chief Eds.), Saunders Elsevier Philadelphia PA. pp 2556-2579, 2010)

of the problem followed by a tendency to blame others and a period of depression before final acceptance of the infertility. The reaction may take years to resolve, and it can threaten the stability of the partnership, interfere with investigation and management of infertility, and lead to futile involvement in expensive “cures” offered by the unscrupulous. Participation in unsuccessful treatments during this phase is often particularly difficult emotionally for the patients. Stresses of ordinary existence are unlikely to influence semen quality. 162 An empathetic approach and involvement of independent counselors or self-help infertility groups may assist some couples. In most, the unpleasantness of the psychological reaction subsides with time.

TIMING OF COITUS

A practical approach is to advise intercourse each day when ovulation might occur. Ovulation can be predicted to occur approximately 14±2 days before a period is due. Knowing the range of menstrual cycle length allows calculation of the days when ovulation is most likely to occur. Symptoms of ovulation including mittelschmerz and mid-cycle mucus changes also help identify the fertile time. 159 Temperature charts may be used to indicate the end of the fertile time as the basal body temperature rises after ovulation. Ovulation timing by measurement of estrogen and progesterone metabolites in urine, urine or serum LH levels, or ovarian ultrasonography may also be used.

GENERAL HEALTH ASPECTS

Although correction of adverse lifestyle factors in the most men seen for infertility is unlikely to produce normal fertility, healthy living has positive long-term benefits. The following are advised: weight reduction for the obese, reduced alcohol intake for the moderate to heavy drinker, avoidance of social drugs including tobacco, avoidance of heat from frequent sauna and spa baths, and management of stress in the workplace, in their relationship, and that engendered by the infertility.

EMPIRCAL TREATMENTS: EVIDENCE-BASED VERSUS UNCONFIRMED TREATMENTS

Treatments of some causes of male infertility are available as discussed previously, but for the majority of patients with abnormal semen analyses, there are no methods of proved effectiveness. 20, 126 A medical or surgical treatment may become established because it is logical and obviously effective, for example, gonadotropin treatment for Kallmann syndrome or vasoepididymostomies for postinflammatory obstructions of the tails of the epididymides. However, in other situations in which semen quality is reduced and there is subfertility rather than absolute sterility, it is necessary to demonstrate that the treatment increases semen analysis results and pregnancy rates by a clinically meaningful amount. This evidence-based medicine approach generally requires controlled clinical trials of promising methods. These trials are usually designed to detect a certain magnitude of difference in the primary responses and thus a positive result supports the use of the method. However, if the trial is negative, it merely does not confirm the magnitude of benefit tested; it does not prove the method is of no value. In time, the results of several trials can be combined by meta-analysis to get better estimates of the overall effects of the method.

In the past, many treatments were used in an uncontrolled fashion for defects of sperm production. 20, 126 Androgens have been given to suppress spermatogenesis in the hope that there would be “rebound” improvement after the treatment is stopped. Low-dose testosterone or weak androgens, such as mesterolone, have been given in the hope of improving epididymal maturation of sperm. Human chorionic gonadotropin has been given for similar reasons. Antiestrogens have been used to increase gonadotropin secretion or gonadotropins (FSH and human chorionic gonadotropin) given to “stimulate” spermatogenesis. Antibiotics and anti-inflammatory drugs have been given for subtle infections or inflammations in the accessory sex organs. Antioxidants, amino acids, vitamins, herbs, and minerals such as zinc, cold baths, and testicular coolers have been used. There are difficulties with the interpretation of the results of these treatments. 20 Marked improvements in semen quality can occur spontaneously (Fig. 10). Semen analysis results also display the phenomenon of regression to the mean. That is, on average, repeated semen analyses improve in men with initially abnormal results. 156 Pregnancy rate data were not analyzed effectively in many early studies. Floating numerator pregnancy rates, in which a percentage of patients pregnant is given without regard for time of exposure, have caused confusion in the infertility literature. Statistical methods for life table analysis and regression analysis with censored data are especially useful for assessing the impact of groups of variables on pregnancy rates, for analysis of prognostic factors, and for testing results of therapeutic trials. 20

Figure 10. Sperm concentration and motility in a man with severe oligospermia and severe hypospermatogenesis included in a therapeutic trial of clomiphene. Semen quality improved and his wife conceived. He was given the placebo! (Baker HWG: Requirements for controlled therapeutic trials in male infertility. Clin Reprod Fertil 4:13-25, 1986.)

Figure 10. Sperm concentration and motility in a man with severe oligospermia and severe hypospermatogenesis included in a therapeutic trial of clomiphene. Semen quality improved and his wife conceived. He was given the placebo! (Baker HWG: Requirements for controlled therapeutic trials in male infertility. Clin Reprod Fertil 4:13-25, 1986.)

The empirical treatments either have not been submitted to adequately controlled clinical trials, or when they have, the trials have not shown consistently positive results. Meta-analyses have also produced conflicting results, probably because of the variable quality of the trials included in the analyses. Until there is sound evidence of the value of a drug or procedure from controlled therapeutic trials, patients should be advised that none of the empirical methods meet the requirements of evidence-based medicine.

AIH is widely practiced with dubious evidence of efficacy in patients who do not have coital difficulties. Ovulation induction with intrauterine artificial insemination probably does increase the pregnancy rates by increasing the number of oocytes exposed to the sperm.[172, 173]Results are lower with timed intercourse and multiple ovulation induction. Generally, the results are poor when the semen analysis is abnormal. Although this may be acceptable in countries where ART is expensive, the risk of multiple pregnancy is substantial. IVF or ICSI would be preferable because the number of embryos placed in the uterus can be controlled and high multiple pregnancies avoided.[172]

In Vitro Fertilization/Intracytoplasmic Sperm Injection for Male Infertility

ICSI has revolutionized the management of male infertility. It involves the injection of a single sperm into the ooplasm (Fig. 11).[174, 175] ICSI can be used with almost any live sperm with an expectation of results similar to those obtained with standard IVF using normal sperm. ICSI may not be needed with mild semen disorders. Provided that more than approximately 2 million motile sperm can be harvested from an ejaculate, IVF can be attempted with an expectation of success close to that of IVF for other indications. The outcome depends particularly on sperm morphology and the ability of the sperm to bind to and penetrate the ZP. ICSI should be offered if there is a chance of failure of fertilization with IVF: less than 2 million motile sperm per ejaculate, less than 4% of sperm with normal morphology, less than 5% of sperm with progressive motility, sperm autoimmunity, and defects of sperm-oocyte interaction.

Figure 11. Intracytoplasmic sperm injection.

Figure 11. Intracytoplasmic sperm injection.

Preparation of the Patients

The couple needs to be counseled carefully about the procedures, predicted chance of a live birth, and the possible complications. Special arrangements for the collection of semen, or for its preparation, may be required. Trial-run sperm preparations help to identify those patients who have difficulty collecting semen. These patients should practice collections before attending for the IVF procedure. Men with many inflammatory cells in the semen could be treated with antibiotics. Those with low motility or sperm autoimmunity may have better sperm motility with short, 1- to 2-day durations of abstinence. Cryopreserved semen can be used as backup if the fresh semen is particularly poor on the day of ICSI in patients with fluctuating semen abnormalities. This is particularly useful when sperm are present in the semen only intermittently. Those patients who produce an unexpectedly poor sample on the day of IVF should provide a second sample later to supplement the first sample. Electroejaculation or needle biopsy of the testes can be used if the man is unexpectedly unable to collect semen. Patients with genital tract obstruction can have sperm retrieved from the testis or epididymis by needle aspiration.

ICSI also allows patients with severe primary spermatogenic disorders to be treated provided some live sperm or elongated spermatids can be recovered from the semen, testes, or genital tract (Table 172-7). If no sperm can be found in the semen, the likelihood of finding elongated spermatids in the testicular tissue can be estimated by the clinical situation and testicular histology. Sperm can be found by biopsies in approximately 50% of men with Klinefelter syndrome. Results are good if any tubules with complete spermatogenesis can be seen in diagnostic biopsies. However, if there are no elongated spermatids, the success rate with open biopsies is low: approximately 25% with Sertoli-cell-only syndrome and rarely with germ cell arrest at the primary spermatocyte stage.[117, 176] Yq microdeletions in the AZFa and b regions are also associated with a complete absence of spermatogenesis. Other factors such as hormone levels and testicular size do not seem to be predictive. The use of donor sperm should be discussed during preparation of the couple if the outlook is poor.

Testicular biopsy techniques should maximize the chance of finding sperm while minimizing damage. Microsurgery with examination of exposed testicular tissue under the operating microscope may allow selection of the larger diameter tubules, which are more likely to contain more advanced spermatogenesis.[176, 177] Alternatively, multiple sampling through small holes in the tunica may be used.[178] Large biopsies, particularly at multiple sites, have higher complication rates and will further impair testicular function (future androgen deficiency). Generally, repeat open biopsies for sperm collection should be performed only after the patient and testes have recovered from the previous surgery, and this may take 4-6 months.

Approaches

The standard approach is to stimulate multiple ovarian follicular development with FSH and collect the oocytes by ultrasound-guided transvaginal needle puncture of the ovaries after administration of human chorionic gonadotropin to mature the oocytes. ICSI or IVF is performed, and the resulting embryos are transferred into the uterine cavity, usually at the four- (day 2) to eight-cell (day 3) or blastocyst (day 5 or 6) stage with cryopreservation of remaining embryos. Although cryopreservation reduces the implantation potential frozen embryos produce good pregnancy outcomes.[179]

Biopsy of blastomeres for detection of chromosomal and genetic abnormalities is used to avoid transmission of serious hereditary diseases or to select embryos with higher cnances of implantation.[180]

Sperm Preparation

Various procedures have been developed for sperm preparation for IVF. Most popular is centrifugation on gradients of colloidal silica because this can be performed with high reproducibility.[181] Cryopreserved samples require especially gentle handling, particularly with dilution of the semen cryoprotectant medium with culture medium. Motility, even just an occasional slight twitch of the tail, is required in selecting sperm for ICSI. The morphology of the sperm cannot be assessed in detail at the magnification used, but obviously grossly abnormal sperm are excluded. If no motile sperm can be found, motility stimulation with pentoxifylline or hypoosmotic swelling can be used to show that the sperm are alive.[181]

Results

With IVF and ICSI, 60% of oocytes fertilize and cleave normally over the first 48 hours. For women younger than 35 years of age, the clinical pregnancy rate (fetal heart positive ultrasound at 6 weeks gestation) for transfer of one fresh 2-day old embryo is approximately 30% and for transfer of two 2 day old embryos 40%. Pregnancy rates are approximately 25% lower with cryopreserved embryos. The cumulative live birth rates with transfer of fresh and cryopreserved embryos from the first oocyte collection are approximately 50% and 80% by the third oocyte collection in women under 35 years of age. Approximately 25% of the births resulting from transfer of two 2-day old embryos are twins. Multiple pregnancy is more frequent if three or more cleave stage embryos or two or more blastocysts are transferred. Many clinics are transferring single embryos (elective single embryo transfer) in patients with good prognosis to minimize the multiple pregnancies. A number of other factors influence the results of ART, including embryo quality and particularly female age. Implantation and pregnancy rates decrease and pregnancy losses increase after age 35, mostly due to increasing abnormalities in the oocytes. For women aged 36-39 years the live birth rate is about 70% of that for women under 36 and for those 40 years and over using their own oocytes, about 30% that for women under 36.

Evaluation of Failed Fertilization

When most or all oocytes fail to fertilize in IVF, the cause is usually defective sperm. Oocytes may not fertilize because of immaturity or abnormality, but this is an unusual cause of total failure of fertilization of all the oocytes retrieved from a woman.[74] Unexpected failures of fertilization should be evaluated by examination of the number of sperm bound on the ZP and penetrating the ZP. Low numbers usually indicate sperm defects.[74] Low fertilization rates may also result from undiagnosed sperm autoimmunity, infected semen, or technical problems in the IVF laboratory. Careful evaluation of the semen quality and screening patients with idiopathic infertility for defects of sperm-oocyte interaction before IVF should allow most couples likely to have low fertilization with standard IVF to be directed to ICSI.[74, 182] ICSI of unfertilized oocytes with the man’s sperm 12 to 24 hours after standard IVF insemination may result in fertilization and pregnancy, but, overall, the results are poor and many clinics do not perform this “ICSI rescue” procedure. Re-insemination of failed fertilization oocytes with donor sperm is also possible for diagnostic or therapeutic purposes. This procedure is not permitted in some countries.

Individual oocytes may not fertilize with ICSI. In these, the sperm head is often only partially decondensed. Failure of fertilization of all oocytes with ICSI is rare. Globospermic, immotile and rarely sperm from patients with severe oligospermia may produce low or zero fertilization rates with ICSI. In these cases there may be a deficiency of an oocyte-activating factor from the sperm. Modified ICSI techniques (assisted activation) may be successful in artificially activating the oocytes in some of these cases.[91]

Complications of In Vitro Fertilization/Intracytoplasmic Sperm Injection

Potential adverse effects of ART include well-understood conditions in the woman and a variety of possible issues for the offspring. In general, the outcome and complications of ART are the same for standard IVF and ICSI.[183, 184] The implantation rate, pregnancy wastage, pregnancy complications, perinatal mortality, and risk of congenital abnormalities are no greater for ICSI than with IVF. Recent results for ART for male infertility are compared with those for infertility of other causes in Table 7.[185] The pregnancy, miscarriage and other outcomes are if anything better not worse for male infertility.

Table 7

Comparison of results of fresh and cryopreserved embryo transfers for male infertility versus other causes of infertility in Australia and New Zealand 2005.[185]

 

Type of infertility

 Male only Other
Fresh embryo transfers

Clinical pregnancy (fetal heart(s))

Live birth

 7880

26.1%

21.0%

 20115

23.2%

18.4%
Cryopreserved embryo transfers

Clinical pregnancy (fetal heart(s))

Live birth

 4708

20.2%

15.8%

 12051

19.5%

14.6%
Clinical pregnancies (fetal heart(s))

Spontaneous abortion

Induced abortion

Ectopic pregnancy

Stillbirth

Preterm (<37 weeks gestation)

 3008

17.6%

0.5%

1.0%

0.8%

16.0%

 7013

19.4%

0.7%

1.7%

0.7%

18.3%

In 2005 48% of embryo transfers were with single embryos and the multiple pregnancy rates were for fresh embryo transfers following IVF: 16.2%, ICSI 14.7% and thawed cryopreserved embryo transfers (FET) 10.9%. The perinatal mortality (stillbirth and neonatal deaths within 28 days of life) was for IVF 18.2, ICSI 15.3 and FET 11.8 per 1000 births. The perinatal mortality for ART singletons was 9.6 and for the general population in 2004: 10.2.

Risks for the Woman

The ovarian hyperstimulation syndrome is a major risk with gonadotropin stimulation of multiple follicular development.[186] Careful monitoring of the patients is necessary. If many follicles develop, embryo freezing rather than transfer avoids pregnancy and allows the ovaries to recover, reducing the risk of severe complications such as thromboembolism, renal failure, and death. Surgical complications including bleeding and infection from the oocyte collection procedure are rare. There is also a small risk of complications from anesthesia and sedation. Maternal complications of pregnancy increase in frequency with multiple pregnancy. Cesarean section is more frequent for singleton ART births than in the general community. There are also concerns about the ovarian stimulation drugs predisposing to breast or gynecologic cancers.[187]

Risks for the Child

Multiple Pregnancy

The risks of multiple pregnancy for the child, prematurity, low birth weight, increased perinatal mortality and morbidity, less parental attention during childhood, are well known and can be controlled by reducing the numbers of embryos transferred together.[183, 186, 188, 189]

Transmission of Genetic and Chromosomal Disorders

The known genetic risks were covered previously (see Table 4). For conditions such as cystic fibrosis and myotonic dystrophy, pre-implantation genetic diagnosis can be used so that only unaffected embryos are transferred. Balanced chromosomal translocations may become unbalanced in embryos and result in miscarriage or rarely in the birth of an abnormal child. Pre-implantation genetic diagnosis may also be used to detect unbalanced chromosomal constitution in the pre-implantation embryo.[180]

Defects Possibly Associated with Abnormal Spermatogenesis

There is a correlation between the production of abnormal sperm with poor morphology and motility and abnormal sperm DNA measured by a variety of techniques including acridine orange fluorescence, sperm chromatin structure assay, chromomycin staining, and comet assays. [22] Abnormal sperm produce reactive oxygen species that could damage sperm DNA and result in defects of implantation or pregnancy loss. [22] However, the results of ART do not reveal such problems (see Table 7).[24] A number of mechanisms reduce the likelihood such abnormal sperm would be involved in natural or assisted fertilization such as sperm aggregation caused by heavy coating with clusterin, poor motility, and limited ability to bind to the zona pellucida.[190, 191]

An increase in de novo sex chromosomal aneuploidy and structural autosomal defects reported with ICSI related to increased rates of chromosomal nondisjunction as a general association with abnormal spermatogenesis has not been confirmed by all studies.[109, 192-194]

Embryo Defects Possibly Caused by Laboratory Conditions

Laboratory conditions or procedures on gametes could affect embryo development and health of the child. In domestic animal IVF, there is a syndrome of large offspring that results from stress-induced changes in gene expression that may involve changes in DNA methylation and gene imprinting in embryos cultured to blastocysts.[195] Increased frequencies of rare conditions caused by disorders of imprinting such as Beckwith-Weidemann syndrome and Angelman’s syndrome have been reported in children born from ART procedures.[196] Increased frequencies of tumors in children born after ART have also been claimed, for example, retinoblastoma, but other studies do not support a general increase in childhood cancer rates.[197]

Surveillance Studies

In most studies the fertilization, implantation, and pregnancy failure rates and congenital malformation rates are no greater with ICSI than with standard IVF.[183, 184] However, overall results are different from those in the general population: preterm birth (6%), low birth weight (5%), major congenital malformations (2%), and perinatal mortality (1%)(see Table 7). The differences are partly explained by the high multiple pregnancy rate with IVF and ICSI, and perhaps female age and infertility factors. Closer surveillance and more accurate reporting may also contribute. The lower birth weight also affects singletons and there is a difference between the results of fresh and cryopreserved embryo transfers the low birth weight being more frequent in babies born after fresh embryo transfers.[179]

Studies of children born as a result of ART have not revealed any consistently associated congenital malformations, but there is data suggesting generally increased congenital malformation rates.[198-200] However, these studies may be biased. For example, if IVF and ICSI babies are examined more thoroughly and reporting to health registers is more complete, the malformation rates may appear higher than for naturally conceived children who are less carefully examined and reported. A higher uptake of prenatal screening for Down syndrome and other triploids in ART patients may also influence the reported rates of birth defects. Even with perfect reporting, differences in malformation rates could be caused by other factors, such as age, parity or health of the mothers, which have not been adequately allowed for in the statistical analysis.

USE OF DONOR SPERM

Donor insemination is a common method of managing male sterility.[201-204] Donor sperm may be involved in approximately 1 in 200 births in countries where it is permitted. The main indications for donor insemination are untreatable sterility in the man or when treatments and ICSI for severe or chronic subfertility have failed. The couple may choose donor insemination as the primary method of managing their infertility. Donor insemination is also used to avoid transmission of a severe genetic or infectious disease in the man. Women without a male partner may use donor insemination to have children. Donor sperm may also be used in IVF when there is a combination of female infertility and male sterility. Because of the higher pregnancy rates, IVF with donor sperm may be used if donor insemination fails.[203] Donor sperm may also be used in IVF procedures as a backup, for example, when there is a high risk of failure with of sperm extraction with a severe spermatogenic defect.

Cryopreservation of Semen

Donor insemination can be performed in the setting of a specialist infertility clinic with all donor and patient management available. Alternatively, the sperm bank may only supply semen for the patient and be separate from the clinics or physicians performing the artificial insemination. Because of the risk of transmission of infectious diseases, particularly HIV, and also for convenience, donor insemination services now use only cryopreserved semen.[201, 203] Semen cryopreservation with glycerol–egg yolk cryoprotectant and either vapor freezing or controlled-rate freezing in plastic straws or vials produces pregnancy rates equal to those with fresh semen. Importantly, cryopreservation allows the semen to be quarantined for 6 months for donors to be recalled and retested for infectious diseases before it is used.

Selection of Donors

Prospective donors have their medical and family histories evaluated and a physical examination to exclude the possibility of transmitting serious genetic diseases such as hemoglobinopathies or sexually transmissible infections. Donors sign a lifestyle declaration to indicate that they are not involved in any practices that might expose them to serious infections, such as HIV. There usually is an upper age limit of 40 to 45 years because of the increasing frequency of genetic abnormalities in sperm with age. Semen quality is selected to be in the upper part of the normal range, particularly for concentration and motility.[201, 202] The semen is cultured for bacteria and blood is tested for hepatitis and HIV antibodies. Genetic screening for hemoglobinopathies and cystic fibrosis or other conditions may be included depending on their prevalence in the community. The freezing of semen does not appear to cause any increase in the frequency of congenital abnormalities.[201, 203, 205]

It is usual to match the physical characteristics of the recipient’s husband and the donor including race, complexion, build, height, and hair and eye color. In addition, blood groups may be matched. In some programs, the recipient couple may be able to choose the donor on other information such as occupation and education. Known donors may also be used; these may be friends or relatives of the infertile couple. In this situation, special counseling of the donors and recipients is necessary. Also, there should be a full workup of the known donor as for an anonymous donor, including cryopreservation and quarantining of the semen.

Donor factors relevant to the success of donor insemination are mainly to do with the quality of the semen. Post-thaw motility has the strongest predictive value for high fertilization rates, but sperm morphology, motility, and concentration are also significant.[201, 203] Despite selection of high-quality semen, there remains considerable variability in the pregnancy rates between donors. A policy to discard semen from a donor who produces no pregnancy after a certain number (e.g., 20–40) of inseminations is necessary.[202]

Counseling

The special nature of the use of donor sperm is discussed in detail with the couple so that they are fully aware of the implications for the child and their family. Donor insemination is forbidden in some religions. There may be local legislation or regulations to control the use of donated gametes. In some countries, special laws have been enacted that may either allow or prevent the child from obtaining identifying information about the donor. The legal status of the child may also be specified in various ways. The couple needs to decide how and when to disclose the child’s donor sperm origin. What and how much they should tell their friends and relatives about their infertility treatment should also be discussed, as should their reaction to acquaintances questioning the paternity of the child. The possibility that in the future half-siblings may unwittingly find each other and attempt to have children is of concern to some prospective parents and donors. This needs to be discussed carefully and the risks explained in view of the number of pregnancies permitted per donor by the clinic. Studies of donor families in which there has been expert pretreatment counseling indicate no physical or emotional problems with the children, and greater marital stability than average.[205]

Procedures and Results

The prospective recipients are screened for HIV, hepatitis B and C, rubella immunity, blood group, and genetic conditions if necessary. Tests of tubal patency are performed if the history suggests pelvic pathology. The inseminations are timed to coincide with natural ovulation. Careful monitoring of ovulation and timing of intrauterine insemination of prepared motile sperm suspensions (as for IVF) appear to increase the pregnancy rate. Pregnancy rates are approximately 10% to 25% per month for the first 4 to 6 months and then 5% to 10% thereafter, so that approximately 50% of women are pregnant by 4 to 6 months.[201, 203, 204] Female age affects the pregnancy rates.[201, 204] Women with subfertile male partners have on average lower pregnancy rates than those with sterile male partners, indicating the presence of female factors contributing to the infertility when the male partner is subfertile.[201, 204] Cumulative pregnancy rates for women who have had more than one pregnancy by donor insemination indicate higher conception rates over the first few months for the second pregnancy, approximately 33% pregnant in the first cycle and 55% by the second cycle.[201]

Multiple ovulation induction and intrauterine insemination may increase the pregnancy rates but at the risk of multiple pregnancy.[201, 204] IVF may be used if no pregnancy has occurred after a reasonable number of inseminations (e.g., 4–6).[203] Live birth pregnancy rates with IVF in such patients are high, and women in good health can be advised that they have an 80% chance of having a child within 2 years.

PREVENTION OF INFERTILITY

Prevention is difficult because of the lack of understanding of the causes of most types of male infertility. Mumps orchitis was an uncommon cause of infertility, and childhood immunization for this disease should make it very rare. It is important to recognize that subfertility often is a couple problem, with both partners contributing. Therefore, general factors that would change a society’s attitude to child bearing could have an important impact on the frequency of infertility, for example, a trend toward having children at earlier ages. On the other hand, toxins and environmental factors known to cause defects of sperm production, such as heat, dibromochlorobenzine, lead, benzene, ionizing radiation, and microwaves, are probably well controlled by environmental health measures.

Preventable Diseases and Conditions

Sexually Transmissible Infections

Post-gonococcal epididymal obstructions appear to be the most important cause of infertility from sexually transmitted diseases. In countries where gonorrhea is treated promptly, post-gonococcal epididymal obstruction is rare. On the other hand, it remains a common preventable cause of infertility in other countries.

Undescended Testes

Although undescended testes have been sought and treated aggressively over the past 50 years, previously undescended testes remain a common association of male infertility, affecting approximately 7% of the men seen. It is therefore uncertain whether early surgery for undescended testes has any impact on subsequent fertility. It is possible that the failure of normal descent is a feature of testicular dystrophy and that the sperm production will be poor whether or not the testes are placed in the scrotum.

A randomized controlled trial of orchiopexy for unilateral palpable maldescended testis at 9 months versus 3 years of age showed that surgery at 9 months was followed by significant growth of the testis up to age 4 years but there was no change in testis size in those treated at age 3 [50]. This has led to clinical guidelines for treatment of maldescended testes that recommend orchiopexy for congenital forms between 6 and 12 months of age and as soon as possible for those discovered later and for acquired maldescent. Hopefully this will reduce the frequency of subsequent testicular tumours and spermatogenic defects.

Varicocele

The effectiveness of varicocelectomy for sperm defects is controversial. Varicoceles are common and usually appear about the time of puberty. Although some groups believe that varicoceles should be sought actively and treated in adolescence to prevent infertility, this approach could pose a major burden on the health resources because at least 15% of men have varicoceles. Long-term prospective trials are needed.

Vasectomy

Vasectomy reversal and treatment for continuing infertility after attempted vasectomy reversal are now common. Better counseling about the limited effectiveness of vasectomy reversal is needed and cryopreservation of semen before vasectomy in men who are uncertain about their need for future fertility should be promoted.

Androgen deficiency

In patients with symptomatic androgen deficiency, testosterone therapy should be deferred until fertility issues have been addressed. Alternatively, Human chorionic gonadotropin (hCG) preparation can be given in order to stimulate testosterone production without inhibition of spermatogenesis.

If fertility is not immediately sought, sperm cryopreservation allows testosterone replacement to be commenced.

Options for fertility preservation in males

Advances in the treatment of cancer in young patients have led to great improvements in life expectancy which approaches 80% 5-year survival rate. As a result, fertility preservation and desire for paternity have become a significant issue in this group. A major concern is the negative impact of chemo and radiotherapy on fertility. Up to two thirds of patients are azoospermic following chemotherapy. Recovery of spermatogenesis is strongly dependent upon the chemotherapy and radiation regimen and the patient’s baseline reproductive function. Alkylating agents seem to have the most profound reproductive effects. Thus men about to have treatment for malignant conditions may have sperm cryopreserved before commencing chemotherapy or radiotherapy.[206] Although pretreatment semen quality may be too poor for AIH, ICSI now has improved the outlook for successful pregnancies. Semen collected during chemotherapy or radiotherapy must not be used because of the likelihood of induced mutations.[207]

Infertile men with conditions such as orchitis or severe primary spermatogenic disorders that might involve progressively declining semen quality such as Klinefelter syndrome, when sperm can be recovered, should also store any live sperm that can be obtained as insurance for the future. A similar approach could be extended to adolescents with risk factors for infertility such as undescended testes in childhood, testicular torsion, and possibly a family history of infertility or a father with a Yq microdeletion. Semen may also be stored after treatment of gonadotropin deficiency or surgery for genital tract obstruction in case of re-stenosis. Storage of sperm before premature death or after a sudden unexpected death is also possible. Although the use of gametes from a dead person is surrounded by complex ethical and legal issues it is permissible in some countries. Semen cryopreservation can also be offered to adolescents. Sperm may be obtainable from the semen or testis after mid puberty. Although only a small proportion of men who store semen may use the frozen sperm, the service provides insurance for future fertility.

Methods of Collection

Sperm for fertility preservation may be collected in a variety of ways. Ejaculated sperm cryopreservation is the most common technique used as enough sperm is usually found for freezing in majority of men. Unfortunately, some men with testicular and other malignancies may present initially with oligospermia or even azoospermia. This may be related to a basic infertility condition which puts them under higher risk to develop testicular cancer or the stress effect of the disease on spermatogenesis. Some patients will not be able to produce a sample. Some patients are unable to ejaculate for social, religious or medical reasons or may be unfamiliar with masturbation for example peri-pubertal boys. For them, sperm can be retrieved by penile vibratory stimulation, electroejaculation or surgically from the epididymis or the testis as discussed above.

Fertility Preservation in Prepubertal Boys

Preserving fertility in postpubertal boys facing chemotherapy can be achieve similar success rates to those in adults. However fertility preservation in prepubertal boys presents a great challenge as sperm banking is not possible. Alternative strategies have been developed but all are currently experimental. These strategies are based on immature spermatogenic cell cryopreservation as cell suspensions or whole testicular tissue for future fertility restoration using autografting, xenografting or in-vitro spermatogenesis. [208] More research is needed to establish the best approach to generate spermatozoa from immature stem cells via in-vivo or in-vitro maturation. In the meantime, prepubertal tissue preservation should be discussed with the boys and their parents and samples should be banked only after careful counseling emphasizing the experimental nature of this approach.

Post Chemotherapy Sperm Recovery

Various barriers to sperm banking such as prepubertal age, under-referral or inadequate understanding of the sterilizing effect of chemotherapy and defective spermatogenesis before the treatment may have prevented sperm banking.[209, 210] In these patients an approach similar to that used for men with severe primary spermatogenic disorders can be used as recovery of spermatogenesis after gonadotoxic treatments is highly variable. Men with persistent azoospermia following chemotherapy or radiotherapy can be offered microsurgical testicular sperm extraction (TESE) and ICSI as there is some chance of success. Schlegel et al. reported 84 microdissection TESE procedures performed in 73 patients with 43% having sperm retrieved and these produced an average 57% fertilization rate with ICSI and 42% had live births.[211]

DNA FRAGMENTATION

The study of sperm DNA fragmentation has become topical in recent years. It has been proposed that DNA damage may contribute to infertility in a way that is not revealed by simple morphological evaluation of spermatozoa. Sperm showing a high proportion of fragmented DNA has been blamed for a lower fertilization rate [212], poorer embryo development and reduced implantation rates. [213-215]. It is now assumed that a significant proportion of infertile men have clinically important levels of DNA damage in their spermatozoa. [216]

Basically, once a sperm nucleus has been introduced into the oocyte, a rapid de condensation should occur to allow the DNA formation of the paternal pronucleus. Abnormalities in the structural DNA organization can cause delays and errors in the paternal DNA delivery. Animal studies demonstrated that sperm DNA damage can cause embryo development compromise.[217, 218].

Apoptosis plays a key role and is considered to be the main pathway leading to DNA breakage in sperm.[219] The known main external inducers of sperm DNA fragmentation are chemotherapy [220], advanced age [216], environmental factors such as cigarette smoking [221], genital tract inflammation and varicoceles [222-224] and the presence of leukocytes in the semen [225]

Three main mechanisms have been proposed as contributors to the generation of DNA fragmentation; 1) DNA nicks occurring to promote the remodeling of sperm chromatin are not completely repaired due to an impairment of the sperm maturation process, [226]; 2) DNA cleavage produced by a process of apoptosis first triggered and later interrupted [227, 228] and; 3) reactive oxygen species (ROS) , acting both in testis and in posttesticular sites [229]

Based on the above understanding, strategies to identify fragmented sperm gained popularity.[230] Novel sperm selection techniques like annexin V–magnetic activated cell sorting (annexin V–MACS), zeta potential selection, electrophoretic systems for the rapid isolation of sperm exhibiting high levels of DNA integrity and hyaluronic acid binding techniques, have been recently described.[231] Currently, the evidence is insufficient to recommend one specific method of sperm selection in the case of high sperm DNA fragmentation.

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Surgical Treatment of Obesity

ABSTRACT

Obesity is one of the most prevalent pathogens in the developed world, causing numerous common and lethal diseases. Non-surgical treatments to date have failed to provide an effective, durable solution. Bariatric surgery includes the procedures of gastric bypass, sleeve gastrectomy, gastric banding and biliopancreatic bypass. These procedures have been shown to produce substantial and durable weight loss yet are provided annually to less than 0.2% of eligible obese people, making current bariatric surgery largely irrelevant to public health.

The principal mechanisms of effect vary between procedures and include control of hunger, change of appetite, restriction of intake, diversion of food from the proximal small intestine, malabsorption of macronutrients, increased energy expenditure, food aversion and possibly changes to the gut microflora and changes to serum bile acid levels.

Weight loss outcomes are typically 50-60% of excess weight loss (EWL) at 10 years for gastric bypass, 45-55% EWL for gastric banding, 70% EWL for biliopancreatic bypass. There are no long-term weight loss data for sleeve. In association with the weight loss there are significant and sustained improvements in the length of life, the quality of life and in many of the comorbidities of obesity. In particular, all procedures have been shown by randomised controlled trials (RCT) to induce remission of diabetes better than non-surgical therapies in the short-term. Medium and long term data from RCTs are not yet available.

The mortality risk reflects the type of surgery and varies between 0.1% for gastric banding to 1-2% for other procedures.

The criteria for consideration of bariatric surgery include the presence of obesity (BMI > 30), a history of multiple attempts at weight reduction by non-surgical means, an awareness of the potential risks and a commitment to attend the follow up program. The decision on which procedure should be used is based on patient or surgeon preference, availability of appropriate aftercare and the patient’s tolerance of risk and permanent anatomical change. For complete coverage of this and related areas of endocrinology, plese see our on-line free web-book, www.endotext.org.

BACKGROUND

Obesity is one of the most significant pathogens in the developed world. It causes or exacerbates numerous common and lethal diseases. It can markedly reduce the quality of life and it competes with smoking as the commonest cause of premature death. The prevalence of obesity has increased markedly in the last thirty years. A systematic review of 199 countries in 2008 estimated 502 million people worldwide were obese1. According to the World Health Organization, obesity has more than doubled worldwide since 1980 and more than 600 million or 13% of the adult population were obese in 2014.

The epidemic is quite recent. In the United States, between 1960 and 1980, there was a relatively modest rise in the number of adults with obesity, from 12% to 14% only. This number has more than doubled since 19802. Currently, approximately 80 million people in the USA, 34% of the adult population, are obese3 and this number is estimated to increase to more than 140 million by 20304. The direct health care costs were estimated to be $75 billion per year in 2003. These would rise to $140 billion per year and it would be an associated loss of productivity (indirect costs) of $580 billion4 Based on the Health and Wellness Survey in 2008 there is estimated to be loss of 1.7 - 3.0 million productive person-years in the USA, representing a cost of $390 - $580 billion due to the current state of obesity4 .

The morbidity caused by obesity makes it our greatest current health challenge because of its direct contribution to many chronic, debilitating and life-threatening diseases. These include type 2 diabetes, cardiovascular diseases such as ischemic heart disease, stroke, hypertension and dyslipidemia and several common cancers. The health care costs of obesity are now a major component of health budgets across the developed world. Currently there is no non-surgical method for predictably achieving major weight loss in the obese and maintaining that weight loss for an extended period. Current programs involving diet, behavioral modification, exercise and activity, with or without drug supplementation, are able to achieve a modest weight loss which is generally sustained only for the duration of the program.

In this chapter I will review the surgical options, their strengths and weaknesses, to provide a framework of evidence that enables a logical treatment approach to this major healthcare challenge. In doing so, I will seek an alignment of the views of health professionals and the general community so that they recognize obesity as a disease and not a sign of personal failing, they acknowledge the severity of this disease and they recognize the spread of invasiveness and risk and effectiveness associated with options for surgical treatments.

Surgical methods have been known to achieve substantial and durable weight loss for more than half a century and yet they have not achieved a significant impact on community health. Currently, less 1 in a 1000 world-wide and less than 1 in 250 in the USA and Australia of those who would benefit by treatment are having bariatric surgery. In terms of the public health, bariatric surgery is used so infrequently as to be largely irrelevant. In its early days the risks were high, undoubtedly discouraging many. But these risks have been reduced markedly5. The risk to benefit ratio has now become much more favorable and the cost to benefit ratio is also favorable. It is time for a change and the data are there to justify a change.

AN HISTORICAL PERSPECTIVE OF BARIATRIC SURGERY

Although the weight loss that accompanied surgery, particularly if it involved the stomach or small intestine, had been noted for as long as the procedures have existed, a direct use of surgery for the specific purpose of weight loss - bariatric surgery - really began in 1954 with the small bowel bypass procedure. It can be seen to pass through three phases so far. The small bowel bypass phase was replaced by the gastric stapling phase in the late 1960s and then, in the early 1990s, the introduction of laparoscopic surgery and gastric banding led to the third phase.

The Initial Phase (1950 – 1970) – Small Bowel Bypass.

Surgical management of obesity began with the introduction of the jejunoileal bypass (JIB) in the 19546. In this procedure the proximal jejunum was diverted to distal part of the gut, leaving a long segment of excluded small intestine and a marked reduction in absorptive capacity. Many variations existed. In a typical procedure, the proximal 35 cm of proximal jejunum was joined end-to-side to the last 10 cm of ileum. The JIB procedures represented the best and the worst of bariatric surgery. Major and sustained weight loss was achieved and there were impressive health benefits, particularly in relation to lipid metabolism. However it was associated with serious side-effects including copious offensive diarrhea, electrolyte imbalances, oxalate calculi in the kidneys and progressive hepatic fibrosis with eventual liver failure7-11. For these reasons this group of procedures was generally abandoned by the 1970s in favor of stomach stapling procedures

The Middle Phase (1970 – 1990) – Stomach Stapling.

The Roux-en-Y gastric bypass (RYGB) operation was introduced by Edward Mason in 196012. In this procedure the stomach was completely partitioned into a small upper gastric pouch, draining into a Roux–en-Y limb of proximal jejunum of variable length from 40 to 150 cm, and a distal excluded stomach. This procedure provided a hybrid between the malabsorptive approach of JIB and later, more purely restrictive, operations. It has undergone various modifications over the subsequent 40 years and still serves us well as an effective anti-obesity operation. However its drawbacks of perioperative death and significant perioperative and late morbidity, although markedly less threatening than those of JIB, have been nevertheless sufficient to cause most of the obese to stay away.

Dr Mason and his colleague, Dr Printen, then introduced a purely restrictive operation of gastroplasty in 197313. The procedure involves partitioning the stomach into a small upper pouch draining through a narrow stoma into the remainder of the stomach. Numerous variations of this procedure have followed, the most significant variant being the vertical banded gastroplasty (VBG) which was first described by Dr Mason in 198214 . It was hoped that this group of operations would provide greater short and long term safety and yet retain the power of gastric bypass. Unfortunately both randomized controlled trials and observational studies have consistently shown that it has failed in both aspirations15-18.

In the meantime there was a resurgence of malabsorptive surgery with Italian surgeon, Nicola Scopinaro, introducing the biliopancreatic diversion procedure (BPD) in 197619. It too has undergone change with time and experience. The basic procedure involves distal gastrectomy leaving a proximal gastric pouch of 200 – 500 ml, a 200 cm length of terminal ileum anastomosed to the gastric pouch and the biliopancreatic limb entering at 50 cm from the ileocecal valve20. The most notable remodeling of the procedure has been the so-called duodenal switch variant (BPD-DS) proposed by Picard Marceau’s group in 199321, 22 in which a longitudinal gastrectomy (sleeve gastrectomy) enabled retention of the gastric antrum for controlled gastric emptying, and the ileal limb was anastomosed to the proximal duodenum. The benefit of this variation remains controversial.

The Current Phase (1990 - Present) – Laparoscopic Procedures.

This phase is characterized by the advent of the laparoscopic approach to bariatric surgery particularly gastric bypass23 and biliopancreatic diversion24, introduction of the laparoscopic adjustable gastric band (LAGB) with the particular features of minimal anatomical disturbance, adjustability and potential reversibility25, 26 and growth of the sleeve gastrectomy as a stand-alone procedure27. The reduced invasiveness and increased safety of these approaches has led to a major rise in the use of bariatric surgery for obesity across the world. In the United States the estimated total bariatric procedures in 1990 was 30,000. The total for 2008 was estimated to be 220,000 cases28

Adjustable gastric banding had first been proposed by two Austrian surgical researchers, Szinicz and Schnapka, in 198229. The idea was brought into clinical practice as an open operation by Lubomyr Kusmak in 198630. It did not attract major interest until the advent of the technology that enabled the performance of complex laparoscopic surgical procedures became widespread in the early 1990s. The BioEnterics® Lap-Band ® system (LAGB) was specifically designed for laparoscopic placement and was introduced into clinical practice by Mitiku Belachew from Huy, Belgium in September 1993. Because of the dual attractions of a controlled level of effect through adjustability and of laparoscopic placement without resection of gut or anastomoses, through the 1990s this procedure rapidly became the dominant bariatric procedure in all regions of the developed world except the USA, where its introduction was delayed until regulatory requirements were completed in June 2001. More recently, the challenges of maintaining good aftercare for the banded patient and the relative simplicity of the sleeve gastrectomy has seen this latter procedure take a dominant position.

In the meantime, laparoscopic approach to the RYGB was introduced by Wittgrove and Clark in 199431. The technical challenge of completing the gastrojejunostomy laparoscopically has been variously managed by transoral passage of a circular stapler, end-to-end or side-to-side stapling transabdominally or a simple hand-sewn anastomosis. As the technical challenges of the laparoscopic approach were overcome, this procedure became the dominant approach in the USA until it was overtaken by gastric banding around 2008 and then by sleeve gastrectomy which, in 2015, is the most used procedure world-wide.

Laparoscopic BPD or its DS variant has remained clinically challenging. The mortality can be high24 and few are now performed28. Most procedures are still performed by open technique and as they represents less than 2% of bariatric surgery worldwide, it is unlikely they will grow beyond that.

CURRENT METHODS IN BARIATRIC SURGERY

Sleeve Gastrectomy

The sleeve gastrectomy involves excision of approximately 80% of the stomach by using multiple firings of a linear stapler/cutter to separate a narrow tube or sleeve of the lesser curve of the stomach from the greater curve aspect. The antrum is preserved to maintain gastric emptying. A bougie is placed in the lesser curve segment during the resection to maintain adequate lumen yet achieve a standardised gastric remnant. The optimal size of this bougie is not yet agreed upon. A 36 French is the most common size but sizes from 30F to 50F are reported. The procedure is non-adjustable and non-reversible.

The procedure is a laparoscopic procedure, taking between 30 and 90 minutes. Some patients are treated on an outpatient basis but a one to two day stay is more common.

Figure 1. Sleeve gastrectomy

Figure 1. Sleeve gastrectomy

Gastric imbrication is a non-resectional variant of the sleeve gastrectomy. The greater curve vascular pedicles are ligated and then the gastric wall is imbricated using two rows of sutures to create a narrow lumen, similar in size to the sleeve gastrectomy. The costs are reduced by avoiding the use of multiple firings of stapling devices and the risks are expected to be lower by the absence of the stapled closure of divided stomach.

Figure 2. Gastric imbrication. The upper panel shows the placement of the initial continuous suture after division of the greater curve vascular pedicles. The lower panel shows completion of the second row of sutures.

Figure 2. Gastric imbrication. The upper panel shows the placement of the initial continuous suture after division of the greater curve vascular pedicles. The lower panel shows completion of the second row of sutures.

Laparoscopic Adjustable Gastric Banding (LAGB).

The LAGB is the safest of the surgical options and therefore has tended to be considered ahead of other bariatric procedures on the hierarchy of risk because of its effectiveness in the setting of a better safety profile, minimal invasiveness and complete and easy reversibility. Its requirement for skilled follow up and adjustments has proved challenging for many.

The procedure was introduced into clinical practice by Dr Mitiku Belachew with the laparoscopic placement of a Lap-Band in a patient in Huy, Belgium on the 3rd September, 1993. Others quickly followed and soon it was being embraced as an important new procedure. However initially there were significant gaps in the knowledge of the process of care the LAGB required and skills in LAGB placement, optimal aftercare and management of late adverse events. It was not known how the band worked or even if it would work. There were no data on optimal placement and fixation. Protocols for the aftercare process, the adjustment protocols and the education of the patient into the specific requirements for eating and activity after the band were not present.

There has been important growth in knowledge since that time with more than 1,000 peer-reviewed papers defining the LAGB process and outcomes. It is now more studied than any bariatric procedure with better knowledge of its mechanisms and a higher quality evidence base than all the other bariatric options.

There are a number of adjustable gastric bands available. The principal two, which have approval of the US Food and Drugs Administration for use in the USA, are the Lap-Band ™     (Apollo Endosurgery, Austin, Tx) and the Realize Band™ (Ethicon Endosurgery, Cincinnati, OH). Others include the Mid-Band, the Heliogast band, the Minimizer band and the AMI band. These are in use principally in Europe. They lack adequate published data attesting to their effectiveness, they are not approved for use in the USA and so their uptake has been modest. The discussion below will focus primarily on the data from the Lap-Band.

The current version of the Lap-Band, known as the Lap-Band AP (for Advanced Platform), is shown in figure 3.

Figure 3. The band consists of a ring of silicone with an inner balloon. The balloon is connected to an access port.

Figure 3. The band consists of a ring of silicone with an inner balloon. The balloon is connected to an access port.

The LAGB is placed laparoscopically. Commonly 3 x 5mm, 1 x 10mm and 1 x 15 mm ports are required. A path is developed from the top of the lesser curve of the stomach to the angle of His, the band is placed around this path, closed and then stabilized at this site with some sutures. The tubing passes to an access port, placed through a 3 cm incision over the left rectus abdominis muscle. The procedure takes 30 -50 minutes to complete and the patient is able to go home at a mean of 2 hours after completion of the procedure32.

Figure 4. The LAGB is placed over the cardia of the stomach within 1cm of the esophago-gastric junction.

Figure 4. The LAGB is placed over the cardia of the stomach within 1cm of the esophago-gastric junction.

Roux en Y Gastric Bypass (RYGB)

Gastric bypass combines a marked reduction in the size of stomach available for food with a narrow stoma passing from the gastric pouch to a Roux-en-Y loop of jejunum diverting food from the duodenum and proximal jejunum. In a typical current laparoscopic version (figure 5), the stomach is divided completely by multiple firings of a device which places two rows of staples and cuts the gastric wall in between. This creates a small proximal gastric pouch of volume of 50ml or less and a large residual stomach, now excluded from the food. A Roux limb of jejunum is formed by dividing the proximal jejunum completely at about 50cm from the duodeno-jejunal flexure, taking the distal aspect of this point of division up to form a small anastomosis with the small gastric pouch and anastomosing the proximal aspect to the more distal jejunum at 50 cm below the gastro-jejunal anastomosis. The procedure takes 90 -150 minutes and the patient stays in hospital for 2 - 4 days.

Figure 5. RYGB showing a small gastric pouch, a narrow gastrojejunostomy and exclusion of foods from the duodenum and proximal jejunum.

Figure 5. RYGB showing a small gastric pouch, a narrow gastrojejunostomy and exclusion of foods from the duodenum and proximal jejunum.

Single anastomosis Gastric Bypass (SAGB)

This form of gastric bypass is increasingly popular as an alternative to the RYGB. It is variously known as the SAGB, OAGB (one anastomosis gastric bypass), OLGB (Omega loop gastric bypass) or MGB (Mini gastric bypass). It differs from the RYGB by having a loop of small bowel rather than a Roux limb, a long and narrow lesser curve gastric pouch and a longer bypass of the duodenum and proximal jejunum (typically, 150 cm rather than 40 cm) (figure 6).

Figure 6. SAGB showing the gastric pouch as a sleeve of lesser curve of stomach and the loop gastrojejunostomy.

Figure 6. SAGB showing the gastric pouch as a sleeve of lesser curve of stomach and the loop gastrojejunostomy.

There are now extensive published data on outcomes which include large patient groups, long-term follow up and systematic literature reviews33-36. Early concerns regarding bile reflux leading to intractable gastritis and esophagitis have not proven to be valid.

In a prospective randomized controlled clinical trial performed on 80 patients with a two year follow up37, the SAGB proved to be simpler and safer than RYGB, yet achieved similar outcomes for effects on the metabolic syndrome and quality of life and demonstrated no disadvantages over RYGB.

Biliopancreatic Diversion / Duodenal Switch (BPD/DS)

The BPD contains a "restrictive" component and a "malabsorptive" component. The restrictive component is a partial gastrectomy leaving a large proximal gastric segment of between 200 and 500ml. The actual size is only loosely defined. The originator of BPD, Dr Nicola Scopinaro, moved to the "ad hoc gastrectomy" with its wide variability of gastric pouch size after he had treated nearly 1000 patients. He recommended tailoring the gastrectomy to the patient's weight and some physical characteristics. He also recommended tailoring the intestinal lengths38. The validity and effect of these variations has not been reported.

The malabsorptive component consists of division of the small intestine, usually at 250cm proximal to the ileocecal valve, anastomosis of the distal side of this division to the gastric pouch and end-to-side anastomosis of the proximal side of the division to the terminal ileum, usually at 50cm proximal to the junction with the cecum.
In the duodenal switch variant of the BPD (figure 7) a sleeve gastrectomy is performed, preserving the pylorus and proximal duodenum. The distal end of the transected small bowel is anastomosed to the duodenum. This structure is designed to avoid dumping syndrome but any particular benefit of one version over the other is unclear.

Figure 7. The DS variant of BPD with a sleeve gastrectomy, retention of the gastric antrum, diversion of food into the mid small gut and diversion of pancreatic and biliary secretions to the distal small gut .Note both limbs are passing behind the transverse colon and a colour difference is added to help follow the respective pathways.  The common channel is the normal ileum terminating at the ileo-caecal junction.

Figure 7. The DS variant of BPD with a sleeve gastrectomy, retention of the gastric antrum, diversion of food into the mid small gut and diversion of pancreatic and biliary secretions to the distal small gut .Note both limbs are passing behind the transverse colon and a colour difference is added to help follow the respective pathways.  The common channel is the normal ileum terminating at the ileo-caecal junction.

It is the most metabolically severe of the current options and therefore hasn’t proved to be popular with patients or surgeons in spite of favorable published outcomes. BPD has been available for 30 years19 and yet remains a very minor part of bariatric surgery. Worldwide, it constitutes less than 2% of bariatric surgery28. However, it does generate good weight loss and should be considered on occasions as a second line bariatric surgical option.

MECHANISMS OF ACTION IN BARIATRIC SURGERY

Historically, bariatric surgical procedures were all subdivided into two groups. Some were classified as restrictive, with the creation of a small pouch that limited the size of a meal and a small opening from that pouch into the rest of the gut so that the transit of food was abnormally delayed. The best model of a restrictive procedure was the vertical banded gastroplasty used during the 1970s and 80s. Others were described as malabsorptive, in which the changed anatomy reduced normal macronutrient absorption. The best example of this was the original jejunoileal bypass. Some procedures are considered to be a hybrid containing both elements, the BPD being an example. Other possible mechanisms were not considered. This narrow dichotomous concept has happily faded as better research and more careful consideration has led to a much broader understanding of the mechanistic options39. Table 1 provides a list of current known mechanisms. It is undoubtedly incomplete but provides a more up-to-date view of how bariatric surgical procedures can achieve weight loss.

Table 1. Possible mechanisms of bariatric surgical effect.

Induce satiety, reduce appetite, control hunger
Change of taste preference - less sweet foods; lower fat content;
Restrict Intake
Diversion from upper GI tract
Malabsorption   of macronutrients
Increased energy expenditure; Increased diet-induced thermogenesis
Aversion to food through side-effects
Inhibition of the metabolic adaptation to weight loss
Changes to the gut microflora
Changes to plasma bile acid levels
Changes in gut hormones: candidates include the incretins (GLP-1; GIP), ghrelin, CCK, Peptide YY,
Central mechanisms: Modify hedonics; central appetite control; altered liking and wanting;

Mechanisms in Sleeve Gastrectomy

Although primarily seen as a gastric restrictive procedure, it is likely that satiety induction is centrally important and occurs because of removal of most of the source for ghrelin. Further, with rapid gastric emptying the increase of nutrients into the small intestine may lead to change in distal gut hormonal responses. The sleeve gastrectomy is the first element of the duodenal switch variant of the BPD. It has lately become popular as a single procedure because of ease of surgery, early effectiveness and perceived lack of need for close follow up ("sleeve and leave").

Mechanisms of Gastric Bypass (RYGB and SAGB)
RYGB is a complex procedure both anatomically and physiologically. There are several mechanisms of action involved. First, there is early satiation after eating a small amount of food due to the small volume and slow emptying of the gastric pouch. The small stoma provides a restrictive component through delayed gastric emptying. The diversion of food away from the distal stomach, duodenum and proximal jejunum reduces the digestion and possibly the absorption of food by this area of the gut. However, as most of the small bowel remains in the absorptive pathway, it is unlikely the absorption of macronutrients is affected and so the standard RYGB should not be regarded as having a malabsorptive component. The long-limb version of RYGB where food is diverted from the digestive enzymes of the pancreas for 150cm or more40 is more likely to contain a malabsorptive component. Micronutrients which are normally absorbed in the upper gut, such as calcium, are changed. Finally it can have an aversion effect with the symptoms of dumping syndrome occurring if there is ingestion of simple sugars or small osmotically-active molecules, leading to reduce sweet-eating. The diversion of food from the duodenum and proximal jejunum may mediate gut hormonal effects with increased release of GLP-1 and GIP from the distal gut. These hormones act as incretins, increasing release of insulin from pancreatic β cells.

Mechanisms in Laparoscopic adjustable gastric banding (LAGB)

The band lies at the very top of the stomach, around the cardia and within one cm of the esophago -gastric junction. The access port is place in the subcutaneous layer of the anterior abdominal wall and is accessed by a percutaneous injection. The primary mechanism of action of the gastric band is by the induction of a sense of satiety, a lack of appetite or hunger41. There are two components to this - satiety and satiation.
Satiety is the state of not being hungry. It is achieved for the LAGB patient by adding or removing of fluid from the system to change the degree of compression of the band on the gastric wall. When this compression is optimal, it induces a sense of satiety which is present throughout the day. Although some hunger may develop at times during the day, there is a general reduction of appetite, less interest in food and less concern about not eating.
Satiation is the resolution of hunger with eating. For the LAGB patient, it is induced by each bite of food as it passes across the band. When the band is optimally adjusted each bite is squeezed across by esophageal peristalsis, generating increased pressure on that segment of the gastric wall. This reduces any appetite that may have been present and induces a feeling of not being hungry after eating a small amount. The combination of these effects allows the person to eat three or less small meals per day. The mean energy intake of the banded patient should be between 1,000 and 1,200 kcals per day42.

Figure 8. The Lap-Band AP with and without added fluid.

Figure 8. The Lap-Band AP with and without added fluid.

Figure 8 shows two views of the Lap-Band AP. On the left, it contains only the basal volume 3ml of saline and on the right it is well inflated band containing 7 ml of saline. The space within which is occupied by the cardia of the stomach. With 3ml added the internal space has an area of 357cm2. This is reduced to an area of 139cm2 with 7.0ml is present. These two areas represent the limits within which the LAGB is set for most patients. This ability to titrate the level of adjustment against the level of satiety is central to the effectiveness of the band.The optimally adjusted band modifies the normal transit of a food bolus into the stomach. With normal swallowing, a food bolus is swallowed and carried by oesophageal peristalsis down the esophagus. The lower esophageal sphincter (LES) relaxes and the bolus passes intact smoothly into the stomach. The LES facilitates the final transfer with an aftercontraction. With the band in its correct place with only 1-2 cm of cardia above the upper edge of the band and with the band optimally adjusted (exerting a pressure of between 25 and 35 mm Hg on the gastric lumen43), the esophagus must generate stronger peristalsis and the aftercontraction of the LES become more important. The bolus is squeezed through by these forces. It takes between two and six squeezes to achieve complete transit of a single small bite. This may take up to one minute.

Figure 9. A small bite of food is being squeezed across the band, thereby compressing the vagal afferents and generating a feeling of satiety.

Figure 9. A small bite of food is being squeezed across the band, thereby compressing the vagal afferents and generating a feeling of satiety.

The aftercontraction of the LES is evident. Just part of each bite will transit on each peristaltic sequence. The remainder will reflux into the body of the distal esophagus, generate a secondary peristalsis wave and a further squeeze will occur. After several squeezes the bite will have passed. Importantly, each squeeze generates signals to the satiety centre of the hypothalamus. A second swallow should not commence until all of the previous bite has passed totally into the stomach below the band.

The signalling of both satiety and satiation to the arcuate nucleus of the hypothalamus does not appear to be mediated by any of the hormones known to arise from the cardia as none has been shown to be increased in a basal state after band placement and none increases post-prandially44. Vagal afferents are the more probable mediators and, among these, the intraganglionic laminar endings (IGLEs) demonstrate the characteristics needed to subserve this role45, 46

Figure 10 shows the components of the lower esophageal contractile segment (LECS), an entity described by Dr Paul Burton from extensive study of the "physiology" of the gastric band47. It brings together the key elements that together generate early onset of satiation after eating. The distal esophagus squeezes each bite of food to the stomach proximal to the band. The lower esophageal sphincter relaxes to allow passage and then contracts to maintain the forward pressure. The proximal segment of stomach maintains tonic contraction and detects the pressure increase. The band maintains an optimal compression to provide sufficient resistance to stimulate afferent signals but not sufficient to stop transit. There should be no restrictive component for normal functioning of the LAGB.

Figure 10. The four components of the Lower Esophageal Contractile Segment (LECS)

Figure 10. The four components of the Lower Esophageal Contractile Segment (LECS)

Mechanisms in Biliopancreatic bypass (BPD)

The initial element of the BPD is a partial gastrectomy either as a standard Bilroth II procedure for the standard BPD or as a sleeve gastrectomy in the DS variant of the BPD which generates a restrictive element and also through reduction of the ghrelin sources, and sense of satiety is induced. The gastric volume is reduced to 200 -500ml thus limiting food intake and this is considered to be the initial mechanism of effect48. Subsequently the separation of ingested food from the digestive enzymes until the last 50 cm of terminal ileum leads to malabsorption of all macronutrients. A number of positive and negative secondary effects occur. Through interruption of the enterohepatic circulation of bile and the bypassing of the proximal small intestine, there is a marked reduction of total cholesterol, triglycerides and LDL-cholesterol. Insulin sensitivity, as measured by HOMA, is improved49. Malabsorption of amino acids can lead to hypoproteinemia. Malabsorption of micronutrients and minerals can lead to osteoporosis and anaemia.

OUTCOME FROM BARIATRIC SURGERY

Mortality And Adverse Events

Perioperative mortality. There is a mortality risk with any surgery and this risk was strongly evident for bariatric surgery prior to the general use of laparoscopic approach. Pories et al50 reported a perioperative mortality of 1.9% of the 605 patients treated by open gastric bypass. In a major series from Richmond, Virginia there were 31 perioperative deaths (1.5%) in 2011 patients having RYGB between 1992 and 2004. The mortality occurring at the level of community surgery is probably higher than from the major academic centers. Flum and Dellinger51 used the Washington State Comprehensive Hospital Abstract Reporting System database and the Vital Statistics database to evaluated 30 day mortality of all people having a RYGB procedure in that state during the period 1987 to 2001. Of 3,328 procedures there were 64 deaths, a mortality rate of 1.9%. This period included both laparoscopic and open surgery and could be seen to reflect community practice.

The overall mortality has decreased in more recent years, particularly with the widespread use of a laparoscopic surgical approach as reported in the systematic reviews of the published data. Death after LAGB is rare and in most reports is 10-15 times less likely than after RYGB52, 53. At the Centre for Bariatric Surgery in Melbourne, we have performed more than 9000 primary LAGB procedures and have performed revisional LAGB surgery on more than 1500 of these or other patients without any 30-day mortality or any later death related to the LAGB procedure.

The most definitive evaluation of mortality currently available is derived from the Longitudinal Assessment of Bariatric Surgery (LABS) study report in 20095. This NIH-sponsored study of bariatric surgery involved 10 sites, carefully selected for their expertise and experience. The 30-day rate of death was monitored closely. Of the 4776 patients studied, 3412 had RYGB, 1198 had LAGB and 166 had other procedures unspecified in the report. There were 15 deaths in the RYGB group (0.44%), 6 after a laparoscopic approach and 9 after an open approach. There were no deaths in the LAGB group of patients. The difference was highly significant.
Sleeve gastrectomy was not included in the LABS study. In a recent systematic review54 there were 26 deaths in 8922 patients (0.3%) after sleeve gastrectomy, an outcome similar to RYGB in the LABS study.

Early adverse events. The LABS study serves also to inform on early adverse events for the two major bariatric procedures of RYGB and LAGB. Not surprisingly, the incidence of adverse events mirrored the perioperative mortality rates. Using a composite end-point of death, DVT or pulmonary embolism, reintervention or failure to be discharged by 30 days, they identified 189 who were positive to that end-point, 177 in the RYGB group (5.2%) and 12 in the LAGB group (1.0%), a difference that was highly significant. The complication rate for sleeve gastrectomy is at least comparable to RYGB. In the systematic review of all reports available on the sleeve gastrectomy reported by Brethauer in 200955, the quality of the data prevented careful analysis but those papers that included complications declared rates from 0% to 24%. Postoperative leaks from the staple line after sleeve gastrectomy are relatively frequent (2-3%) and appear to be independent of surgeon experience56. In contrast to leaks after RYGB, they tend to be slow to close thereby generating mortality, greater morbidity, long-term patient suffering and high hospital costs.

Late adverse events. There is, and always will be, a maintenance requirement with any bariatric procedure as we are treating a chronic disease. The procedure needs to remain effective over decades rather than years. It is inevitable that there will be the need to correct or repair. Whilst reversal of a bariatric procedure should be counted as failure, revision to correct or repair should not. It is a part of the process of care.
All bariatric procedures have been shown to have a maintenance requirement. The revisional surgery rate of the studies which report 10 or more years of follow-up, as shown in table 3, was a median value of 24% and it was not different between procedures. Sleeve gastrectomy is not included as there are no 10 year follow-up studies available to date.

The median rate for the six RYGB reports that provided data was 22% with a range of 8% - 38%. All of the LAGB sets provided data on revisional surgery. The median value was 26% with a range of 8% - 60%. As almost no details of the type of revisional procedures were provided and, as there were too few reports at ten years on other procedures, a more detailed analysis is not possible.

Weight Loss Outcomes

Weight loss can be described in different ways, each of which has its advantages and drawbacks. In bariatric surgery the percent of excess weight lost (%EWL) is the preferred method. The excess weight approximates the excess fat within the body. That is what we are trying to reduce and therefore a measure of that effect has more relevance in this clinical setting than measures of total weight change as are commonly used in the non-surgical weight loss literature. It could be argued that percent of excess BMI lost (%EBL) is the most relevant to health status. However as it has a fixed linear relationship to %EWL and it currently lacks broad usage, it has not yet been embraced. Most reports in the bariatric surgical literature provide %EWL, and thus allow comparison between studies.

As obesity is a chronic disease, for treatments to be effective they must also be effective in the long term. Short-term studies (1 - 3 years) are plentiful but simply suggest a potential effectiveness. Medium term studies (3 -10 years) are far fewer but are more assuring of real effectiveness. Long-term studies (greater than 10 years) are very few and yet are the only ones that truly enable rational decision making on effectiveness. There have been a number of systematic reviews bringing these data together. These provide perhaps the best understanding available of the reasonable weight loss expectations for bariatric surgery in general and for specific bariatric procedures. I will deal with these in order of the duration of follow up provided.

One year outcomes: The most quoted of all the systematic reviews is the report by Buchwald et al57 published in JAMA in 2004. There was 47.5%EWL at one year after LAGB, 61.6%EWL for RYGB, 68.2%EWL for gastroplasty, principally vertical banded gastroplasty, and 70.1%EWL for biliopancreatic diversion. In dealing with the outcome data with one year follow up only, the study gave advantage to the stapling procedures as the weight loss generally peaks at this time for these procedures. However, for the LAGB procedures, weight loss continues for 2 -3 years and therefore assessment at one year was premature. Note that in this review gastroplasty, a procedure now largely discontinued, appeared to provide better weight loss than RYGB and was equivalent to BPD in this study.

Short-term (1 - 3 yr)outcomes: Chapman et al52 performed a systematic review of the literature available up to mid-2001 and compared the published reports on LAGB, RYGB and VBG. They found that, although LAGB showed less weight loss at 1 and 2 years, all three groups of procedures produced comparable weight loss at 3-4 years, that being the longest follow up available on LAGB at that time. They noted the lower mortality associated with LAGB.

Maggard et al53 reviewed the literature up to mid-2003 and identified 89 reports that provided weight loss data after bariatric surgery. Weight loss data were available at or beyond three years in 57 of these reports. Table 2 shows a summary of their weight loss findings for 3 or more years of follow up.

Table 2. Summary of published data providing three or more years of follow up (adapted from Maggard, 200553). Note that they identified only one report for laparoscopic RYGB and for BPD at that time.

Procedure Weight Loss (kg) 95% CI No. of Studies No. of Patients
Open RYGB 41.6 37.4 - 45.8 20 1266
Laparoscopic RYGB 38.2 28.0 - 48.6 1 15
VBG 32.0 27.7 - 36.4 18 1877
LAGB 34.8 29.5 - 40.1 17 3076
BPD 53.1 47.4 - 58.8 1 50

Based on the confidence intervals, only BPD appears to offer better weight loss than the other procedures.

Medium-term (3 - 10 year)outcomes: A single systematic review has focused on the medium term-term outcomes and thus included only reports that provided at least three year data58. A total of 43 reports were included, 18 related to LAGB, 18 related to RYGB and 7 on BPD or its DS variant. Figure 11 shows the %EWL for these three procedures.

Figure 11. The pattern of weight loss over time after RYGB, LAGB and BPD ( from reference 58, with permission).

Figure 11. The pattern of weight loss over time after RYGB, LAGB and BPD ( from reference 58, with permission).

The most significant single finding was that each of these procedures was effective in achieving substantial weight loss over the medium term. RYGB was significantly more effective than LAGB at years 1 and 2 but not beyond that point. BPD appeared to be more effective than the other procedures but the difference was shown to be statistically significant at 5 years follow up alone.

Long-Term ( >10 Year) Outcomes:

It is not hard to achieve substantial weight loss in the short term. Most lifestyle programs can do it. Showing weight loss after bariatric surgery in the short term cannot justify its costs or risks. The key weakness of the lifestyle programs is not their impotence but their lack of durability. For bariatric surgery to be worthwhile the effects must last. Yet most of the published studies deal with the short-term outcomes only.

Data published up to the end of 2011 on weight loss at 10 or more years after bariatric surgery have been subjected to systematic review59. At that time there were 9 reports of RYGB, 7 reports of LAGB, and 4 reports of BPD/DS with data beyond 10 years. No long-term outcome data for sleeve gastrectomy are yet available.

Table 3 summarizes the findings. Notably, all three procedures have generated substantial long-term weight loss. BPD/DS appears to be the most effective with 72% EWL. RYGB (54.0% EWL) and LAGB (54.2% EWL) are identical.

Table 3. Bariatric surgical procedures: systematic review of long-term outcomes (Adapted from ref 59).

Procedure RYGB LAGB BPD / DS
No. of Reports 9 7 4
No. of patients – initial 3194 6369 3408
Perioperative mortality 21/2102 (1.01%) 1/6369 (0.002%) 27/3066 (1%)
% Follow up achieved 64% 82% 83%
% EWL at 10+ yr:
Weighted mean % EWL (range)

 

54.0 (28-68)

 

54.2 (33-64)

 

71.7 (69-75)

Revisional procedures:

Range as % of total followed up

 

8-38

 

8-60

 

NR

NR = not reported.   As perioperative mortality was not reported in all studies, the denominator for mortality rates may be lower than the initial number of patients.

The results emphasize the laudable effectiveness of all of these procedures, each of which has been able to maintain more than 50% EWL beyond 10 years. Bariatric surgery does work and RYGB and LAGB appear to be equal in the long-term.

Sleeve Gastrectomy Outcomes.

Adequate weight loss reporting for sleeve gastrectomy remains a problem with no long term data, poor medium term data and major loss to follow up in all series providing this information. Brethauer et al provided a literature review of the 36 published reports involving 2570 patients that were available in 200955. Only one report gave data beyond 3 years. There was a mean weight loss for the full group of 55% EWL. The only study exceeding three years showed a weight regain of approximately 40% from 3 - 5 years60.

In 2014, Diamantis et al61 reported a systematic review of the literature which included 16 studies, involving a total of 492 patients, which reported more than 5 year follow-up. There was 62% EWL at 5 years, 54% at 6 years, 43% at 7 years and 54% at 8 years. Not included in this review were the data from Weiner from Frankfurt who has a large series with longer follow up62. He has reported the outcomes of 746 patients with up to 8 year follow up. The %EWL was 59% at 2 yr, 45% at 5 yr and 36% at 8 yr.

Gastric imbrication is too new to know much of the benefits or risks. Very few reports of weight loss outcomes are available. Brethauer et al report a 53%EWL in 6 patients at one year63. The anticipated freedom from perioperative complications needs to be shown. Just as with sleeve gastrectomy, there is a high likelihood of failure of initial weight loss or weight regain in the medium term. It remains an experimental procedure at this time.

Type 2 Diabetes and Bariatric Surgery

Type 2 diabetes mellitus is the paradigm of an obesity-related illness and improved control for diabetes and other metabolic disorders represents the greatest potential strength for bariatric surgery. The metabolic effects of weight loss following bariatric surgery have been well documented and provide clinicians with an obvious path for the prevention and treatment64-66. The effects of bariatric surgery on type 2 diabetes have been subject of 2 systematic reviews64, 66. Buchwald reported 86% of 1835 patients from multiple case studies showed remission or improved control64. Maggard et al66 reviewed 21 case series and reported a range of 64% - 100% showing remission or improvement.

Sadly, few patients with diabetes have been offered this benefit to date. Arguments against the broad application of bariatric surgery have included: Safety concerns, lack of evidence from RCTs, insufficient understanding of mechanism, lack of proven durability of effect and lack of proven reduction of the complications of diabetes in association with long-term remission. All of these arguments are losing substance as new data continue to appear. However, not all have yet been answered fully. The question regarding long-term outcomes, in particular, needs to be better supported by data.

Safety.

The safety of bariatric surgery has improved greatly in recent years with almost no perioperative mortality risk associated with LAGB and, in expert centres, a mortality rate of 0.44% after RYGB5. Compared to the mortality risk of diabetes, bariatric surgery is a safe path.

Remission rates in RCTs.

Until recently, the lack of high levels of evidence has been the most valid criticism of reports of bariatric surgery and diabetes. The medical literature had been littered with numerous observational studies. These studies often carried major deficiencies including poor definition of both diabetes and its remission and extensive or unreported loss to follow up. Systematic reviews have sought to resolve these deficiencies through the pooling of data64, 66.
We have now been provided with at least five high-quality randomised controlled trials67-71. Their findings should over-ride the past observational studies. All studies have compared one or more bariatric procedures with a group having non-surgical treatment (NST). However the studies are not directly comparable due to extensive heterogeneity, including different criteria for patient selection, various treatment durations and various definitions of remission of diabetes, particularly the cut-off values for HbA1c. Nevertheless, they serve to provide key comparisons with NST, the current offering to more than 99% of people with diabetes.
The key outcome of remission of diabetes from each of these studies is shown in figure 12. The first of the studies was performed by our group at the Centre for Obesity Research and Education (CORE) in Melbourne67. 60 patients were randomised to LAGB or non-surgical therapy (NST). They were required to have a BMI between 30 and 40 and to have been known to have type 2 diabetes for less than 2 years. Inter alia, remission was defined as a HbA1c of less than 6.2%. The results reported were after 2 yr of follow up.
The study by Schauer et al68 from the Cleveland Clinic randomised 150 patients and included NST, RYGB and sleeve gastrectomy. They defined remission, in part, as a HbA1c of less than 6.0%. They reported their results at one year and then at 3 years72.
Mingrone et al69 randomised 60 patients to three groups – NST, RYGB or BPD by open laparotomy and reported their outcomes after a two year follow up. They used a HbA1c cut-off of 6.5% to define remission. They have recently reported the five year follow up of these groups of patients73.
Ikamuddin and coworkers70, from Minnesota and other sites, performed a similar study to the Cleveland Clinic study except for restricting it to a comparison of NST with RYGB. It involved 120 patients and was multicentered, including a group from Taiwan. They defined remission as HbA1c of less than 6.0% and to date have reported the one70 and two year74 follow up data of a planned 5 year study.
Courcoulas et al from Pittsburgh randomised 69 patients into 3 groups – NST, RYGB and LAGB. They defined partial and complete remission, in part, as HbA1c values of less than 6.5% and 5.7% respectively. They have published the one year71 and three year75 outcomes to date. The data shown in figure 11 below are for partial remission.

 

Figure 12. Comparison of remission of type 2 diabetes in five RCTs of bariatric surgical procedures versus non-surgical treatment.

Figure 12. Comparison of remission of type 2 diabetes in five RCTs of bariatric surgical procedures versus non-surgical treatment.

Three of the studies included a RYGB arm and report remissions rates of 44% at one year in 2 studies68, 70 and 75% at two years in one study69. The remission rates for LAGB were 73% at two years67, for biliopancreatic diversion (BPD) 95% at two years69 and for VSG 37% at one year68. The studies contained sufficient heterogeneity to not permit head-to-head comparison between procedures but have established the significantly greater effectiveness of all bariatric surgical options over non-surgical treatment.

Mechanisms of effect.

This remains an area of contention and learning and is linked to the general study of mechanisms reviewed above. Optimal selection of the patients and the procedure requires more knowledge of what drives better outcomes. The two primary effects of bariatric procedures is an immediate and sustained reduction of nutritional intake and a reduction of insulin resistance associated with loss of weight. Additional factors include improvement on β cell function, changes in appetite and changes in the gut-associated incretins.

The relative effectiveness of different bariatric procedures has been subject to considerable debate. RYGB is associated with the incretin effects with increased release of GLP-1 and GIP from the distal gut, increasing β cell function, at least in the short term after the procedure76. However comparison with non-surgical patients taking a diet equivalent to the early post-operative RYGB patient suggests that an improvement in insulin resistance immediately after RYGB is primarily due to caloric restriction, and the enhanced incretin response after RYGB does not improve postprandial glucose homeostasis during this time77.

The improvement in diabetes following weight loss is, in part, related to the dual effects of improvement in insulin sensitivity and pancreatic beta-cell function78. As beta-cell function deteriorates progressively over time in those with type 2 diabetes, early weight loss intervention should therefore be a central part of initial therapy in severely obese subjects who develop type 2 diabetes. For obese patients with type 2 diabetes, weight loss provides benefit not equalled by any other therapy, and may prove to be the only therapy that substantially changes the natural history of the disease50, 79.

Durability of remission. Recent reports derived from several of the RCTs suggest a possible lack of durability of effect. For RYGB there has been reasonable stability for three of the four studies. In the Cleveland clinic study, at three years the remission rate for RYGB did not change significantly (43% to 38%). Partial remission for the RYGB patients in the Pittsburgh study changed from 50% at one year to 40% at three years. The multi-centered study based in Minnesota also showed stability of the RYGB effect between one and two years74. In contrast, for RYGB, the Rome study, found the remission rate fell from 75% at 2 years to 37% at 5 years, a relapse rate of 53%.

Sleeve gastrectomy was measured only in the Cleveland study and between one and three years it showed a marked fall from 37% to 24%, a 50% reduction72. The remission rate for the BPD group of the Rome study fell from 95% to 63% a 37% relapse rate73 between the two and five year study points. LAGB partial remission rate in the Pittsburgh study was unchanged at three years (27% at year 1 and 29% at year 3).In the Pittsburgh study, comparing the relative effect of RYGB and LAGB, complete remission of diabetes had occurred in 17% of RYGB patients and 23% of LAGB patients at year 3.

Macrovascular and microvascular complication rates.

The durability of remission and the reduction of complications has been demonstrated at a fifteen year follow up in the Swedish Obese Subjects (SOS) study, a prospective matched cohort study80. They reported that the remission rate for the surgical group, predominantly gastroplasty, at two years was 72% and at 15 years was 31%. This remission rate, though reduced with time, was significantly better than their control group and indicates an important long-term benefit. Furthermore, and arguably more important than the remission rate, they found the macrovascular and microvascular complications of diabetes were fewer at 15 years in the surgical group than in the controls.

Earlier surgery improves outcome.

The duration of diabetes prior to bariatric surgery is a consistent and strong predictor of remission72, 80. A commitment to substantial weight loss must be a component of the early management of the obese person with diabetes. In a recent report from the SOS study, the patient who had bariatric surgery within the 1st year of diagnosis had more than twice the likelihood of remission of diabetes at two years and approximately six times the likelihood of remission at 15 years when compared to the patients who was diagnosed more than 4 years before surgery. If an obese patient with diabetes cannot achieve substantial weight loss by lifestyle change, early referral for consideration of bariatric surgery is an essential responsibility of the health care provider.

 

OTHER HEALTH OUTCOMES AFTER BARIATRIC SURGERY

Dyslipidemia Of Obesity

Increased fasting triglyceride and decreased high-density lipoprotein (HDL)-cholesterol concentrations characterize the dyslipidemia of obesity and insulin resistance81. This dyslipidemic pattern is highly atherogenic and the most common pattern associated with coronary artery disease 82. Weight loss surgery produces substantial decreases in fasting triglyceride levels, an elevation of HDL-cholesterol levels to normal, and improved total cholesterol–to–HDL-cholesterol ratio 83-85. Although elevation of total cholesterol is not obesity-driven, hypercholesterolemia can be controlled by malabsorptive procedures such as BPD86 and, to a lesser extent, RYGB87.

Hypertension

There is evidence of a reduction in both systolic and diastolic blood pressure (BP) following weight loss in association with a bariatric procedure88. We have studied the outcome of 147 consecutive hypertensive patients at 12 months after LAGB. Preoperatively, only 17 of these patients had BP within the normal range, all on therapy. Hypertension was present in 130 patients preoperatively; 101 of these were taking antihypertensive medications and the remaining 29 were not on therapy. Mean BP for these patients was 156/97 mm Hg prior to surgery. At 12 months after LAGB, 105 patients had normal BP, 42 remained hypertensive, and only 42 were taking any antihypertensive medication at that time. Mean BP was 127/76 mm Hg. From these data, we found that 80 patients (55%) had resolution of the problem (i.e., normal BP and taking no antihypertensive therapy), 45 patients (31%) were improved (less therapy and easier control), and 22 patients (15%) were unchanged89. We have demonstrated that the fall in blood pressure is sustained to at least 4-years after surgery but durability of blood pressure reduction over a longer period is uncertain90.

Gastro Esophageal Reflux Disease (GERD)

GERD is much more common in the obese. The community adult prevalence is estimated to be 8-26% whereas 39 - 53% of an obese population have been shown to suffer the disease91. The reasons for this increase are still unclear but suggestions include reduced basal pressure at the lower esophageal sphincter, increased transient lower esophageal sphincter relaxations, hiatal hernia and abnormal motility patterns in the esophagus or stomach. Multiple observational studies of LAGB and RYGB have reported strong benefit with improvement in LES function, reduced acid reflux and a level of symptom relief comparable to standard anti-reflux surgery91, 92. However it appears that to date no RCTs have been performed directly comparing the two approaches in a well-constructed study. We studied 87 patients who had moderate or severe GERD and have been followed for at least 12 months after LAGB. 73 (89%) have had total resolution of the problem, as defined by the absence of symptoms without treatment for the previous month. Preoperative and postoperative pH study and manometry have been performed on 12 of these patients who had severe symptoms preoperatively. The mean DeMeester score was 38 +/- 15 preoperatively and 7.9 +/- 8 at follow-up (p<0.001). In all but one of these patients symptoms had resolved completely93.

Asthma

There is a positive relationship between asthma and obesity with a possible dose-response effect in evidence94, 95. The Nurses' Health study identified a five-fold increase in the relative risk of asthma with a weight gain of 25kg from age 18 when compared to a weight stable group96. In the setting of obesity, asthma is more difficult to control97, 98. Weight loss by non-surgical means has shown to improve asthma. In an RCT from Finland99 the weight loss group (14% of total body weight lost) showed improved lung function, symptoms, morbidity, and health status. However lack of durability of the weight loss programs limits the clinical application of the non-surgical approach.

Several observational studies have report major improvement in asthma after bariatric surgery. A study of 32 consecutive patient with asthma treated by LAGB reported improvement in all measured aspects of the disease, including symptoms, severity, need for asthma medications (including corticosteroids), and hospital admissions100. The asthma severity score fell from 44.5 before operation to 14.3 at 12 months after operation. Other studies have also reported benefit after LAGB101 and community studies indicate an important reduction of asthma medications after bariatric surgery in the state of Michigan at a time when RYGB was the dominant procedure 102, 103. No RCTs of bariatric surgery versus best medical care appear to have been performed to date.

Obstructive Sleep Apnea

A range of sleep disorders is associated with obesity. The most serious of these is obstructive sleep apnea (OSA). Severe obesity is the greatest risk factor for the development of sleep apnea, with a 10-fold increase in prevalence. Excessive daytime sleepiness, a disabling and potentially dangerous condition, is very common in the obese population and is not necessarily related to OSA104. There are major improvements in sleep quality, excessive daytime sleepiness, snoring, nocturnal choking, and observed OSA with weight loss following LAGB surgery105.

Obstructive sleep apnea and other sleep disturbances has been studied in 313 patients prior to LAGB and repeated at one year after operation in 123 of the patients105. There was a high prevalence of significantly disturbed sleep in both men (59%) and women (45%). Observed sleep apnea was decreased from 33% to 2%, habitual snoring from 82% to 14%, abnormal daytime sleepiness from 39% to 4% and poor sleep quality from 39% to 2%.

Depression

Depression is common in the morbidly obese. Does the presence of obesity cause the person to be depressed or does depression cause the person to eat too much? We have investigated the effect of weight loss induced by LAGB on depression as measured by the Beck Depression Inventory (BDI)106. Preoperative BDI on 487 consecutive patients was a mean of 17.7+/- 9.5, a level within the range for moderate depression. Weight loss was associated with a significant and sustained fall in BDI scores with a mean score of 7.8 +/- 6.5 (N=373) at one year after surgery. By four years after surgery, the 134 patients studied had lost 54% of excess weight and had a BDI of 9.6 +/- 7.7. Although a small number remained in the major depressive illness category, the shift of the majority to normal values for BDI strongly indicates that most of the depression of obesity is reactive to the problem of obesity rather than a cause of obesity and is resolved by weight loss.

IMPROVEMENT IN QUALITY OF LIFE

Improvement in QOL is one of the most gratifying outcomes of bariatric surgery. A number of studies clearly demonstrate major QOL improvements following bariatric procedures 107-111. A large prospective study of QOL after weight loss surgery employed the Medical Outcomes Trust Short Form-36 (SF-36). The SF-36 is a reliable, broadly used instrument that has been validated in obese people. In this study, 459 severely obese subjects had lower scores compared with community normal values for all 8 aspects of QOL measured, especially the physical health scores. Weight loss provided a dramatic and sustained improvement in all measures of the SF-36. Improvement was greater in those with more preoperative disability, and the extent of weight loss was not a good predictor of improved QOL. Mean scores returned to those of community normal values by 1 year after surgery, and remained in the normal range throughout the 4 years of the study. It is significant that patients who required revisional surgery during the follow-up period achieved the same improvement in measures of QOL. Similar improvements in QOL have been demonstrated in patients having LAGB for previously failed gastric stapling112.

Studies of appearance orientation and appearance evaluation indicate that the severely obese usually have quite normal pride and investment in their appearance and presentation but they evaluate their appearance as being very poor113. Weight loss following LAGB has been shown to produce major improvements in self-evaluation of appearance, although it does not return to community normal levels. The extent of the improvement in appearance is related to the percent of excess weight loss. The discrepancy between one’s pride and investment in appearance and presentation and one’s self evaluation of appearance is lower with weight loss, reducing psychological stress113.

IMPROVEMENT IN SURVIVAL

The ultimate test of effectiveness of a treatment is the reduction of mortality. A comparison of the long-term mortality of bariatric surgical patients with obese controls shows improved survival. A systematic review by Pontiroli and Morabito114 identified 6 relevant trials, shown in table 4. They reported a reduction of risk of global mortality with an odds ratio of 0.55 (95% CI = 0.49 - 0.63) . The overall survival advantage was not different between LAGB (odds ratio 0.57) and RYGB (odds ratio 0.55).

Table 4. The relative risk of mortality compared to controls after weight loss following bariatric surgery.

Study Operation Relative Risk Source of controls
Christou (2004)115 RYGB 0.11 Medical
Flum (2004)51 RYGB 0.67 Medical
SOS study (2007)116 Various 0.71 Medical
Busetto (2007)117 LAGB 0.38 Medical
Adams (2007)118 RYGB 0.40 Community
Peeters (2007)119 LAGB 0.28 Community

The source of the control patients should be known and considered in the interpretation of the trials as sourcing a control group from a medical setting can preselect those who already have an existing disease that will shorten their life expectancy. As an example, the study by Christou115 used patients admitted to the Quebec provincial health insurance database with a diagnostic profile which included obesity as their control group. The reasons for admission were not known. This group had a mean age of 47 yr and had a mortality rate of 12.6 deaths per 1000 patient.years. By comparison, the Peeters' study (mean age of 55 yr) and Adams study (mean age of 39 yr) used true community controls and had mortality rates for the controls of 8.6 and 5.7 deaths per 1000 patient.years respectively. Adjusting for age, the mortality rate or the Christou patients was approximately twice those using community control, suggesting they included a number with late-stage illness

The study by Adams118 had sufficient numbers and details to provide some sub-group analysis. They found that mortality was significantly reduced for deaths from diabetes, heart disease and cancer whereas the rates of death from accidents and suicide were higher in the surgical group.

In spite of some weaknesses in the data and the inability to do a matched controlled study, the consistent pattern of the various reports indicates that global mortality is reduced in association with weight loss after RYGB and LAGB

THE HIERARCHY OF OBESITY THERAPIES

Resolution of the disease of obesity requires substantial and durable weight loss. The therapeutic options available are listed in table 5 in order of their risk, side effects, invasiveness and costs. We should always begin with the simplest and safest and work down the list. Only when a simpler and safer option has failed should we seek a more complex or risky option.

Table 5. Hierarchy of weight loss techniques in order of rating for invasiveness /risk / complexity /potential problems.

Therapy Rating
Lifestyle - Eat less and do more 1.0

Cognitive behavioural therapy

Very low calorie diets

Drug therapy

 2.0
Endoscopic - gastric balloons et al 4.0
Laparoscopic adjustable gastric banding (LAGB) 5.0
Sleeve gastrectomy 6.0
Laparoscopic gastric bypass (RYGB and SAGB) 7.0
Open RYGB 8.0
Open Biliopancreatic diversion (BPD) 9.0
Laparoscopic BPD 10.0

Lifestyle therapies (diet, exercise, behavioral change) should always be the first line of management. Multiple RCTs have shown that a modest weight loss of between 2 and 5 kg can be achieved at 12 months120-123. This level of weight loss is associated with a clinically valuable reduction of metabolic risk124-126 but generally will not solve the problems of obesity. Lifestyle therapies should be applied optimally and sought to be maintained permanently. If however they fail to resolve the obese patient’s problems, the next level of therapy should be considered. Current drug therapies add little further benefit127. Very low energy diets can be effective if taken correctly but are inevitably short term. The recent versions of the intragastric balloon have yet to show effectiveness by RCT and remain short term options. In spite of vigorous research effort, no additional endoscopic approaches are yet available which can provide even medium term benefit.

In general, this table provides a path along which the obese patient seeking treatment should travel. The procedure of gastric imbrication has not been placed on the list as its safety and effectiveness are not yet known.

Comparison of bariatric surgery with non-surgical therapies

We have performed three randomised controlled trials (RCT) in which we have compared gastric banding with optimal non-surgical programs. The initial RCT was of mild to moderately obese adults (BMI 30-35). We compared optimal non-surgical therapy, including lifestyle measures, drug therapy and very low energy diets with the gastric band and showed significantly better weight loss, health and quality of life for the banding group128. Adverse events were similar between groups. The gastric band patients had lost 86% of their excess weight (%EWL) compared to 21% EWL in the non-surgical group. The effects are durable as the substantial weight loss, health benefits and improved quality of life have remained at 10 yr follow up129.

Figure 13. RCT outcomes at ten years (adapted from reference 129). Weight loss outcomes for the surgical group for the ten year period are shown by the continuous line. After completion of the two year randomised study, 17 of 40 patients in the non-surgical arm elected to cross-over to the LAGB. Their weight loss following cross-over is shown by the dotted line.

Figure 13. RCT outcomes at ten years (adapted from reference 129). Weight loss outcomes for the surgical group for the ten year period are shown by the continuous line. After completion of the two year randomised study, 17 of 40 patients in the non-surgical arm elected to cross-over to the LAGB. Their weight loss following cross-over is shown by the dotted line.

The second study was of obese adults (BMI 30 – 40) with type 2 diabetes. There was 73% remission rate of diabetes in the gastric band group and 13% in the lifestyle group130. Again, the metabolic syndrome was significantly improved in the banding group alone. The third RCT was of obese adolescents (BMI > 35; age 14 -18yr)131. The gastric band group lost 79% of their excess weight and showed a significant improvement on the metabolic syndrome which reduced from 36% to zero. There was also an improved quality of life.

Comparison between bariatric surgical procedures.

Table 6 lists a range of comparators and the position of each current option against these comparators.

Table 6. Comparison of attributes of the principal bariatric procedures

Attribute LAGB RYGB Sleeve Gastrectomy BPD +/- DS
Safe +++ ++ ++ +
Weight loss:
1. Short term
2. Medium term3. Long term

 

++

++

++

 

+++

++

++

+++++

Not known

 

+++
+++
?+++
Durable ++ ++ ? ++
Side effects ++ ++ + ++
Reversible easily Yes No No No
Minimally invasive +++ ++ ++ +
Controllable/adjustable Yes No No No
Low revision rate + ++ ? ++
Requires follow up +++ ++ ++ ++

 

The key outcome comparators between procedures are weight loss and safety. Short term data (<3 yr) are largely irrelevant as durability is essential. Systematic review of the medium term weight loss outcomes have shown no difference between RYGB and LAGB and suggest there is a better weight loss to be achieved with biliopancreatic diversion. Figure 10 shows the relative % EWL for the three principal procedures. Long term (> 10 years) weight loss as shown in table 3 also appears to be similar for RYGB and LAGB but several studies have a major potential for bias, particularly those with low numbers of patients.

WHO SHOULD BE CONSIDERED AND WHO SHOULD NOT?

BMI Criteria

There is level 1 evidence supporting a better outcome for using LAGB in the mild to moderately obese (BMI 30-35) when compared with lifestyle therapy128, 130. This approach is cost-effective132, 133. When the two treatment paths are modelled over time, the LAGB approach is dominant i.e. it provides increased number of quality-adjusted life years at a lower cost than the existing option of non-surgical therapies. Any person who is obese (BMI > 30), is suffering from the medical, physical or psychosocial consequences of the obesity and has diligently sought a solution through a range of lifestyle options over time, should be considered for LAGB.

Because the stapling group of surgical options lack level 1 data, are of greater risk, and are not controllable or reversible, maintenance of existing cut-off of BMI >40 or BMI > 35 with major comorbidities should remain for these procedures.

Age criteria.

We now have the results of a RCT showing the clear benefit for LAGB for the obese teenager134 and we offer this option to severely obese teens from age 14 years. We do not consider offering an irreversible procedure to teens when a safe, effective and reversible option is available.
We are generally hesitant to offer bariatric surgery above the age of 65 but do at least consider people between age 65 - 70 who do not have chronic cardiovascular or pulmonary disease.

Contraindications.

Bariatric surgery is contraindicated in those with portal hypertension. Sleeve gastrectomy is considered by many to be contraindicated in those with severe gastro-esophageal reflux disease. LAGB is unsuitable for those who are mentally defective or otherwise unable to engage in the “partnership” needed for optimal outcome. Because of its particular aftercare needs, LAGB is also contraindicated in those who live remote from a follow-up center.

NEEDS AND CHALLENGES

Bariatric surgery is never a quick fix. It is a process of care that begins with a careful initial clinical evaluation and detailed patient education and it continues beyond the operative procedure through a permanent follow up. All procedures have the potential for perioperative complications and death. Revisional surgery is relatively common as maintenance of the correct anatomy is intrinsic to effectiveness. But bariatric surgery does provide a solution to the problem of obesity. It achieves substantial weight loss, improved health and quality of life and a longer life. We need to optimize these benefits and minimize the risks and the costs. The following are some of the areas for further research and development:

  • A better understanding of the mechanisms of action of each procedure is required to enable optimum surgery and follow up.
  • Accurate and comprehensive data management for all patients. Bariatric surgical procedures should be incorporated into national clinical registries to enable objective assessment of the risks and benefits across the community.
  • More randomized controlled trials to define the benefits of weight loss on various comorbidities of obesity. More study is needed in particular for the patients with metabolic diseases – type 2 diabetes, metabolic syndrome, non-alcoholic steatohepatitis, the dyslipidaemias, polycystic ovary syndrome and obstructive sleep apnoea.
  • Better definition of who is suitable and who is not suitable for bariatric surgery.
  • Definition of safe and efficient pathways for assessment, surgery and aftercare.
  • Cost-effectiveness evaluation of the bariatric surgical approach to disease management in comparison with existing options.

Bariatric surgery has the potential to be one of the most important and powerful treatment approaches in medicine. High quality of clinical care, good science and comprehensive data management will allow optimal application of this approach to be realized.

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  105. Dixon JB, Schachter LM, O'Brien PE. Sleep disturbance and obesity: Changes following surgically induced weight loss. Archives of Internal Medicine. 2001;161:102-106
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Autoimmunity to the Thyroid Gland

ABSTRACT

This discussion stresses the normal occurrence of immune self-reactivity, the genetic and environmental forces that may amplify such responses, the role of the antigen-driven immune attack, secondary disease-enhancing factors, and the important contributory role of antigen-independent immune reactivity. Research on thyroid autoimmunity has benefited greatly by knowledge of the specific target antigens and easy access to blood cells and involved target tissue. As research moves apace in realm of molecular genetics and investigation of environmental factors that cause disease, we may look for rapid progress in understanding and controlling these common illnesses. For complete coverage of this and all related ares of Endocrinology, please see our FREE web-book www.endotext.org.


A Brief Review of Immunologic Reactions

The human immune system is comprised of about 2 X 1012 lymphocytes containing approximately equal ratios of T and B cells. B lymphocytes synthesize immunoglobulins that are first expressed on their membranes as clonally distributed antigen-specific receptors and then secreted as antibodies following antigenic stimulation. The ability of the immune system to recognize antigens is remarkable. A human being can produce more than 107 antibodies with different specificities. The concentration of antibodies in human serum is 15 mg/ml, which represents about 3 x 1020 immunoglobulin molecules per person!   Since each B cell has approximately 105 antibody molecules of identical specificity on its surface, the human humoral immune system scans the antigenic universe with about 1017 cell bound receptors. To maximize the chances of encountering antigen, lymphocytes recirculate from blood to lymphoid tissues and back to the blood. The 1010 lymphocytes in human blood have a mean residence time of approximately 30 minutes, thus an exchange rate of almost 50 times per day.

 

T lymphocytes develop from precursor stem cells in fetal liver and bone marrow and differentiate into mature cell types during residence in the thymus. Mature T lymphocytes are present in thymus, spleen, lymph nodes, throughout skin and other lymphatic organs, and in the bloodstream. B lymphocytes (immunoglobulin producing cells) develop from precursor cells in fetal liver and bone marrow and are found in all lymphoid organs and in the bloodstream. The ontogeny and functions of these cells have been identified in a variety of ways, including morphologic and functional criteria, and by antibodies identifying surface proteins which correlate to a varying extent with specific functions. Lymphocytes develop through stages leading to pools of cells which can be operationally defined, and be recognized by acquisition of specific antigenic determinants (1) (Fig. 7-1, Table 7-1).   Human B and T cells normally express class I (HLA-A, B, C) major histocompatibility complex (MHC) antigens on their surface, and B cells express class II antigens (HLA‑DR, DP, DQ). Activated T cells also express class II antigens on their surface, and are then described as DR+.

TABLE 7-1

KEY DIFFERENTIATION ANTIGENS WHICH CHARACTERIZE SPECIFIC LYMPHOCYTE SUBSETS

Primary
Antigen Synonyms Distribution Comment
CD2 LFA-2 Cells T cells Cytoadhesion molecule; NK Cells cognate to LFA-3
CD3 T3, Leu 4 All peripheral T Cells T Cell reseptor complex cells
CD4 T4, Leu 3
(L3T4 in mice)		 Class II restricted T Cells CD4 binkds to MHC clas II (55-70% of peripheral T cells)
CD8 T8, Leu 2 Lyt 2 Class I restricted T Cells CD8 binds to MHC class I (25-40% of peripheral T cells)
CD11a LFA-1 chain Leukocytes LFA-1 chain adhesion molecule, binds to ICAM-1
CD14 LPS Receptor Monocytes Marker for monocytes
CD16 Fc R111 NK cells,
Granulocytes		 Low affinity Fc receptor
CD20 B1 B cells Marker for B cells
CD25 TAC, IL2 Activated T and B cells and monocytes Complexes with chain; T cell growth
CD28 Tp44 Most T cells T cell receptor for B7-1
CD29 - 40-45% of CD4+ and CD8+ cells 1 chain of VLA protein, an "integrin" type of adhesion molecule
CD40 - B Cells B cell activation
CD45RO - 25-40% of peripheral T cell subsets Expressed on naive T cells
CD54 ICAM-1 T and B Cells Cognate to LFA-1
CD56 NKH1 NK Cells, some T cells Neural cell adhesion molecule; NK marker

 

7-1

Figure 7-1:  Development of T Cell Subsets. In the thymus, undifferentiated precursors give rise to CD4+ and CD8+ cells. In the peripheral lymphoid tissues CD4+ cells (CHO) differentiate following activation by exposure to cognate antigen into two subsets (TH1 and TH2), which are well characterized in the mouse, less so in man. Development of these cells is to some extent reciprocally controlled by cytokines, and the cytokines secreted are also distinct. CD8+ cells similarly mature after antigenic stimulation into less well defined subsets. or = effect on subset proliferation. = cytokines produced.

 

Lymphocyte Surface Molecules

T cells have on their surface T cell antigen receptors (TCR) which recognize an antigen/HLA complex, accessory molecules which recognize HLA determinants, and adhesion molecules which recognize their counterpart ligands on antigen presenting cells (APCs). After activation, T cells also have new receptors for cytokines, the hormone products mainly produced by macrophages, T cells and B cells, which control other T or B cells (2) (Table 7-2). The T cell antigen recognition complex consists of disulfide-linked TCR heterodimers, usually the TCR-a and TCR-b chains, plus five or more associated peptides making up the CD3 complex (3). A small proportion of T cells have TCRg and TCRd chain instead of a and b chains. TCR-a and b peptides and gd peptides are derived from rearranged genes coding for proteins which are unique in each cell clone.   The germline TCR genes are very large, containing 40 - 100 different V (variable) segments, D (diversity) segments (in genes), many J (junctional) segments, and one or two C (constant) segments (Fig. 7-2).

TABLE 7-2

CYTOKINES

Cytokine Cell Source Targets Primary Effects On Targets
Type 1 IFN (IFN-α, Β) Mononuclear phagocyte, fibroblast All Antiviral, antiproliferative, increased class I MHC expression
Tumor necrosis factor Mononuclear phagocyte, T cell

Neutrophil

Liver

Muscle

Hypothalamus

Inflammation,

Acute phase reactants,

Catabolism,

Fever
Interleukin-1 Mononuclear phagocyte

Thymocyte

Endothelial cell

Hypothalamus

Liver Muscle

fat

Costimulator

Inflammation

Fever

Acute phase reactants

Catabolism (cachexia)
Interleukin-6 Mononuclear phagocyte, endothelial cell, T cell

Thymocyte

Mature B cell

Liver

Costimulator, Growth,

Acute phase reactants
Interleukin-2 T cells

T cell

NK cell

B cell

Growth; cytokine production,

Growth, activation,

Growth, antibody synthesis
Interleukin-4

CD4+

T cell, mast cell

B cell

Mononuclear

phagocyte T cell

Isotype switching,

Inhibit activation,

Growth
Transforming growth factor- β T cells, mononuclear phagocyte, other

T cell

Mononuclear phagocyte

Other cell types

Inhibit activation,

Inhibit activation

Growth regulation
Interferon-γ T cell, NK cell

Mononuclear

phagocyte

Endothelial cell

All cells

Activation

Activation

Activation.

Increased class I and class II MHC
Cytokine Cell Source Primary Effects On Targets
Lymphotoxin T cell

Neutrophil

Endothelial cell

NK cell

Activation

Activation

Activation
Interleukin- 10 T cell

Mononuclear phagocyte

B cell

Inhibition

Activation
Interleukin-5 T cell

Eosinophil

B cell

Activation

Growth and activation
Interleukin- 12 Macrophages

NK cells

T cells

Activation

Activation

Adapted from tables in Cellular and Molecular Immunology, Edition II by AK Abbas, AH Lichtman, and JS Pober, WB Saunders Company, Philadelphia

 

Cartoon of the human T cell receptor and its subunits.

Figure 7-2:  Cartoon of the human T cell receptor and its subunits. Part A shows subunit composition of the human T cell receptor. The TCR subunits are held together by S-S bonds and are closely associated with either the CD4 or CD8 molecule and chains of the CD3 complex. The subunits are anchored in the cell membrane. The CD3 complex consists of three subunits referred to as gamma, delta, and epsilon. Associated in the TCR complex is another pair of 16 kD homodimer (32 kD nonreduced), subunits existing as homodimers of zeta or heterodimers of zeta and eta. Part B shows the structure of the Ti subunits. The predicted primary structure of the -chain subunit after translation from the cDNA sequence is depicted, as are the variable region leader (L), V, D, and J segments, a hydrophobic transmembrane segment (TM) and cytoplasmic part (Cyt) in the C region, potential intrachain sulfhydryl bonds (S-S), and the single SH group (S) that can form a sulfhydryl bondwith the subunit. Part C shows a scheme of the genomic organization of human -and -chain genes. In the locus, V indicates the V gene pool located 5', at an unknown distance from the D 1 element, the J 1 cluster, and the C 1 constant-region gene. Further downstream, a second D 2 element, J 2 cluster, and C 2 constant-region gene are indicated. A similar nomenclature is used for the Ti locus, in which only a single constant region is found. ?D indicates the uncertainly about the existence of a putative Ti -diversity element. (From Reference 1).

During development of each T cell, segments of the germline gene are rearranged so that one TCR gene V segment becomes associated with one D (in the case of TCR-b), one J, and one C segment to produce a unique gene sequence. This random combination of different V, D, and J and C segments, and additional variations in DNA sequence introduced in the J and D region during recombination, provides the enormous diversity of specific TCRs required to recognize the entire universe of T cell antigens. This process also means that all individuals have (before clonal deletion) preformed TCRs able to recognize thyroid autoantigens as well as thousands of other autoantigens.

 

Each TCR recognizes one specific antigenic peptide sequence termed an epitope (5), which consists of 8 - 9 amino acids for class I restricted T cells, and 13 - 17 amino acids for class II restricted T cells. However, T cells respond to several portions epitopes of any one antigen; these may represent overlapping peptide segments of the epitope. Thus the response of each individual T (and B) cell is extremely specific, but the combined effect of many T (and B) cells acting together is observed in the typical final polyclonal response.

 

T cells recognize antigen presented by an MHC-molecule; CD4+ T cells (often functioning as helper cells) recognize MHC class II molecules plus antigenic epitope, and CD8+ T cells (often functioning as cytotoxic cells) recognize MHC class I molecules plus antigenic epitope. The epitope fits within a cleft in the HLA-DR molecule and the TCR functions to recognize this complex (Fig. 7-3). The five associated peptides of the CD3 complex are believed to be signal-transducers and to initiate intracellular events following antigen recognition. The normal response proceeds via TCR antigen recognition, then activation of the T cell through the combined effect of antigen recognition and costimulatory signals (see below) leading to T cell IL-2 secretion and IL-2 receptor expression, followed by proliferation of the T cell into an active clone.

In this diagram the antigen is depicted in a cleft of the HLA-DR molecule on an APC, being recognized by the T cell TCR.

Figure 7-3:   In this diagram the antigen is depicted in a cleft of the HLA-DR molecule on an APC, being recognized by the T cell TCR. "Adhesive" peptide segments may augment close contact. A CD4 molecule is associated with the TCR. Presumably the APC surface is normally covered with many DR molecules, each studded with an antigen. T cells must somehow scan these complexes in order to find the one that best fits their TCR.

Lymphocyte development is controlled by cytokines released by macrophages, dendritic cells, lymphocytes, and many other cells. Both T and B cells release a large array of cytokines which carry out their effector functions and alter the function of other cells (Fig. 7-1, Table 7-2). As lymphocytes mature in the thymus, and become activated on exposure to antigen, the types of cytokines to which they respond -- and produce -- become altered. In animals, and to a lesser extent in man, types of lymphocytes can be operationally defined by the cytokines produced. For example, Th1 T cells produce IL-2, IFN-g and TNF and are predominant in delayed hypersensitivity type reactions, whereas Th2 T cells produce IL-4 and IL-5, stimulate B cells, and are involved especially in antibody-mediated reactions. Cytokines produced by Th1 cells enhance the activity of this subset but inhibit Th2 cells, and vice versa. This type of regulation may be critical in determining an immune response and in suppressor phenomena. Additional Th subsets are now recognized, including Th17 cells which secrete IL-17 see below), as well as Th9 and Th22 cells which also have discrete pathological roles.

 

As well as cytokines and their receptors, T cells express a number of receptors for chemokines, integrins and selectins which are involved in the sequential stages of cell adhesion which leads to T cell homing to tissues (7).   A word of caution is necessary however in terms of translating these findings into the human situation where boundaries between the subsets are less clear. It is also increasingly recognized that the simple dichotomy of T cells into two types is over-simple, with cytokines such as IL-12 being assigned to the Th1 subset although not being secreted by T cells, and production of this cytokine is stimulated by the Th2 cytokines IL-4 and IL-13, which will drive the immune response from Th2 towards Th1. The blurring of pattern that is seen in many autoimmune diseases challenges the dogma of an easy divide in the type of immune response.

 

Each B cell produces a unique immunoglobulin (Ig) programmed by an Ig gene which has also been rearranged from the germline V, D, J, and C segments (as for the TCR) (8). The TCR and Ig genes are, not surprisingly, members of one gene superfamily.   Further diversity is provided by antigen-driven somatic mutations which occur during amplification of the progeny of a stimulated B cell, causing the production of a family of antibodies with slightly different sequences. B cells secrete their unique antibodies into surrounding fluids, and also express surface Ig which is therefore a B cell receptor for antigen (Fig. 7-4). The recognition process by antibodies involves the shape of the epitope - i.e. it is conformational and for B cells normally involves unprocessed antigen. Thus B cell and T cell epitopes for the same antigen are usually different segments or forms of the molecule.

The B cell surface is studded with specific Ig molecules which function as high affinity receptors for specific antigen epitopes which match the shape of the Ig recognition idiotype.

Figure 7-4:  The B cell surface is studded with specific Ig molecules which function as high affinity  receptors for specific antigen epitopes which match the shape of the Ig recognition idiotype.

Antigen Presentation On Mhc Molecules

The genes for the HLA‑A, B, C and HLA‑DR, DP, DQ molecules are on chromosome 6, and comprise some of the genes in a large immune response control complex (Fig. 7-5). Each cell surface HLA molecule is made up of 2 peptide chains; an a chain and b2 microglobulin for class I molecules, and a and b chains for class II. Each individual inherits from each parent one HLA-A, B, and C, one DRa and 3 DRb genes, a pair each of DP and DQa and b genes, and other related genes which are not expressed, including DX and DO (Fig. 7-5). The genes are expressed in a co-dominant manner, and (in contrast to TCR and Ig molecules) are invariant in individuals. However, the genes are all highly polymorphic, that is, many alleles may exist for each gene. The actual evolutionary drive for this diversity is unknown. While TCR gene rearrangement provides the T cell repertoire to respond to individual antigens, HLA diversity guarantees that different individuals will have different T cell repertoires, which confers evolutionary advantage to the species in terms of responding to new pathogens.

Partial map of the short arm of human chromosome 6 showing the molecular organization of the area containing the MHC loci, with details of the HLA Class I, II, and III genes. Map distances

Figure 7-5:  Partial map of the short arm of human chromosome 6 showing the molecular organization of the area containing the MHC loci, with details of the HLA Class I, II, and III genes. Map distances in kilobases were determined by pulsed-field gel electrophoresis. Genes are not drawn to scale. Expressed genes are designated by filled boxes _ (|_|). (From Trowsdale, J. and Campbell, R.D. Physical map of the human HLA region. Immunology Today, 9:34, 1988.)

The HLA molecules play a central role in T cell clonal selection during fetal development, in normal immune responses, and in presentation of self-antigens. In many instances -- including autoimmune thyroid disease (AITD) as detailed below -- inheritance of a specific HLA gene correlates with increased susceptibility to disease. In some cases this can be related to a gene coding for a specific amino acid in the HLA molecule which is believed to control epitope selection (often called determinant selection) and thus to be associated with disease susceptibility.

 

Antigen can be presented to CD4+ T cells by conventional (or "professional") APCs, particularly dendritic cells (9), and also by B cells and activated T cells, and less effectively by a variety of other cells (fibroblasts, glial cells, thyrocytes), when these normally HLA-DR-negative cells are altered and express HLA class II molecules on their surface. This is because non-classical APCs cannot provide the necessary costimulatory signals, including the B7-1 (CD80) and B7-2 (CD86) molecules, which bind to CD28 on the T cell and are necessary for activation of certain T cells. If B7 molecules bind instead to CD152 (CTLA-4) on the T cell, the immune response is terminated. The individual roles of CD80 and CD86 are not clearly established, although some functions appear to be distinct (e.g. CD80 appears to stimulate CD152) and some overlapping (e.g. both stimulate CD28), and the tempo of their involvement at different times of the immune response is likely to be critical to the type of response produced. The maturation state of the dendritic cell is another determinant of immune homeostasis. Semi-maturation, induced by proinflammatory cytokines like TNF-a, allows the development of a tolerogenic stage for these cells. Full maturation, induced by signaling through toll-like receptors, complement receptors or antibody Fc receptors, induces proinflammatory cytokine production by the dendritic cell and allows them to generate T cell immunity.

 

T And B Cell Responses

Antigen presentation to T cells leads to a variety of responses which include proliferative or suppressive functions, development of cell cytotoxic responses, control of Ig secretion, and many more. In addition, under specific circumstances, antigen presentation may cause the T cell to become non-responsive or anergic (10). Presentation of antigen and the accompanying second signal are required to activate a naive T cell and initiate an immune response; previously activated T cells are much less dependent on B7-mediated costimulation. Antigen recognition and APC-produced cytokines (Table 7-2) together cause T cell stimulation. This activates the T cell to express IL-2 receptors and to secrete IL-2 itself. Increased T-cell secreted IL-2 induces the responding T cell, and nearby ("bystander") T cells to proliferate. T-cell secreted IL-2, IL-6 and other cytokines and IL-4 cause B cells to be stimulated and proliferate and cell surface receptors such as CD40 on B cells and its ligand on T cells are also involved in B cell activation (11).

 

B cells themselves secrete distinct profiles of cytokines, in response to the engagement on CD40, and these cytokines can upregulate or downgregulate an immune response in a manner which depends on whether the B cell is simultaneously stimulated by antigen (12). Intimate T-cell to B-cell contact may account for antigen-specific help for T cell and B cell responses, whereas the effect of T cell-secreted cytokines on bystander T or B cells may account for stimulation of non-antigen-specific responses by these lymphocytes. The beneficial effects of rituximab, a CD20 specific, B depleting monoclonal antibody, in autoimmune conditions including Graves’ disease (13) is related to its effects on inhibiting this interaction between T and B cells.

 

Th1 cells function as inflammatory cells, typical of a delayed hypersensitivity type reaction, while Th2 cells are more specifically helper cells for B cell immunoglobulin synthesis. A number of factors including TCR affinity and ligand density, and non-T cell-derived cytokines such as IL-4 and IL-12, determine whether the outcome of an immune response is predominantly by Th1 or Th2 cells. A third population of T helper cells has been defined recently, based on their secretion of the pleiotropic proinflammatory cytokine IL-17, and are so called Th17 cells. The differentiation and expansion of these cells depends on the coordinate effects of IL-6, transforming growth factor beta (TGFβ) and IL-23 (14). These Th17 cells are responsible for defense against certain micro-organisms such as Klebsiella, Borrelia and fungi. Of relevance to this discussion, they also have important roles in tissue inflammation and organ-specific autoimmunity.

 

Although the concept of suppressor cells fell into disrepute during the late 1980s, there has been resurgence in interest with the recognition that CD4+ cells expressing high levels of the IL-2 receptor, CD25, act in a way entirely in keeping with the previously defined suppressor population. These CD4+, CD25+ T cells have been termed regulatory or Treg cells. Such cells can prevent autoimmunity when transferred from healthy, naïve animals and their depletion results in autoimmune disease. Such cells express Foxp3 which encodes a critical transcription factor for their function: mutation of this gene in man results in the lethal immunological disorder IPEX syndrome that includes autoimmune hypothyroidism amongst its manifestations (15).

 

APCs have a central role in controlling Treg cells, with resting APCs (including thymic epithelial cells) promoting their development through the induction of the transcription factor Foxp3 (16). Activation of APCs, for instance through their T cell-like receptors, has the opposite effect, and at least one component responsible for the suppression of Tregs then is the cytokine IL-6; this pathway allows effector T cells to predominate over Tregs, thereby shifting the dynamic equilibrium in favor of an immune (or autoimmune) response. Another critical molecule in the Treg cell pathway is the costimulatory signal receptor, CD28, which is required for both development and maintenance of Treg function. TGFβ exposure induces Tregs, but when combined with IL-6, Th17 effector cells are generated. The absence or presence of IL-6 is thus critical to determining whether there is a regulatory milieu or a proinflammatory response mounted by Th17 cells. Both Th1 and Th17 cells are potent inducers of organ-specific autoimmunity, but their relative roles in each type of disease remain to be clarified.

 

It is increasingly clear that Treg are more complex a group of cells than originally clear. T regulatory cells can be classified as those which arise within the thymus and express Foxp3, and a Th3-like population which probably does not express this molecule and which develops in the periphery. More recently described regulatory cells have been phenotyped as CD4+CD69+ and CD4+NKG2D+ T cells. The glucocorticoid inducible tumor necrosis factor receptor (GITR) is expressed by both populations but CD25bright expression is not a requirement for regulatory T cell function.

 

The reciprocal relationship between Th1 and Th2 cells, exerted through secretion of cytokines, serves as another model of suppressor function. This paradigm is conceptually useful but is almost certainly too simplistic, not least because there may exist within the Th2 compartment different types of T cells, some with pathological effector function and others which act as physiological regulators of Th1 responses. Endeavors to manipulate the entire Th2 population, to deviate an immune response away from Th1 cells, may therefore lead to exacerbation of the immune response, and may explain the reciprocal relation between the prevalence of infectious disease and autoimmunity (18).

 

Killer (K) And Natural Killer (Nk) Cells

In addition to the standard T cell function described above, other cells participate in immune responses. Macrophages may destroy cells having immune complexes on their surface through recognition of the Fc portion of bound Ig. Other cells which do not bear the CD3 marker of T cell lineage exist (K and NK cells) and have the ability to spontaneously kill other cells (especially those expressing HLA antigens). NK cells can be detected by specific monoclonal antibodies such as anti-CD16, and are recognized phenotypically as large granular lymphocytes. Like T cells, NK cells can have a type 1 or 2 pattern of cytokine release. Macrophages, T, K, NK, or other cells also kill cells coated with immune complexes in the process of antibody-dependent-cell-cytotoxicity (ADCC) (Fig. 7-6).

Some of the proposed mechanisms which could produce thyroid damage in AITD.

Figure 7-6: Some of the proposed mechanisms which could produce thyroid damage in AITD. Emperipolesis is the movement of lymphocytes and macrophages between epithelial cells and occurs in many organs such as gut, bronchus, and thyroid. The existence of interepithelial cells with immunoreactive potential is obviously relevant to an understanding of how autoantigens at the luminal surface of the thyroid cells may be exposed to allow recognition.   (From Weetman AP, McGregor AM. Endocrine Rev 5: 309-355, 1984 ).

Self-Non-Self Discrimination

The immune system, which evolved to defend us from invading foreign proteins, normally tolerates (i.e. does not develop recognizable responses to) self‑antigens. The level of this control is variable. For example, self‑reactivity to serum albumin is not seen. However, antibodies to thyroid antigens exist in up to 20% of adult women, and their presence must be considered effectively normal. The development of tolerance is closely associated with the restriction of TCRs to recognizing an antigen only when presented by an HLA molecule. The process, which for T cells occurs in the fetal thymus, leads to elimination of some T cells, and retention of others with TCRs having desirable features. Self-antigens are believed to be presented on HLA molecules to T cells developing in the thymus. This implies that antigen must be in the thymus or in the circulation for tolerance to develop and indeed we now know that specialized cells in the thymus can express a panoply of autoantigens during development. T cells bearing autoreative TCRs are largely inactivated or destroyed. T cells which have the capacity to react with foreign antigens presented by self MHC molecules are allowed and retained (Fig. 7-7). This system is imperfect however and some T cells which react with MHC molecules plus self-antigen are not deleted, which is the fundamental explanation for autoimmunity.

Fetal Thymus

Figure 7-7:  Left: Fetal Thymus; T cells strongly activated by DR alone, or strongly reactive to self-antigen presented by HLA molecules, are selectively destroyed. T cells, with a weak or absent response to DR alone, or to DR+ self-antigen, survive. Center: Normal Adult Immune Reaction; T cell TCR and APC-DR interaction is normally a weak or neutral signal. The presence of allo-antigen serves to switch the signal to positive. Right: Allo-MLR; Allogeneic DR is sufficiently different from autologous DR to act as a positive signal with or without antigen present.

The best evidence that thymic T cell deletion prevents autoimmunity in man comes from autoimmune polyglandular syndrome (APS) type 1, which is the result of an autosomal recessive mutation in the AIRE (AutoImmune REgulator) gene. Such patients have multiple autoimmune disorders, principally Addison’s disease and hypoparathyroidism but including thyroid autoimmunity. The AIRE protein is expressed in the thymus by medullary epithelial cells and regulates the surprising expression of an array of self proteins (normally confined to extrathymic tissues) by these cells during fetal development. When through the AIRE mutation such self-antigens cannot be expressed to allow clonal deletion, autoimmunity ensues and this accounts for the early onset multiple autoimmunity found in this syndrome (reviewed in 19). Recently, dominant mutations in AIRE have been identified and such patients have later-onset, milder phenotypes (19b). During maturation in the thymus, probably 95% or more of the lymphocytes produced are negatively selected, and die through a process described as programmed cell death or apoptosis. This process involves several genes including those required for apoptosis, such as Fas. A similar process is thought to ensue whenever a T cell is stimulated by its cognate antigen but does not receive a "second signal", and during induction of anergy by other mechanisms. Defects in Fas lead to preservation of autoreactive T cells in some models of animal autoimmune disease.

 

This trade-off between perfection in clonal deletion and repertoire maintenance allows a limited number of autoreactive T cells to survive, and thus sets the stage for autoimmune disease. The main mechanism to prevent autoimmunity by these escaped cells and also to induce tolerance to autoantigens not present in the fetal thymus or circulation is termed peripheral (i.e. non-thymic or central) tolerance, and is mainly effected by the Tregs described above.

 

B cells undergo a similar selection process in fetal bone marrow or liver, except for the participation of MHC molecules.   If exposed to antigen during this early stage of development, B cells are permanently inactivated. As for T cells, the selection process is not perfect, and leaves some B cells having the ability to make antibodies directed to self‑antigens in the adult. However, B cells require T cell help in order to proliferate and differentiate into mature Ig secreting cells. In the absence of self‑reactive T helper cells, these B cells remain dormant and expanding clones do not develop.   Although such clonal ignorance may be an important pathway in preventing B cell autoreactivity, it is not the only mechanism, and physiological concentrations of autoantigen may induce anergy of B cells, even when their affinity for autoantigen is low.

 

Tolerance to self‑antigen can be overcome ("broken") in animals by injecting the antigen in an unusual site on the body, especially in the presence of adjuvant compounds such as mycobacterial fragments and oil, or alum, or by slightly altering the antigen structure, or by altering the responding immune system (for example, by whole body irradiation, or depletion of suppressor T cells). An additional mechanism for the inflammatory component of many autoimmune disorders has recently been proposed based on the evolutionary origins of mitochondria from bacteria. Given that the prime function of the immune system is to defend the organism from microbes, it is possible that the immune system may mistake mitochondria released from damaged tissue through pattern-recognition receptors and thereby induce a ‘mistaken’ inflammatory response (20).

 

The Syndromes of Thyroid Autoimmunity

 

The three syndromes classically comprising autoimmune thyroid disease are (1) Graves' disease with goiter, hyperthyroidism and, in many patients, associated ophthalmopathy (2) Hashimoto's thyroiditis with goiter and euthyroidism or hypothyroidism; and (3) primary thyroid failure or myxedema. Many variations of these syndromes are also recognized, including transient thyroid dysfunction occurring independently of pregnancy and in 5 - 6% of postpartum women, neonatal hyperthyroidism, and neonatal hypothyroidism. The syndromes are bound together by their similar thyroid pathology, similar immune mechanisms, co-occurrence in family groups, and transition from one clinical picture to the other within the same individual over time. The immunological mechanisms involved in these three diseases must be closely related, while the phenotypes probably differ because of the specific type of immunological response that occurs. For example, if immunity against the TSH receptor leads to production of thyroid stimulating antibodies, Graves' disease is produced, whereas if TSH blocking antibodies are formed or a cell destructive process occurs, the result is Hashimoto's thyroiditis or primary myxedema.

 

Associated with autoimmune thyroid disease in some patients are other organ specific autoimmune syndromes including pernicious anemia, vitiligo, myasthenia gravis, primary adrenal autoimmune disease, ovarian insufficiency, rarely pituitary insufficiency, alopecia, and sometimes Sjögren's syndrome or rheumatoid arthritis or lupus, as manifestations of non-organ specific autoimmunity. There has also been a description of pituitary antibodies and growth hormone deficiency in around a third of patients with autoimmune hypothyroidism, implying the existence of a substantial reservoir of pituitary autoimmunity in these patients but further work is needed to confirm these findings and to understand the basis for the autoimmune response against the pituitary (21).

 

The Antigens in AUTOIMMUNE THYROID DISEASE

 

Thyroglobulin

The three most important antigens involved in thyroid autoimmunity are clearly defined. First to be recognized was thyroglobulin (TG), the 670 kD protein synthesized in thyroid cells and in which T3 and T4 are produced. Four to six B cell epitopes of TG are known to be involved in the human autoimmune responses and epitope recognition is similar in both Graves’ disease and Hashimoto’s thyroiditis (22). Animal studies suggest that antigenicity of the molecule is related to iodine content, but studies on human antisera do not consistently bear this out: these species differences and the role of measuring TG antibodies in thyroid disease are reviewed elsewhere (23).

 

Mouse experiments suggest that, to induce autoimmunity to TG, initial tolerance to dominant epitopes must be overcome, and the immune response then spreads to cryptic epitopes that are the major inducers of thyroidal T cell infiltration (24). One particular TG T cell epitope, Tg.2098, has been identified which is a strong and specific binder to the MHC class II disease susceptibility HLA-DRβ1-Arg74 molecule, and stimulates T cells from both mice and humans that develop AITD (25). This could be a major T cell epitope which might be involved in pathogenesis through initiating an immune response that then spreads to involve other autoantigens. Furthermore, screening a diverse library of small molecules has identified one, cepharanthine, which blocked Tg.2098 peptide binding and presentation to T cells in mice with experimental autoimmune thyroiditis; such an approach has obvious therapeutic potential (25a).

 

Tsh Receptor

The second antigen to be identified was the TSH receptor (TSH-R), a 764 aa glycoprotein. Antibodies to TSH‑R mimic the function of TSH, and cause disease by binding to the TSH‑R and stimulating (or inhibiting) thyroid cells, as described later. The human TSH-R is a member of a family of cell surface hormone receptors which are characterized by an extra-membranous portion, seven transmembrane loops, and an intracellular domain which binds the GS subunit of adenyl cyclase (26, 27). Uniquely among G-protein-coupled receptors TSH-R undergoes post-translational cleavage to comprise a 53kD extracellular A subunit (53 kDa) and transmembrane and intracellular B subunit coupled by disulfide bridges. The A subunit may be shed provoking speculation on the role of this in stimulating autoimmunity. Recent evidence indicates that in Graves' disease TSHR antibody affinity maturation is driven by A-subunit multimers rather than monomers (27a). Human TSH-R B cell epitopes are conformational and composed of several segments of the protein.

 

The initial description of mouse and hamster monoclonal TSH-R antibodies was significant for several reasons (28-30). Firstly, these antibodies confirmed that a single antibody was sufficient to activate the receptor, rather than two or more simultaneously. Secondly, they have permitted epitope mapping. One antibody preferentially recognized the free A subunit, not the holoreceptor, suggesting that free A subunit, shed from thyroid cells, may initiate or amplify the autoimmune response. Another antibody, in contrast to TSH, did not enhance post-translational TSH-R cleavage, which may extend the receptor half-life and thus account for the prolonged thyroid stimulation seen following antibody binding. Finally, these antibodies paved the way for the development of human monoclonal antibodies which have allowed a greatly improved understanding of the mechanisms involved in Graves’ disease.

 

The first human monoclonal TSH-R stimulating antibody bound to the TSH-R with high affinity, either as IgG or as Fab fragment, and the monoclonal had similar features in all respects to known TSAb (thyroid stimulating antibodies) (31). This observation indicated that only a single species of antibody is needed to stimulate the receptor. More conventional approaches based on different methods of expressing the TSH-R have shown that TSAb preferentially recognize the free A subunit rather than the holoreceptor, either because of steric hindrance from the plasma membrane or membrane spanning region of the receptor or because of TSH-R dimerization (32). The epitopes for TBAb overlap with those for TSAb but are more focused on the C terminus and are able to recognize holoreceptor more efficiently. These observations have provided support from the hypothesis that shedding of free TSH-R A subunits may be critical in initiating or amplifying the autoimmune response in Graves’ disease. Further evidence comes from immunization of mice with adenoviruses expressing different structural forms of the TSH-R: goiter and hyperthyroidism occur more frequently when mice are given virus that expresses the free A subunit rather than a receptor with minimal cleavage into subunits (33).

 

Patients with autoimmune thyroid disease may have both stimulating and blocking antibodies in their sera, the clinical picture being the result of the relative potency of each species. Switching between one type of antibody and another in unusual patients, involving changes in concentration, potency and affinity, may be caused by a number of factors including levothyroxine treatment, antithyroid drug treatment and pregnancy, and can lead to difficulties in clinical care (35). TSH-R neutral antibodies have also been identified which do not block TSH binding and are unable to stimulate cAMP production; these antibodies are capable of inducing thyroid cell apoptosis in vitro and therefore could conceivably play a role in pathogenesis by inducing release of thyroid autoantigens (36).

 

Identification of the critical T cell epitopes has proved elusive although peptides 132-150 do appear to constitute one key epitope; there is poor correlation between binding affinity and T cell immunogenicity in experiments to attempt such localization (37). In animal studies, however, there is clear evidence of epitope spreading when mice are immunized with TSH-R peptide epitopes or TSH-R cDNA, indicating that dominant TSH-R epitopes are, at best, elusive (38). TSH-R mRNA transcripts and protein have been identified in retrobulbar ocular tissue, particularly the preadipocyte fibroblast, suggesting that TSH-R expression in the orbit could well be involved in the development of autoimmunity and ophthalmopathy, and similar TSH-R-expressing fibroblasts have also been found in the thyroid gland itself (39). Further support for involvement of the TSH-R comes from experiments showing that activation of the TSH-R stimulates early differentiation of preadipocytes, but terminal differentiation is not induced (40). An animal model with some features of similarity to human ophthalmopathy has been induced in mice by immunization with TSH-R A subunit plasmid given by a specific electroporation protocol (41). Oddly there was no thyroid lymphocytic infiltrate to accompany these orbital changes, which were very heterogeneous between immunized animals. It should also be noted parenthetically that an alternative pathway for fibroblast involvement in ophthalmopathy has been proposed which depends on the production of insulin-like growth factor antibodies in these patients but it is difficult to reconcile these findings with the orbital specificity of the autoimmune process in thyroid eye disease (42). Most recently, TSH-R has been identified in immature thymocytes, which can be stimulated by TSAb. This could in turn explain why thymic hyperplasia is seen in occasional cases of Graves’ disease (42a).

 

Thyroid Peroxidase

The third thyroid antigen was described as "microsomal antigen" was identified as thyroid peroxidase (TPO) in 1985 (43) (Fig. 7-8). DeGroot’s laboratory demonstrated that human antisera reacting to "microsomal antigen" precipitated human thyroid peroxidase (TPO) prepared from Graves' disease thyroid tissue () (Fig. 7-8) and at the same time Czarnocka et al. purified human TPO and confirmed identity with the microsomal antigen (44). The cDNA was cloned and sequenced in several laboratories (45-48). The interaction of human anti-TPO antisera and monoclonal antibodies also indicate the presence of several B cell epitopes which map to two main domains, A and B (reviewed in 49). Further experiments with monoclonal antibodies have defined individual amino acid residues that are critical for the two immunodominant regions (50). The epitopes recognized by antibodies are stable within a patient and may be genetically determined (51). Investigation of TPO epitopes recognized by T cells from patients with AITD has produced conflicting results but certain sequences are beginning to emerge which are shared between reports on various patients (52, 53). There is also debate as to whether patients with autoimmune hypothyroidism differ in their pattern of epitope recognition from healthy controls who are TPO antibody positive, and further work is required to analyze this in detail, as it might allow better prediction of those antibody positive individuals who will progress to overt hypothyroidism (54)

TPO is expressed on the thyroid cell surface as well as in the cytoplasm, and likely represents the cell-surface antigen involved in complement-mediated cytotoxicity as well as antibody-dependent cell mediated cytotoxicity (55).   Intracytoplasmic binding of antibodies to TPO indicates that there is access


Precipitation of peroxidase activity by sera from a patient with autoimmune thyroid disease and positive "microsomal" antibodies, and from a control subject without circulating antibodies.

Figure 7-8: Precipitation of peroxidase activity by sera from a patient with autoimmune thyroid disease and positive "microsomal" antibodies, and from a control subject without circulating antibodies. TPO was precipitated by primary incubation with human sera, and removal of TPO-Ig complexes was achieved by addition of Protein H-Sepharose CL-4B. Residual hTPO activity in the supernatant was assayed in a guaiacol assay.

 

Other Antigens

Antibodies against the sodium/iodide symporter (NIS) were first shown functionally in cultured dog thyroid cells (56). Up to a third of Graves’ disease sera contain antibodies capable of blocking NIS-mediated iodide uptake in cells transfected with the human NIS but the relevance of this for thyroid function is unclear (57). The same antibodies have also been detected using an immunoprecipitation assay (58). Others have found no such blocking activity using assays with cell lines displaying much higher 131I uptake, in turn suggesting that any NIS blocking activity only occurs at limiting conditions (59). This implies that NIS autoantibodies probably have no effect in vivo.   NIS expression on TECs is upregulated by TSH and downregulated by cytokines and the latter could impair thyroid function in the setting of AITD when such cytokines are synthesized in the thyroid (60). Pendrin, an apical protein responsible for mediating iodide efflux from thyroid cells into the follicular lumen, has also been identified as an autoantigen. Autoantibodies were initially found in 81% of patients with AITD by immunoblotting (more frequently and at higher titer in Hashimoto’s than Graves’ patients) and also in 9% of controls (61), but the frequency of these autoantibodies detected using a radioligand binding assay is rather low at around 10% of patients and no controls (62).

 

Antibodies to a variety of other thyroid cell components are also occasionally present in AITD, including antibodies that react with thyroxine or triiodothyronine (63). The insulin-like growth factor receptor has also emerged as a possible autoantigen involved in ophthalmopathy, with antibodies being detected in patients with this complication, and this receptor co-localizes with the TSH-R on both fibroblasts and thyrocytes (64).

 

Immune Reactions in Autoimmune thyroid disease

 

Humoral Immunity

The principal autoantibodies identified in AITD and the methods for detecting them are listed in Table 7-3. Antibodies to the TSH receptor are discussed in detail in Chapter 10, but, in brief, observation of a factor in serum of patients with Graves' disease causing long acting stimulation of thyroid hormone release from an animal's thyroid, in contrast to the short acting stimulation produced by TSH, led directly to our knowledge of TSH-R antibodies. We summarize here a huge amount of clinical and laboratory research. The antibodies directed to the TSH-R are currently separated into three types. Some antibodies bind to an important epitope in TSH-R and activate the receptor, producing the same effects as TSH, in particular causing generation of cyclic AMP. These antibodies may be referred to as TSI or TSAb -- thyroid stimulating immunoglobulins or thyroid stimulating antibodies. Other antibodies bind to different, or the same epitopes and interfere with radiolabelled TSH binding in certain assays -- thus they are known as thyrotropin binding inhibitory immunoglobulins or TBII. Still others bind and prevent the action of TSH -- thus blocking antibodies. These may either interfere directly with TSH binding or have less well characterized

 

TABLE 7-3

ANTIBODIES REACTING WITH THYROID AUTOANTIGENS IN AITD AND TECHNIQUES FOR DETECTION

Antigen Test Used To Identify Antibody
TG Precipitin
Hemagglutination assay
Immunofluorescence on fixed sections of thyroid tissue: colloid localization
localization Solid-phase RIA
Immunoradiometric assay
Hemolytic plaque assay
Colloid component other than TG Immunofluorescence on fixed sections: colloid localization
Microsomal antigen/
TPO		 Complement fixation
Immunofluorescence on unfixed sections;
thyroid tissue cell localization
Cytotoxic effect on cultured thyroid cells
Hemagglutination assay
ELISA
Solid-phase RIA
TPO activity inhibition
TSH-R Bioassay in mice
cAMP production by thyroid cells, TSH- R transfected cells or membranes
Iodide uptake by thyroid cells
Thymidine incorporation by thyroid cells
Inhibition of TSH action on thyroid cells
Inhibition of TSH binding to cells or membranes
Immunoprecipitation
Sodium/iodide symporter Western blotting
Immunoprecipitation
Bioassay using cultured thyroid cells or cells transfected with the symporter

 

TSI cause non-TSH dependent stimulation of thyroid function, which, if of sufficient intensity, is hyperthyroidism. TBII comprise the mixture of TSI and TSH blocking antibodies, and therefore function cannot be predicted from the TBII level. Predominance of TSI characterizes Graves' disease, and TSH blocking antibodies are present in a small proportion of patients with Hashimoto's disease and primary myxedema. Probably a combination is present in most patients with AITD. Recent work indicates that both types of TSH-R antibody are present in Graves’ sera at low concentration with high affinity and similar (but nonetheless subtly distinct) binding epitopes (65). TSI directly cause thyroid overactivity, their level correlates roughly with disease intensity, and a drop in levels correlates loosely with disease remission. Unlike TG and TPO antibodies which are polyclonal and not restricted by immunoglobulin subclass (reviewed in 66), there is evidence that some TSH-R are restricted to particular heavy and light chain subclasses, which may indicate an oligoclonal origin (67), and TSH-R stimulating antibodies are present at much lower concentration than TG and TPO antibodies.

 

Normal subjects can have TSH-R antibodies that bind to but do not activate the TSH-R and that generally have low affinity. These natural autoantibodies may be the precursors of the TSI that cause Graves’ disease and it is possible that affinity maturation, with class switching of immunoglobulin isotype, is critical in determining the clinical consequences of TSH-R antibody production. Conversely, using the most sensitive binding assays, there are still a very small number of patients with Graves’ disease who are apparently negative for these antibodies when their serum is tested; it is likely that the explanation lies in either assay sensitivity or exclusively intrathyroidal production of these antibodies (68).

 

Precipitating antibodies to TG were first detected by mixing antibody and antigen in equivalent concentrations, or by agar gel diffusion, as in the Ouchterlony plate technique. Subsequently, much more sensitive methods were developed, such as solid phase ELISA (69) and RIA (70), although for many years the tanned red cell hemagglutination test remained the assay of choice (71). Immunoradiometric assays (IRMA) used currently involve binding of serum antibodies to solid phase antigen, and secondary quantitation of antibody by binding labelled monoclonal anti-human Ig antibody. These tests are very sensitive but lack specificity as so many healthy individuals are positive, albeit with a future risk of developing AITD.

 

Antibodies directed against TG are rarely present in children without evidence of thyroid disease. The prevalence in healthy persons increases with age, and low levels are frequently present in normal adults (72). The greatest frequency occurs in women aged 40‑60 years. The frequency of antibodies in well persons correlates with the incidence of focal lymphocytic infiltration found on microscopic examination of thyroid tissue form healthy individuals (73). Over 90% of patients with Hashimoto's thyroiditis and primary myxedema have these antibodies. Low to moderate titers are found in half of patients with Graves' disease. TG antibodies are either absent or low in patients with subacute (De Quervain's) thyroiditis, who may present clinically like patients with Hashimoto's thyroiditis. In general human TG and its autoantibody bind complement weakly due to the widely scattered epitopes which are unable to allow antibody cross-linking.

 

The second important antigen-antibody system was originally recognized by antibodies which, by immunofluorescence, were observed to bind to non-denatured thyroid cytoplasm, to fix complement in the presence of human thyroid membranes (microsomes), or to bind to microsome‑coated red cells (the MCHA assay). We now know this antigen is TPO (see previous Section 3) (Fig. 7-8). Almost all patients with Hashimoto's thyroiditis have TPO antibodies. They also occur in the normal population in the absence of clinically significant thyroid disease: in a recent survey of a population followed for 20 years, 26% of adult women and 9% of adult men had TPO and/or TG antibodies (74). However, the presence of such antibodies was shown to be associated with an increased risk of future hypothyroidism, especially if the TSH was also raised (subclinical hypothyroidism). Few sera from AITD contain TG antibodies in the absence of TPO antibodies, but the converse is not always true, so it has been proposed that screening for AITD could be undertaken initially with assays for TPO antibodies (75). This is particularly the case if the hemagglutination assay is used for TG antibodies; sensitive RIAs may detect a very high frequency of TG antibodies in individuals with autoimmune thyroid disease, even more than TPO antibodies (76). Using modern types of assay, TG antibodies occurring in isolation from TPO antibodies are more commonly found, and thus measurement of both antibodies might have clinical utility in certain situations, for instance in diagnosing possible causes for impaired fertility in women (77).

Antibodies detected by these techniques are believed to be similar to antibodies first described in the 1950s that fix complement in the presence of extracts from a thyrotoxic gland (78) and that have cytotoxic effects on thyroid cells (79). Sera from patients with Hashimoto's thyroiditis usually have high cytotoxic activity (80). Complement-mediated sublethal injury probably occurs in vivo since complement containing complexes have been identified in thyroid tissue of patients with Graves’ disease and Hashimoto’s thyroiditis (81). Thyroid cell expression of membrane proteins, especially CD59, helps prevent complement-mediated lysis (82), and this protein is upregulated by IL-1 and IFN-g.

 

The cytotoxicity of circulating antibodies has also been explored using systems to detect antibody-dependent cell-mediated cytotoxicity (ADCC) in which nonimmunized lymphocytes (NK cells) or macrophages act as effector cells and kill antigen-coated target cells, following incubation of the targets with antibody (83, 84). This reaction does not require complement, instead depending on the interaction of antibody on the target cell with Fc receptors on the effector cells. The exact role of ADCC in the pathogenesis of autoimmune thyroid disease is unclear, as it has been investigated only as an in vitro phenomenon. Antibodies capable of mediating ADCC on target cells include those against TG and TPO, but other antigens may also be targets, and sera from patients with Hashimoto’s thyroiditis, primary myxedema and Graves’ disease cause ADCC, although the frequency is lower in Graves’ disease (85). A further possible role for TPO antibodies has been suggested by the finding that these bind to cultured astrocytes and it is therefore possible that the controversial entity of Hashimoto’s encephalopathy is the result of some autoimmune cross-reaction between thyroid and central nervous system (86).

 

Titers for all types of thyroid autoantibody obviously increase during the process of development of AITD. It is possible that one critical step in the production of TG autoimmune responsiveness is the generation of immunoreactive C-terminal fragments during hormone synthesis (which results in oxidative stress); these fragments may also lead to preferential presentation of TG epitopes by thyroid cells (87). Natural autoantibodies against TG may be more important in the initiation of the response than previously thought. These low affinity, mainly IgM antibodies, which are frequent in healthy individuals, can complex TG with complement and such opsonized complexes can be taken up by B cells and presented to CD4+ T cells (88). After first observation, antibody levels tend to be stable over months.

 

Radioactive iodine therapy in Graves' disease leads to a rise in thyroid antibody levels during the first few months after treatment (89), and exposure to high levels of IFN-a in those with pre-existing autoantibodies also does this (90, 91). With treatment of Graves' disease, or replacement therapy in Hashimoto's thyroiditis or myxedema, there is characteristically a gradual reduction in antibody levels over months or years, and some patients with total destruction of thyroid tissue eventually lose detectable antibody titers.

 

There are two major conformational epitopes on the TG molecule that are recognized differentially by sera from healthy subjects and those with AITD; linear epitopes are recognized by polyclonal antibodies from healthy individuals (92-94). Similar studies on TPO have indicated at least eight major domains for human autoantibodies which are probably conformational epitopes. Using recombinant proteins and synthetic peptides, human anti-TPO antibodies are found to recognize apparently linear epitopes in the area of amino acids 590-622 and 710-722 (95) but, again, the important B cell epitopes are conformational.

 

Peripheral blood mononuclear cells (PBMC) and thyroid lymphocytes from patients with AITD have among them activated cells that spontaneously secrete TG and TPO antibodies (96). B cell production of antibodies to TPO and TG is most easily shown using cells incubated with mitogens (97). Specific antibody secretion in response to PBMC stimulation by TG or purified TPO is more difficult to demonstrate (98). In patients with AITD, approximately 50 B cells secreting anti-TG antibodies are found per 106 PBMC (~2% of total Ig secreting cells) by using plaque-forming assays after stimulation of PBMC with pokeweed mitogen. B cells from AITD patients synthesize antibodies in response to insolubilized TG bound to Sepharose (98), which appears to function as an especially good antigen. There are reports of production of anti-TSH-R antibodies in vitro, but in general this response has been difficult to observe.

 

In fully developed AITD, the thyroid is clearly an important source of autoantibody and spontaneous autoantibody secretion by B cells is easily demonstrable (99). This is also supported by the histopathological features, including the demonstration of thyroid antigen-specific B cells and the occurrence of secondary immunoglobulin gene rearrangement in intrathyroidal lymphoid follicles, together with a congruent pattern of adhesion molecule and chemokine expression (100). However, lymph nodes, bone marrow and possibly other organs also contribute to autoantibody production (101) and this explains why patients with apparently destroyed thyroid tissue, or those with resected thyroids, continue to have circulating thyroid auto-antibodies

 

Cell-Mediated Immunity In Autoimmune Thyroid Disease

Techniques for identification of T lymphocyte reactivity to foreign or autologous antigens depend on culturing mixed peripheral leukocytes or semi-purified thyroid or blood lymphocytes with an antigen to which the cells may have been pre-sensitized. Upon re-exposure to antigen, the sensitized cells change to a blast-like immature form, synthesize new protein, RNA, and DNA, and directly or through liberated effector molecules alter the function of target cells. Different endpoints characterize the various assays, including measurement of [3H]-thymidine uptake, assay of migration inhibition factor (MIF), or leukocyte migration inhibition (LMI) (102), assessment of the mobility of lymphocytes, and cytokine assay, all after stimulation with antigen in culture.

 

Numerous reports have shown that T cell immunity can be detected in Graves' disease, Hashimoto’s thyroiditis, and primary myxedema, although responsivity of T cells to thyroid antigens is much less than to exogenous antigens such as tetanus toxoid or tuberculin. Peripheral blood T cells respond to incubation with TG or TPO in the form of a microsomal preparation by thymidine incorporation, the so-called proliferation assay (103, 104). Responses by separated lymphocytes are generally weak; better responses are seen by adding IL-2 to thyroid antigen-stimulated cultures of diluted whole blood (105). Thyroid T cells responding to TG are of the CD4+ T helper type (106), or occasionally CD8+ cells (107). T cells also respond to crude thyroid antigen extracts in LMI assays (102). T cell lines and short term T cell clones (CD4+) are stimulated during co-culture with TECs to incorporate [3H]-thymidine; DR+ TECs are especially effective stimulators (108 - 110). The identity of the antigen recognized on TECs is unknown but may well be TPO and/or TG.

The specific peptide epitope fragments of TPO recognized by lymphocytes of patients with HT were noted previously. T cell epitopes present within the extracellular domain of the TSH-R are also heterogeneous with peptides bearing sequences of aa 158-176, 237-252, and 248-263 and 343-362 being especially important (111) but other epitopes (aa 57-71, 142-161, 202-221, 247-266) have been identified by others using different assay parameters (112). HLA-DR3 molecules bind TSH-R peptides with high affinity, which may explain the genetic association of this HLA specificity with Graves’ disease (113).

 

T cell responses to an antigenic stimulus may use a wide variety of variable (V) TCR gene segments, or the response may be restricted to a few V segments. Restriction of autoreactive T cells to use of one or more V gene segments has been found in some experimental autoimmune models (4). Restricted Va and Vb usage in the whole intrathyroidal lymphocyte population has been reported (114, 115) but not confirmed by others (116, 117). However, intrathyroidal CD8+ T cells do display a degree of restriction although their autoreactive potential is at present not known (118). Presumably at an early stage of disease, the T cell response is clonally restricted, but as it advances, spreading of the immune response occurs, involving many more epitopes, leading to an unrestricted response as demonstrated in an animal model of AITD (119). Evidence has emerged of a combined TG and TPO epitope-specific cellular immunity, with CD8+ T cells reacting against these epitopes rising to 9% in the peripheral blood of patients with long-standing Hashimoto's thyroiditis (120).

 

While T cell immunization is conventionally recognized by a stimulatory effect of antigen, direct T cell cytotoxicity of thyroid cells has been recognized in a few studies. For example, Davies and co‑workers developed a CD8+ T cell clone which was cytotoxic to autologous TEC and was appropriately class I restricted (121). Another potential consequence of T lymphocytic adherence to thyroid cells is the stimulation of thyroid cell proliferation via ICAM-1/LFA-3 interaction, rather than their destruction, which could lead to goiter formation (122).

 

Immune Complexes

In addition to the antibody and T cell responses, circulating immune complexes are found in patients with autoimmune thyroid disease as would be anticipated], although their pathogenic importance appears minimal. In a certain sense this is most fortuitous. Since many individuals have circulating TG antibodies and antigen, if the immune complexes caused serious disease, it would be a catastrophe. Fortunately the immune complexes of TG and its antibody do not bind complement and do not cause serious illness such as immune-complex nephritis, except in rare instances (123, 124). Immune complexes, including complement terminal components, can however be recognized around the basement membrane of thyroid follicular cells (81) and may cause sublethal effects including release of proinflammatory mediators by TECs (125).

 

K And Nk Cell Responses

Many studies have been reported on natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC); their conclusions vary. Endo et al (126) found NK cells were decreased in Graves' disease and Hashimoto's thyroiditis, and presented evidence that this was due to saturation of their Fc receptors by immune complexes. Normal NK effector function was found in Hashimoto's thyroiditis PBMC (127) in one study, although by phenotyping, decreased NK cells in Graves' disease, and increased NK cells in Hashimoto's thyroiditis were reported in another (128). ADCC of thyroid cells, mediated by normal PBMC, was induced by TPO antibody positive sera (129) but other, unknown antibody-antigen systems may also contribute (85). Effector cell activity in ADCC was increased in Hashimoto's thyroiditis and in post-partum thyroiditis, and thought to be related to thyroid cell destruction (130). Other data have indicted that ADCC may be more important in primary myxedema than Hashimoto’s thyroiditis explaining the difference in clinical presentation (131), but this has not been confirmed in studies showing equal ADCC activity in sera from both diseases (132).

 

Cytokines

Cytokines lie at the heart of the autoimmune response and can have a number of direct and indirect effects (Fig. 7-9). For example, IFN-g is produced in the thyroid by infiltrating lymphocytes and causes HLA class I expression on the surface of TECs to increase and initiates class II expression. It also has a direct inhibitory function on TEC iodination and TG synthesis (133, 134). Caveolin-1 and TPO form part of the apical thyroxisome, responsible for thyroid hormone synthesis. Recent studies have shown that Th1 cytokines down-regulate caveolin-1, leading to intracytoplasmic thyroxine synthesis and mislocalization of the thyroxisome. Disruption of the thyroxisome in this manner may then lead to damage by reactive oxygen metabolites and apoptosis in Hashimoto’s thyroiditis (135).

7-9

Figure 7-9: Interactions between thyroid follicular cells and the immune system in autoimmune thyroid disease. Reproduced from Weetman AP, Ajjan R, Watson PF. Bailliere’s Clin Endocrinol Metab 11: 481-497, 1997 with permission.

IFN-g is not essential for the development of AITD in mice but exacerbates disease activity (136). IL-2 can activate lymphocytes to produce IFN-g, and activate NK cells. TNF is produced by infiltrating macrophages and is potentially cytotoxic to TEC. TEC can produce several cytokines, including IL-1, which may activate T cells, IL-6, which stimulates T and B cells and IL-8, a chemokine which attracts inflammatory cells (reviewed in 134). More recently IL-14 (taxilin) and IL-16 production by TECs has been described: the former regulates B cell growth and the latter is a chemoattractant for CD4+ cell (136a). Dendritic cells are important sources of IL-1b and IL-6 in the thyroid and can inhibit thyroid follicular cell growth (137). As an aside, plasmacytoid dendritic cell numbers are decreased in the blood in AITD, together with an alteration in their phenotype, but these cells increase in the thyroid gland, also suggesting that this cell type may be important in pathogenesis (138)

 

IL-1a causes dissociation of junctional complexes between thyroid cells which could expose hidden autoantigens (139). An ever wider array of factors besides the classical cytokines has been implicated in the pathogenesis of AITD, including the finding that thyroid cells can release angiopoietin-1 and -2 (140). These ligands serve as a chemoattractant for monocytes and the angiopoietin receptor, Tie-2, is increased in monocytes form AITD patients, suggesting a role for monocytes in thyroid damage. Vascular endothelial growth factor expression is increased in AITD and is important in angiogenesis in autoimmune goiters (141). Cytokines also seem to play a major role in the pathogenesis of thyroid-associated ophthalmopathy through their stimulatory actions on orbital fibroblasts (142). Exogenous cytokines given therapeutically can also precipitate autoimmune thyroid disease, probably in predisposed individuals. The best described such reaction is α interferon used in hepatitis C and cancer therapy (90). Destructive thyroiditis accounts for the majority of thyroid dysfunction after treatment with this cytokine, and risks are highest in white women, whereas smoking is protective (91).

 

SUMMARY

To summarize, augmented pools of activated and resting T and B cells reactive to thyroid antigen exist in patients with AITD.   The time course of development of these reactive cells, before clinical disease is apparent, has not been established. The cells respond to biochemically normal antigen, and some reactive cells exist in otherwise healthy individuals. Immune complex formation appears to be of limited importance in the disease process. K and NK activity may be reduced in Graves' disease and increased in Hashimoto's thyroiditis and may contribute to the course of the disease: proliferative in Graves' disease and destructive in Hashimoto's thyroiditis. Cytokines have multiple actions in the thyroid in AITD and are likely to determine clinical manifestations such as ophthalmopathy. The role of the TEC in the autoimmune response is not simply passive and, as discussed below, the interaction between TECs and cells of the classical immune system may be critical in determining the outcome of an initially mild thyroiditis.

 

EXPERIMENTAL THYROIDITIS IN ANIMALS

Chronic thyroiditis histologically identical to that in Hashimoto's thyroiditis occurs spontaneously in Obese strain (OS) chickens (143), beagles (144), mice, and rats. It can be induced in dogs (145), mice, rats, hamsters, guinea pigs, rabbits, monkeys (146), and baboons (147) by immunization with autologous or allogenic thyroid homogenate mixed with adjuvants, or by using heterologous TG , or TG that has been arsenylated or otherwise chemically modified. The need for modification of TG or adjuvant to break tolerance can also be overcome by immunization with cDNA (148). An important thyroiditogenic epitope includes a thyroxine residue (aa 2553) in human TG (149, 150) but the role of iodination at this site is unclear and may depend on the type of T cell assay system used, as well as other parameters (151). Mice have been the most frequently used model and have provided key insights into genetic susceptibility, pathogenesis and the development of Treg and autoreactive T cell repertoires (152).

 

Induced thyroiditis leads to formation of humoral antibodies and T cell- mediated immunity. Usually the histologic pattern conforms to that of T cell-mediated immunity (153). The role of TG antibodies is unclear but likely to be minor. An idiotype-anti-idiotype network exists for TG antibodies in mice but the induction of those antibodies does not lead to thyroiditis (154). Furthermore, the intensity of the thyroiditis correlates better with T cell-mediated immunity than with antibody levels, and can be transferred by T cells but not antibodies, and both CD4+ and CD8+ T cells are usually needed for transfer (155). In normal mice, thyroiditis can be produced by immunization with mouse TG in adjuvant, and transferred to isogenic animals by sensitized Ly-1+ T cells. The same cells, given before immunization, vaccinate against the development of thyroiditis during subsequent immunization (156).

 

However, a subpopulation of CD4+ T cells has an important regulatory role in tolerance to murine TG, keeping in check those TG-reactive T cells which escape thymic deletion and peripheral anergy-inducing mechanisms (157). Amelioration of thyroiditis by oral administration of TG (158) operates through enhancing the activity of these regulatory T cells although other mechanisms are possible. More recent studies have emphasized the importance of regulatory T cells in suppression of thyroiditis in animals immunized with TG. In particular, semi-mature dendritic cells, which can be induced with granulocyte-macrophage colony stimulating factor, can induce the function of TG-specific CD4+, CD25+ T cells which can suppress thyroiditis through the production of IL-10 (159, 160).

 

Another model has used homologous (murine) TPO in an immunization protocol and this method established thyroiditis and TPO antibody production although none of the immunized mice developed hypothyroidism (161). HLA-DRB1*0301 (DR3) transgenic mice have been created which are susceptible to thyroiditis induced by TG immunization, unlike DR2 transgenics, thus confirming that HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis, and his model has been extended to study of the immune response to TSH-R, with results again showing the importance of the DR3 specificity (162). However, when modeling has attempted to reproduce Graves’ disease by immunization of mice with adenovirus expressing the TSH-R, it is non-MHC genes which play a major role in controlling the development of hyperthyroidism (163). This concurs with the polygenic susceptibility and rather weak effect of HLA-DR3 in Graves’ disease. The DR3-transgenic model has also been used to show that dietary iodide enhances the development of thyroid disease and depletion of CD4+, CD25+ Tregs exacerbates this iodide-induced thyroiditis (193a)

 

Spontaneous thyroiditis in OS chickens more closely resembles Hashimoto’s thyroiditis than the immunization models just discussed, particularly as the birds develop hypothyroidism as a consequence of the autoimmune process. Some evidence suggests that the thyroid of the newly hatched chick is intrinsically abnormal, since its function is partially non-suppressible by thyroid hormone and this constitutes an important element of the genetic susceptibility of these birds, together with genes controlling T cell responses and possibly glucocorticoid tonus. The MHC conversely has only a limited effect. Iodine plays a critical role in the induction of thyroid injury in OS chickens, most likely through the generation of reactive oxygen metabolites, and this injury is an early event, preceding lymphocytic infiltration (165). Iodination of TG is a second path by which iodine influences disease in OS chickens, as autoreactive T cells respond to the antigen only if it is iodinated (166).

 

Lymphocytic thyroiditis occurs spontaneously in the Buffalo and BB/W rat strains and the NOD (non-obese diabetic) mouse, especially the NOD.H-2h4 line (167). In both species, there are associated abnormalities in the animals' immune system.   As in the OS chicken, administration of excess iodine augments the incidence of rat thyroiditis and iodine depletion reduces it (168). Iodine also enhances the susceptibility of NOD mice to thyroiditis, and further exploration of this model has demonstrated a key role for Th17 cells which accumulate within the thyroid (169). IL-17-deficient mice have a markedly reduced frequency of TG autoantibodies and thyroid lesions. Furthermore, selenium supplementation lowers serum TG antibody levels and decreases the prevalence of thyroiditis and the degree of infiltration of lymphocytes in iodine-treated NOD mice (169a). The susceptibility of NOD mice has also been exploited in a model in which the CCR7 gene was knocked out in this strain: such mice do not develop diabetes but do develop severe inflammation elsewhere including a severe thyroiditis with TG autoantibody formation and hypothyroidism (170). CCR7 is a chemokine receptor which is expressed by Tregs; the CC7-deficient mice had lower numbers of these cells. As well as this effect, it is possible that CCR7 deficiency impaired negative selection of thyroid reactive T cells.

 

Another intriguing aspect of this model comes from long-term observations in NOD.H-2h4 mice which have shown that TG antibodies occur initially and much later TPO antibodies appear, suggesting that tolerance at the B cell and presumably T cell level is broken first for TG and then by spreading (see above) for TPO (171). These results suggest a more important role for TG as an autoantigen in AITD than it is currently assigned. When engineered through CD28 knockout to have a deficiency of Treg cells, NOD mice develop more severe thyroiditis than control animals, with thyroid fibrosis and hypothyroidism. Transferring healthy Treg cells reduces thyroiditis without increasing the total number of Treg cells, suggesting that endogenous Tregs in these mice are functionally defective (172).

 

The iodine-accelerated thyroid autoimmunity which occurs in NOD.H2(h4) mice is associated with TG and TPO but not TSH-R autoantibodies However transgenic animals expressing the human TSH-R A-subunit develop pathogenic TSH-R antibodies which can be detected in standard bioassays, and this is especially the case in female animals (172a). These antibodies only weakly cross-react with the murine TSH-R and so do not cause hyperthyroidism.

A third kind of model is produced by manipulation of T cells. The original description of thyroiditis in genetically susceptible rats by sublethal irradiation and thymectomy (173) has been followed by a number of more refined models in which T cell subsets can be perturbed more or less specifically to induce disease. For instance CD7/CD28 double-deficient mice have impaired Treg function and such animals develop spontaneous thyroiditis after 1 year of age (174). These experiments clearly demonstrate the recurrence of autoreactive T and B cells in normal animals and show that any of a number of factors which can perturb the regulation of these could result in autoimmune thyroiditis (Fig. 7-10). The most elegant model resulting from T cell manipulation is the generation of transgenic mice expressing a human T cell receptor specific for a TPO epitope, which resulted in a spontaneous destructive hypothyroidism and hypothyroidism (175). The CD8 T cells recognizing the epitope in these animals unconventionally were MHC class II rather than class I restricted and it is unclear whether this atypical behavior is significant to the creation of the model, nor is it yet clear what the mechanism is for thyroid cell destruction.

Control of thyroid antigen-specific T cells in experimental autoimmune thyroiditis.

Figure 7-10:  Control of thyroid antigen-specific T cells in experimental autoimmune thyroiditis. Development of disease depends on the balance of these factors, and their sites of operation are shown as dotted lines. Reproduced from (255) with permission.

Another intriguing model is one in which necrotic thyroid cells can induce maturation of dendritic cells in vitro, and when injected back into autologous mice EAT is induced, with a lymphocytic thyroiditis and TG-specific IgG (176). It is not clear whether this protocol yields cryptic TG epitopes which can break tolerance. It is possible that such work could be reversed therapeutically to allow attenuation of EAT by pulsing tolerogenic dendritic cells.

 

Establishing an animal model of Graves’ disease has been surprisingly difficult despite the cloning of the TSH-R. Spontaneous models are not obvious, suggesting that critical differences in the TSH-R receptor between man and other mammals (such as glycosylation) may be necessary to break tolerance (177). However, immunization of AKR/N mice (but not other strains sharing the same MHC haplotype) with murine fibroblasts doubly transfected with the human TSH-R and haploidentical MHC class II genes results in a syndrome similar to Graves’ disease except that thyroid lymphocytic infiltration was not induced (178), whereas thyroiditis is a feature of immunization with the TSH-R (179). This is a promising model although its exact physiological parallel remains unclear, particularly as fibroblasts may behave differently to TECs in terms of antigen presentation. This is because the fibroblasts used express the critical costimulatory molecule B7-1 and also because the procedure causes generalized in vivo immune activation. This model is therefore not evidence that thyroid follicular cells (which do not normally express B7) could initiate thyroid autoimmunity.

 

More recent models include the use of transgenic mice expressing the A-subunit of the TSH-R, which develop lymphocytic infiltration of the thyroid, hypothyroidism and autoantibodies against TG and TPO as well as TSH-R following immunization with the TSH-R expressed in adenovirus and regulatory T cell depletion (180). Although obviously a contrived system, this model does clearly show that spreading of the immune response can occur to include the normal array of antibodies found in patients, and that this can result in a severe thyroiditis. Some of the difficulties in producing reliable animal models of Graves’ disease are seen in the disparity between hyperthyroidism in the animal and the presence of TSH-R antibodies detected by bioassays using human TSH-R. This may be the result of loci in the immunoglobulin heavy chain variable region contributing in a strain-specific manner to the development of antibodies specific for the human or the mouse TSH-R (181). This novel finding of a role for immunoglobulin heavy chain variable region genes in TSI specificity indicates a possible role for them genetic susceptibility to human Graves' disease.

 

One unexpected finding has been the observation that mice with a TSH-R knockout do not differ in their response to immunization with TSH-R when compared to healthy animals, whereas the expectation was that such animals would have no tolerance to this autoantigen (as it had been absent throughout development) and therefore a greater immune response would be predicted (182). This suggests that thymic (central) tolerance is not a critical step in self tolerance to this autoantigen. The same conclusions have been drawn from the finding of similar intrathymic transcript levels of thyroid autoantigens (TPO and TSH-R) in mice which are genetically susceptible or resistant to the development of EAT (183). However the situation may be more complex than originally imagined, as the same group has identified a role of the Aire gene in the response to TSH-R and in Aire-deficient mice, intrathymic transcripts of TSH-R and TG are reduced while the expression of TPO is nearly abolished (184). These results are compatible with the finding of an increase in AITD in autoimmune polyglandular syndrome type 1, but at a much lower frequency than the classical disorders of Addison’s disease and hypoparathyroidism. It is also intriguing that TPO transcripts are so much more affected in the Aire-deficient murine thymus, perhaps explaining (via more rigorous tolerance) the rather weak response to this autoantigen, compared to TG, in the mouse.

 

Balb/c strain mice appeared to develop orbital changes suggestive of ophthalmopathy when given TSH-R primed T cells derived from donor mice immunized with TSH-R protein or cDNA but this model has not proved reproducible by the original authors, for reasons which are not yet clear, although complex histological artefacts may be part of the answer (185). A somewhat more convincing model of ophthalmopathy has been described recently in which deep injection of plasmid containing the TSH-R A subunit into the leg muscles of BALB/c mice followed by electroporation resulted in a wide variety of histological orbital changes and obvious eye signs (41). However the animals developed TSH-R blocking rather than stimulating antibodies and thyroiditis was absent. Nonetheless these finding support a pathogenic role for the TSH-R in the pathogenesis of thyroid eye disease.

 

The clear general concept to be derived from all of these studies is that a genetically controlled balance of helper and suppressor T cell function is needed to prevent autoimmunity, and that a variety of perturbations can lead to onset of the disease.

 

Relation of the Immune Response to THE Thyroid Cell: Stimulation and Destruction

For certain we know that the autoantibodies can stimulate the thyroid and cause overactivity in Graves' disease, and can in select circumstances inhibit thyroid function and cause hypothyroidism in neonates and some adults. Whether thyroid antibodies are primary cytotoxic agents in AITD remains an unsettled issue. TG antibodies are probably not normally cytotoxic, but TPO antibodies can certainly mediate complement-dependent thyroid cell cytotoxicity and ADCC. However, the frequently reproduced natural experiment of transplacental antibody passage from a mother with AITD to her fetus, without evidence of thyroid damage, clearly shows that antibodies alone are not destructive to the thyroid.

 

Cell-mediated immunity is thought to be important in thyroid cell destruction, and T cells have been shown to be reactive to TECs. T cell lines or clones have been shown to react to TECs (108-110), but the nature of the antigen recognized is unknown. One CD8+ T cell clone in man has been shown to be cytotoxic specifically to autologous TECs (121), suggesting that cell-mediated TEC destruction is an important process, and similar activity has been reported in CD8+ T cell lines and clones derived from mice with experimental autoimmune thyroiditis (186). A second type of T cell-mediated cytotoxicity is that mediated by gd TCR-bearing T cells and specific recognition of TECs by such cells has been reported in Graves’ disease, but the exact autoantigen involved is unknown (187). In animals it is clearly shown that there can be a marked dissociation between the extent of histologic thyroiditis and the levels of antibodies, again suggesting that T cells rather than antibodies mediate cell destruction.   However, it must be admitted that the hard evidence for direct T cell-mediated cytotoxicity in thyroid autoimmunity in man is meagre at present.

 

There are 3 mechanisms by which T cells might mediate TEC destruction and evidence for all 3 operating in AITD has accrued. Firstly, cell lysis might be effected via T cell-derived perforin, which leads to pore formation in target cell surfaces, and certainly the thyroid lymphocytic infiltrate contains perforin-expressing T cells in AITD (188). Secondly, T cells expressing Fas ligand, especially the CD8+ subset, can induce apoptosis in TECs expressing Fas (189). Fas is induced by IL-1b on TECs, whereas TSH-R stimulation inhibits Fas expression (190) and this may lead to the involvement of this pathway in Hashimoto’s thyroiditis but not Graves’ disease, as TSI would act like TSH in the latter to diminish Fas expression (and other regulatory molecules). It has been suggested that T cells may not be necessary, as Hashimoto TEC may express Fas ligand, and autocrine/paracrine interaction with Fas may lead to TEC death (191). The mechanisms for this are unclear and as yet there is no consensus on the role this may have in AITD. The picture is complicated by the upregulation of molecules which protect against apoptosis such as Bcl-2. The pattern of expression of this molecule is different in Graves’ and Hashimoto’s diseases, suggesting that TECs are protected in the former and more sensitive to destruction in the latter (192). Whether these differences depend on cytokines, genetics or other factors is unknown (193). Finally, T cell-derived cytokines can injure the TECs directly, leading to functional impairment (133-135), and by triggering other phlogistic pathways such as nitric oxide synthesis (194).

 

Possible Explanations for Autoimmunity

Many reasons for the development of autoimmunity have been advanced, and these are briefly catalogued below. Cross-reacting epitopes, aberrant T or B cell regulatory mechanisms, inheritance of specific immune response-related genes, and aberrant HLA-DR expression on TECs have all at some time been considered important for development and progression of thyroid autoimmunity.

 

  1. Abnormal presentation of antigen could occur due to cell destruction, or viral invasion, so that large amounts of antigen or cell fragments are liberated locally into the lymphatics. Excessive levels of antigen are produced, thereby overwhelming the usual low dose tolerance mechanism.

 

  1. Abnormal antigen could be produced by a malignancy, or damage to the cell by viral attack, or other means. This antigen could be a partially degraded or denatured normal antigen, for example.

 

  1. Cross-reacting bacterial or viral epitopes e.g. Yersinia enterocolitica (195) could induce immune responses that happen to cross-react with a self-antigen having identical conformation. An extension of this concept is that the normal anti-idiotypic control response happens to produce an Ig or T cell that cross-reacts with self-antigen. For example, experimentally produced anti-idiotypic monoclonal antibodies directed to TSH antibodies bind to and stimulate the TSH-R (196).
  2. Somatic mutation of a TCR gene could lead to a clone of self-reactive cells. However, somatic mutation of TCR genes is believed to occur very rarely if at all, and such monoclonal or oligoclonal activation has not been documented in autoimmune disease. Somatic mutation of B cell Ig genes is, as described above, a normal phenomenon during an antigen‑driven proliferative response. Such an event could occur by chance during response to any antigen and this does not effectively introduce any new variable, since B cells capable of producing Igs that can react with self-antigens are already normally present. However, TSI seem to be clonally restricted and, until the V gene usage of these antibodies is documented, it remains possible that Graves’ disease is due to the inheritance of a unique, etiologically critical V gene encoding TSI.

 

  1. Inheritance of specific HLA, TCR, or other genes that code for proteins having especially effective ability to process or present antigen.

 

  1. T cell or B cell feedback control mechanisms could be aberrant due to hereditary or environmental factors.

 

  1. Failure of clonal deletion could leave self-reactive T cells present in the adult. In fact this is clearly normal, as described above.

 

  1. Failure of normal maturation of immune system could allow fetal T and B cells that are autoreactive and of wide specificity to persist.

 

  1. Polyclonal activation of T or B cells, by some unknown stimulus, could lead to B cells producing self-reactive Ig, in the apparent absence of antigenic stimulus. This theory is in a sense impossible to disprove but would need to co-exist with other abnormalities to explain disease remission, genetic associations, associated diseases, etc. Polyclonal activation is not typical of peripheral lymphocytes of patients with AITD (197).

 

  1. TECs could express MHC class II molecules as a primary event and then could function as APCs, including antigens on their cell surface.

 

  1. Environmental factors could distort normal control. For example, stress or steroids may alter immunoregulation, and the potential role of dietary iodine has been mentioned above.

Abnormal Exposure To Thyroid Antigens And The Effects Of Pregnancy

Damage to the thyroid might release normally sequestered antigens, inducing an immune response. Damage to thyroid cells does indeed occur in viral thyroiditis, such as in association with mumps or in subacute thyroiditis of unknown cause, but autoantibodies appear only transiently at low titer, and progressive lesions of the thyroid do not usually occur (reviewed in 198, 199). External irradiation to the thyroid, including that from nuclear fallout, can also lead to an increase in Graves' disease or thyroid antibody production (200, 201), but it is unclear if this is caused by autoantigen release or an effect on the lymphocytes which are radio-sensitive. Even occupational exposure to ionizing radiation appears to be a risk factor for the development of autoimmune thyroiditis (202). Another possible example where exposure to thyroid antigens released by gland injury leads to autoimmunity is the rare case of precipitation of Graves’ disease and ophthalmopathy after ethanol injection of thyroid nodules (203).

 

A powerful argument against the hidden antigen hypothesis is that TG is a normal component of circulating plasma (204). One might turn the first argument around and suggest that thyroiditis results from a lack of exposure to TG at some period, an exposure that is necessary to depress continuously an otherwise inevitable immune response. This suggestion has no clinical or experimental support, and the available evidence indicates that TG is present in the plasma of patients with active immunity. It remains to be seen how sequestered TPO and TSH-R are, but the appearance of T cells capable of proliferating in response to these antigens, in apparently healthy individuals, also argues against any sequestration (205). What is clear is that availability of the thyroid autoantigen is essential to maintain the autoimmune response: complete removal of thyroid antigens following thyroidectomy and remnant ablation with radioiodine leads to disappearance of antibodies to TG, TPO and TSH-R (206). Although this is not surprising, it does suggest that extrathyroidal sources TSH-R are insufficient normally to maintain an autoimmune response.

 

A variant on this theme is that of microchimerism, the persistence of fetal cells in maternal tissues. Studies have found evidence of microchimerism in thyroid tissue from patients with and without AITD (207, 208). Could such sequestered fetal material make the thyroid prone to an alloimmune response, and be responsible for the exacerbation of AITD seen in the postpartum period? If so, this phenomenon would help to explain the high frequency of AITD in women. Twins from opposite sex pairs should have an increased risk of thyroid autoimmunity compared to monozygotic twins if microchimerism has a role, and indeed such twins have been found to have more frequent thyroid autoantibodies (209). However, although parity is associated with an 11% increase in the risk of all female-associated autoimmune disorders, there is no increase with multiple pregnancies, which rather argues against a microchimerism mechanism (210). During and after pregnancy, major changes in Treg function occur and direct effects on the cytokines produced by T cells can also be demonstrated (211). It is these alterations that are most probably the ultimate cause of the increase in autoimmunity after pregnancy.

 

It seems likely that sex steroids play a role in determining the autoimmune response. For instance, in a recent study of an animal model of Graves’ disease, 5α-dihydrotestosterone was given to mice a week before immunization with TSH-R, and this reduced both the severity of the hyperthyroidism that developed and downregulated the Th1 response (211a). Another hypothetical reason for the unequal sex ratio is that skewed X chromosome inactivation could contribute through the failure of some autoantigens expressed on one X chromosome to be expressed at a critical point in the disease pathway. A recent survey of 309 patients with Graves’ disease and 490 with Hashimoto’s thyroiditis found skewed inactivation of the X chromosome in Graves’ disease (odds ratio 2.2) but not Hashimoto’s thyroiditis; when combined with 4 other studies in a meta-analysis, the results remained significant for Graves’ disease and reached significance for Hashimoto’s thyroiditis (odds ratio 2.4) (212).

 

Abnormal Antigens

An abnormal antigen might also serve to produce an immune reaction. The protein abnormality could be either congenital or acquired by an injury such as a virus infection. To date there is no evidence which indicates that TG, TPO, or other proteins of the thyroid of a patient with autoimmunity are abnormal. Minor allelic differences apparently do occur but attempts to associate thyroid disease with polymorphisms of the TPO and TSH-R genes have been unsuccessful.

 

Cross-Reacting Antigens

The theory that immune reactivity to an environmental antigen could lead to antibodies that cross‑react with thyroid antigens has been bolstered by studies which show a relationship between Graves' disease and antibodies to the common enteropathogen Yersinia enterocolitica. An increased incidence of antibodies to Yersinia is found by some, but not all authors, in patients who have Graves' disease, and there are saturable binding sites for TSH on Yersinia proteins (213). After infection by Yersinia, human sera contain Igs that bind to TEC cytoplasm (195), and IgGs which appear to compete with TSH for binding to thyroid membrane TSH receptors (214). The antigens involved may in fact include proteins encoded by plasmids present in the Yersinia, rather than intrinsic Yersinia proteins, but that does not alter the general concept (215). Arguing strongly against a role for Yersinia is the fact that there is no unique pattern of serological immunoreactivity to Yersinia antigens in patients with AITD (216), and most patients with this infection do not develop Graves’ disease. Moreover, there was no association between Yersinia infection and autoimmune thyroid disease in a large prospective study of individuals developing AITD (217).

 

In theory an initial response to one antigen might proceed by reacting to the other antigen, and thereby spread and augment the autoimmune process. In the context of T cell autoreactivity there is much greater scope for molecular mimicry whereby a response to an exogenous epitope leads to a cross-reactive response to an endogenous autoantigenic epitope. Simple sequence homology is insufficient to predict this, as shown elegantly by the cross-reactivity of two TPO epitopes showing a similar surface but not amino acid sequence (218). This makes the prediction and study of molecular mimicry much more difficult than is generally appreciated (219). For these reasons, it may be naïve to believe that the putative orbital antigen responsible for ophthalmopathy has to be an identical protein to that expressed in the thyroid.

 

Virus Infection

Virus infection has for years been speculated to be an etiological factor in most autoimmune diseases, by causing cell destruction and liberating antigens, by forming altered antigens or causing molecular mimicry, by inducing DR expression, or by inducing CD8+ T cell responses to viral antigens expressed on the cell surface. Thyroid autoantibodies are elevated transiently after subacute thyroiditis, which is thought to be a virus-associated syndrome, but no clear evidence of virus-induced autoimmune thyroiditis in humans has been presented. In this regard it is of interest that persistent, apparently benign virus infection of the thyroid can be induced in mice (220), and that infection of neonatal mice with reo virus induces a polyendocrine autoimmunity (Fig. 7-11). These agents could work by liberating thyroid antigens. Virus infection might also augment autoimmunity by causing non-specific secretion of IL-2, or by inducing MHC class II expression on TEC. Despite many attempts to implicate retroviruses in AITD, results to date remain inconclusive (221). Human T lymphotrophic virus-1 has been repeatedly associated with various autoimmune disorders, including Hashimoto’s thyroiditis; presumably the virus alters immunoregulatory pathways allowing autoimmunity to emerge (222).

7-11

Figure 7-11:   Autoantibodies to thyroid in sera of reovirus-infected mice detected by indirect immunofluorescence. (a) Frozen section of normal mouse thyroid incubated with sera obtained from mice 21 days after infection, showing staining of colloid characteristic of antithyroglobulin antibody (original magnification, X200). (b) Section of normal mouse thyroid (fixed in Bouin's solution) incubated with sera obtained from mice 21 days after infection, showing staining of thyroid acinar cells (original magnification, X 200). Reproduced with permission from I. Okayasu and S. Hatokeyama, Clin. Immunol. Immunopath., 31:334, 1984.

Lymphocyte Mutation And Oligoclonality

Apart from the evidence that some TSI may have an oligoclonal origin (67, 223), there is no evidence to support a clonal B cell abnormality in AITD. V gene usage by TSI will need to be analyzed to determine whether Graves’ disease has a unique pathogenesis determined by germ-line immunoglobulin genes. Thyroid-reactive T cells are present in healthy animals and man, as noted above, and therefore a defect at the clonal T cell level is less likely as a primary event in etiology than previously thought. A few autoreactive T cells can be expected to escape tolerance normally, particularly if the autoantigen in question is not available to delete T cells in thymus during fetal development. Stochastic events later in life affecting such undeleted T cells could readily explain the lack of complete concordance for AITD in genetically identical twins (224), and this lack of such concordance argues against an inherited pathogenic TCR as a primary event in AITD.

 

Genetic Predisposition

A role of heredity in AITD is clearly demonstrated by family studies (225, 226). The role of heredity in AITD is clear, since there is an increased frequency of AITD among family members, first degree relatives, and twins of patients with the illness (227). Indeed a detailed analysis of concordance in Danish twins with Graves’ disease came up with the estimate that 79% of the liability for this disorder was attributable to genetic factors (228). Another strand of evidence is the variation of disease with race, although of course this is complicated by environmental influences too. Analyzing military personnel in the USA, it has been shown that HT is more frequent in white individuals, and lowest in black and Asian/Pacific Islander individuals (229). Despite some shared genetic susceptibility factors (see below), in Graves’ disease the opposite is true. It is unknown why these ethnic differences occur and this is clearly an area that could be fruitfully explored further.

 

In an investigation of the relatives of a group of propositi with high circulating antibody levels and clinical thyroid disease, approximately half of the siblings and parents (first‑order relatives) were found to have significant titers of thyroid antibodies, many being without clinical thyroid disease (230) but the transmission of thyroid autoantibodies is a more complex trait than the dominant inheritance originally thought (231, 232).

 

Together, such observations suggest that these diseases have a common genetic defect, although other genes are likely to be disease-specific in their effects, as reviewed extensively elsewhere (233).   The most important susceptibility factor so far recognized is the inheritance of certain MHC class II genes. Inheritance of HLA-DR3 causes a 2 to 6-fold increased risk for the occurrence of Graves' disease or autoimmune thyroiditis in Caucasians, and inheritance of HLA-DR4 and DR5 has been found in some studies to increase the incidence of goitrous hypothyroidism (234). In post-partum painless thyroiditis an association with HLA-DR5 has been reported (235). HLA-DQA1*0501, which is often linked to DR3, may have an even more pronounced predisposing effect in Caucasians with Graves’ disease (236), whereas HLA-DRB1*07 may be protective (227). A large series of 991 Japanese patients with AITD has been studied and the HLA susceptibility to Graves’ disease differentiated from that to Hashimoto’s thyroiditis, while 3 common haplotypes were identified which conferred protection against Graves’ disease; one of these acted epistatically with the HLA-DP5 susceptibility molecule and another also conferred protection to Hashimoto’s thyroiditis (238). It is noteworthy also that the relative risks conferred by HLA alleles is rather modest, borne out by the relatively low concordance for Graves’ disease in HLA-identical siblings of patients with Graves’ disease (239). This suggests the operation of other genetic susceptibility loci, also emphasized by the weak lod scores for linkage with the HLA region in family studies of AITD (240, 241).

 

The nature of these other loci is unclear and their identification is likely to require an extensive analysis involving thousands of families in studies using modern molecular techniques. Association studies have been the method of choice until recently, investigating various candidate genes, but with mixed success. Inconclusive results have been reported for associations of AITD with TCR polymorphisms, immunoglobulin allotype and TSH-R polymorphisms. The most consistent non-HLA association is between polymorphisms in the CTLA-4 gene and both Graves’ disease and Hashimoto’s thyroiditis (242, 243). Despite claims to the contrary, there appears to be no additional risk conferred by CTLA-4 (or HLA) polymorphisms in Graves’ patients with clinical evidence of ophthalmopathy (244), but these CTLA-4 polymorphisms may partially determine outcome after antithyroid drug (245, 246). Given the most important role of the interaction between CTLA-4 on T cells and the B7 family of molecules on APCs, it is possible that this association represents a genetic effect on immunoregulation, although, as with HLA-DR3, this is not specific for thyroid autoimmunity; the same polymorphism is also associated with type I diabetes mellitus and several other autoimmune disorders. Fine mapping of the CTLA-4 region has confirmed that it is indeed this gene, rather than those in linkage disequilibrium, which is responsible for the associations, and the polymorphisms may exert their effects by causing variation in levels of soluble CTLA-4, which in turn may after T cell activation, especially in Treg cells (247).

 

Polymorphism of the vitamin D receptor has been linked with Graves’ disease, an association which has some biological plausibility as vitamin D has immunological effects (248). However a large survey comprising 768 patients with Graves’ disease from the UK, compared to 864 controls, found no evidence of an association (249) and there is not yet any prospective evidence yet for vitamin D deficiency being associated with AITD (250).   Polymorphisms in genes encoding molecules involved the NFkB inhibitor pathway modulating B cell function (FCRL3 and MAP3K7IP2) are more likely to be involved in susceptibility to Graves’ disease (251, 252).

 

Another genetic susceptibility locus in Graves’ disease is polymorphism in the lymphoid tyrosine phosphatase LYP/PTPN22 gene, which has been associated with functional changes in T cell receptor signaling. A study of 549 patients and 429 controls found that a codon 620 tryptophan allele conferred an odds ratio of 1.88 (253), although it should be noted that similar effects have been seen in many other autoimmune diseases. This result has recently been confirmed (254) and another likely locus is the IL-2 receptor alpha (CD25) gene region, which is also associated with other autoimmune diseases like type diabetes (255).

 

As well as genes controlling the immune response, genes that control the target organ susceptibility to autoimmunity are logical candidates for investigation. There is to be conclusive proof from both linkage disequilibrium and association studies, that polymorphisms in the TSH-R gene confer susceptibility to Graves’ disease but not autoimmune hypothyroidism (256, 257). This is one of the few susceptibility factors that segregates with one rather than both types of thyroid autoimmunity, although polymorphisms in the PDE10A and MAF genes (which have many actions, including immune regulation) may also influence whether patients develop Graves' disease or Hashimoto's disease (257a). Although not thyroid-specific in tissue location, selenoproteins (SEP) are central to thyroid hormone deiodination and a significant association of HT with SNP in SEPS1 (odds ratio 2.2) has been reported in a series of 481 Portuguese HT patients (258).

 

A different approach to chasing candidate genes has been genome scanning, although huge effort is required to undertake such studies. Based largely on this approach, other loci which may be important have been identified on chromosomes 14q31, 20q11 and Xq21 (241, 259), and the importance of a gene on the X chromosome is supported by the increased frequency of AITD in women with Turners syndrome, especially those with an isochromosome-X karyotype (260). However in a genome scan involving 1119 relative pairs, there was no replication of these findings (261). A more impressive genome wide scan of thousands of individuals with Graves’ disease confirmed susceptibility loci in the major histocompatibility complex, TSHR, CTLA4 and FCRL3 and identified two new loci; the RNASET2-FGFR1OP-CCR6 region at 6q27 and an intergenic region at 4p14 (262). Seven new loci for AITD, including MMEL1, LPP, BACH2, FGFR1OP and PRICKLE1, have been uncovered by using a custom made SNP array across 186 susceptibility loci known for immune-mediated diseases (263). In another study of almost 10000 Chinese patients with Graves’ disease, five additional novel loci were identified and polymorphism in the TG gene was also confirmed to be associated with Graves’ disease (264). Thus the genetic factors involved in AITD are increasingly more complex and their interactions with each other and with environmental factors in disease pathogenesis will be a major task to uncover.

 

Further developments in genetic analysis will no doubt bring even greater complexity to this area, albeit with the prospect of better defining patient subsets (265). It is now clear that to detect common, low-risk variants with reliability, huge sample sizes are essential facilitated by the haplotypic data available from the HapMap project, which means that genome wide variability can be detected using half a million single nucleotide polymorphisms (266). These studies present considerable logistical challenges, and many older studies of genetic associations in AITD have produced conflicting results as because of lack of power or population stratification issues. However a good example of the utility of such studies is a massive genome-wide association study in which a new set of SNPs, which includes polymorphism in MAGI3, has been associated with an increased risk of progression from TPO antibody positivity without hypothyroidism to the development of hypothyroidism (267).

 

As an aside, it should be noted that low birth weight, a known risk factor for several chronic disorders, has not associated with clinically overt thyroid disease or with the production of thyroid autoantibodies in one study (268) but others have come to an opposite conclusion, with prematurity irrespective of birth size being another risk factor (269, 270).

 

Co-Occurrence Of Autoimmune Diseases

The co-existence of AITD and other diseases possibly of autoimmune cause has often been reported, and suggests some intrinsic abnormality in immune regulation. An extensive review of these associations has been published (271) and extensive population data bases have clarified the strength of the various associations (272). A striking association is with pernicious anemia. Perhaps 45% of patients with autoimmune thyroiditis have circulating gastric parietal cell antibodies (273), and the reverse association is almost as strong (274). Up to 14% of patients with pernicious anemia have primary myxedema, and pernicious anemia is increased in prevalence in patients with hypothyroidism (275).

 

Another strong association is with celiac disease, which is found 3 times more commonly in patients with AITD. Intriguingly the autoantibodies which are the hallmark of celiac disease, directed against transglutaminase, can bind to thyroid cells and thus could be implicated directly in thyroid disease pathogenesis (276). The association of Sjögren's syndrome and thyroiditis is not uncommon and both systemic lupus erythematosus (SLE) and rheumatoid arthritis are also significantly associated with AITD (277, 278). A high frequency of antibodies to nucleus, smooth muscle, and single-stranded DNA (26-36%) is found in AITD (279). Although multiple sclerosis has stood out as a putative autoimmune disease which is not obviously associated with AITD, meta-analysis has revealed there is an odds ratio of 1.7 for AITD in these patients (280).

 

Autoimmune Addison's disease and/or type I diabetes mellitus and AITD occasionally co-exist and this forms the autoimmune polyglandular syndrome (APS) type 2 (281). This is an autosomal dominant disorder with incomplete penetrance and is often associated with other disorders, such as vitiligo, celiac disease, myasthenia gravis, premature ovarian failure and chronic active hepatitis (282, 283). AITD is an infrequent feature of the much rarer APS type I (284) and there is no association between mutations in the AIRE gene, which causes APS type I, and sporadic AITD (285).

 

Together these data provide convincing proof of an association of other autoimmune phenomena with AITD. Most typically, this immunity is organ specific, but in one subset of patients, thyroid autoimmunity develops in association with the non-organic-specific collagen diseases. A syndrome of running together, of course, does not prove a causal association.   Nevertheless, the plethora of associations and their familial occurrence indicates that a defect in the immune system may be more likely than primary defects in each organ. This in turn suggests a shared immunoregulatory defect, which is at least partly genetically determined, as these diseases often share similar genetic associations, including HLA, CTLA-4, PTPN22 and CD25 gene polymorphisms. Recently, analysis of HLA molecules has shown a pocket amino acid signature, DRβ-Tyr-26, DRβ-Leu-67, DRβ-Lys-71, and DRβ-Arg-74, that was strongly associated with type 1 diabetes and AITD (286). This could confer joint susceptibility to these diseases in the same individual by causing significant structural changes in the MHC II peptide binding pocket and influencing peptide binding and presentation.   It is also clear however that there is a difference in the kind of clustering of other autoimmune disease in Hashimoto’s thyroiditis and Graves’ disease, presumably related to differences between these two types of thyroid disease in genetic predisposition (287).

Immunoregulation: Phenomena And Mechanisms

Possible abnormalities in immunoregulation have been addressed in hundreds of studies. The basic hypothesis of this work is that a deficiency of functional T suppressor cells, now termed regulatory cells, may allow uncontrolled T and B cell immune responses to thyroid (or other) antigens. As noted above, this concept is a major theme in experimentally induced or naturally occurring thyroiditis in animal models. Most of the studies to define immunoregulatory responses in AITD have relied on phenotyping (which may relate poorly to effector function in vivo) or in vitro assays done in unique conditions; as we have previously noted, T cell antigen expression and function can vary depending on source of cells, stage of disease, the use of any stimulating agent in vitro, culture conditions, etc.

Sridama and DeGroot found decreased suppressor cells, defined as CD8+ peripheral blood T cells in patients with Graves' disease (288, 289). These results have been challenged, and some investigators have reported depression of CD4+ cells in AITD (290). However, overall, there is now agreement that, in thyrotoxic patients with Graves' disease, a decrease in CD8+ T cell number (291, 292) is characteristically present, and that a similar abnormality exists in the thyroid. CD8+ cells return gradually toward normal during therapy, and are usually but not always normal at the end of therapy (292) (Fig. 7-12). The phenomenon is present but less evident in Hashimoto's thyroiditis patients. It has been attributed by some workers to increased thyroid hormone levels (293), although this issue is clouded, since there are reports disproving the idea that hyperthyroidism per se induces suppressor cell abnormalities in humans, and reduced suppressor T cells (Ts) are found present long after thyrotoxicosis is cured (294). Our interpretation is that the abnormality is not due specifically to excess T4 in blood, but is a manifestation of ongoing active autoimmunity, for reasons which are unclear. Reduced nonspecific "suppressor" T cell function may be in part an inherited abnormality, and is probably also a manifestation of the augmented immune reactivity ongoing in AITD patients. It may be largely a secondary phenomenon, but one which augments and continues the immunological disease. The mechanism causing such reduced Ts number and function is unclear.


Influence of a 6 month course of carbimazole on peripheral blood T cell subsets of 29 patients with hyperthyroid Graves' disease (Mean SD).

Figure 7-12: Influence of a 6 month course of carbimazole on peripheral blood T cell subsets of 29 patients with hyperthyroid Graves' disease (Mean SD). OKT4 = CD4+ OKT3 = CD3+ OKT8 = CD8+ ** = p < .001 vs. zero time value (From Reference 265)

These older findings need to be related to recent developments in understanding Treg function. One study has found that despite increased numbers of CD4+ T cells bearing the T regulatory cell markers CD25, Foxp3, GITR and CD69, in both thyroid and PBMC of patients with AITD, there is a non-specific defect in regulatory function in vitro, which in turn must explain somehow why the increased number of regulatory T cells are so patently ineffective (295). For example there is an increase in circulating CD69+ regulatory lymphocytes in AITD, and numbers are even higher in the thyroid glands of these patients and yet they are functionally deficient in vitro (295a). The existence of a functional rather than numerical deficiency in regulatory T cells has also been suggested in a study of AITD patients, in which the defect was found to be detectable only when optimal in vitro conditions were achieved (296). Analysis in the earliest phases of disease may of course yield different results and unlocking how T regulatory cells can be activated seems an obvious but at present unrealizable therapeutic strategy. The finding that many thyroid infiltrating lymphocytes, early on in the disease process, are in fact recent thymic emigrants does suggest that there is a problem with central tolerance that allows autoreactive T cells to accumulate in the gland where the strength of local immunoregulation could be critical in determining whether disease progresses (297).

 

Thyrotoxic Graves' disease patients and those with active Hashimoto's thyroiditis have a high proportion of DR+ T cells in their peripheral circulation (291, 298), which indicates the presence of activated T cells. It is unlikely that these cells (> 20% of circulating T cells) are all responsive related to thyroid antigens, so they must include DR+ T cells with TCRs for many other antigens.   There is also a marked increase in circulating soluble IL-2 receptors in thyrotoxic Graves' disease, but this appears to be typical of thyrotoxicosis per se, and not specifically Graves' disease (299). Nevertheless, there is no evidence for a generalized ongoing immune hyper-responsiveness in thyrotoxic patients. Perhaps these T cells (for many different specificities) are stimulated by IL-2, but in the absence of the required second signal provided by antigen exposure, do not induce B cell proliferation or cytotoxic responses.

 

Diminished, non-specific suppressor cell function is also observed in many autoimmune diseases including lupus, and multiple sclerosis and the results in AITD are equally non-specific. The most likely explanation for many “suppressor” phenomena is the reciprocal inhibition of Th1 and Th2 cells by their cytokine products, and powerful evidence shows how important this regulatory mechanism is in exacerbating or inhibiting autoimmune disease, at least in animal models. However regulatory phenomena utilizing cytokines are much more complex, and include both Th17 cells and invariant NKT (iNKT) cells. The latter share receptors with T and NK cells, with the α chain of the T cell receptor being invariant gene segment-encoded, and are notable for releasing cytokines when stimulated by antigen, thus endowing them with regulatory properties which may be either stimulatory or inhibitory. Recently iNKT cell lines have been identified that can be stimulated with TG to induce EAT (300).

 

In keeping with the importance of the Th17 subset in inflammatory autoimmune diseases discussed earlier, there is an increased differentiation of circulating Th17 lymphocytes and an enhanced synthesis of Th17 cytokines in AITD, mainly in those patients with Hashimoto thyroiditis (301). Nonetheless a recent study has found an increase in both Th22 and Th17 cells and the levels of plasma IL-22 and IL-17 in patients with Graves’ disease; the magnitude of these increases correlated TSH-R antibody levels (302). Circulating platelet-derived microvesicles are significantly raised in AITD patients and these can inhibit the differentiation of Foxp3+ Treg cells and induce differentiation of Th17 cells (302a). Another newly recognized T cell subset involved in the regulation of antibody production, comprising follicular helper T cells, is increased in the circulation of patients with AITD and correlates with autoantibody levels (303).

 

Many studies have examined T cell subsets in thyroid tissue of patients with active AITD. For example, Margolick et al (304) found increased CD8+ cytotoxic/suppressor cells and also increased CD4+ T helper cells, and a normal Th/Ts ratio. Canonica et al (305) found increased proportions of cytotoxic/suppressor T cells in thyroids of Hashimoto's thyroiditis patients. Infiltrating cytotoxic/suppressor cells in Hashimoto's thyroiditis were found usually to be activated and to express DR antigen, whereas this response was not so obvious in Graves' disease (306). Canonica et al (305) reported an increased proportion of activated T helper/inducer cells in both Graves' disease and Hashimoto's thyroiditis, and increased cells thought to represent cytotoxic T cells in Hashimoto's thyroiditis. Chemokine expression within the thyroid is likely to be an important determinant of this infiltration (307).

 

Increased CD8+CD11B- cells, presumed to be cytotoxic cells, were found in Graves' disease thyroids (in comparison to PBMC of Graves' disease or normal subjects), whereas "dull" CD8+CD11B+ natural killer cells were diminished (308). Other studies have suggested a reduction in NK cells in Graves' disease and an increase in Hashimoto's thyroiditis. Tezuka et al found decreased NK cells in Graves' disease thyroid tissue, no differences in the NK activity of PBMC between Graves' and normal patients, and that the NK cells in Graves' disease did not kill autologous thyroid epithelial cells (309). We have already indicated other reports of normal NK and ADCC in Hashimoto's PBMC, and of increased ADCC in Hashimoto's thyroiditis. Most studies that have looked at Graves' disease tissues also indicate an increased proportion of B cells compared to peripheral blood subsets.

 

Cell cloning has also been used to examine thyroid and peripheral blood lymphocyte subsets. Bagnasco et al (310) found a predominance of cytolytic clones, releasing IFN-g, in Hashimoto's thyroiditis but not in Graves' disease. Del Prete et al (311) found a high proportion of cytolytic cells with the CD8+ phenotype in clones from thyroid tissue, and felt these results may relate to autoimmune destruction of TEC but the non-specific methods used to derive such cytotoxic T cells raises questions about any pathophysiological relevance.

 

There is no clear predominance of Th1 or Th2 cytokines in the thyroid of patients with Graves’ disease or Hashimoto’s thyroiditis (312), although Th1 clones seem to predominate in the retrobulbar tissues in ophthalmopathy (313). It might simplistically be thought that Graves’ disease represents a Th2 response, but the fact that some patients end up with hypothyroidism itself indicates the likely presence of a Th1 response too. This is supported by evidence from an animal model of Graves’ disease: immune deviation away from a Th1 response, in g-IFN knockout mice, did not enhance the response to TSH-R cDNA vaccination (314).

 

One situation in which it is likely that perturbation the cytokine milieu is responsible for the emergence of Graves’ disease is during reconstitution of the immune system following lymphopenia induced by alemtuzumab treatment for multiple sclerosis, bone marrow or stem cell transplantation or after highly active antiretroviral therapy for HIV infection (315, 316). In these situations there is an initial increase in the Th1 response flowed by a Th2 response at the time when Graves’ disease becomes apparent. Defects in T regulatory cells may also contribute.

 

A general summary of these data is difficult. The results probably at least indicate there are increased B cells, increased DR+ T cells, increased CD4+DR+ T helper cells, decreased CD8+DR+ T suppressor/cytotoxic cells, and possibly lower NK cells in Graves' disease AITD tissue and in blood than among normal subjects' PBMCs. The intrathyroidal T cells are a mix of Th1 and Th2. Such studies have been performed primarily on patients with well developed and often treated disease, and do not bear directly on early stages of the disease, or on whether the changes represent primary or secondary phenomena. To date there has been no certain indication that a non-specific or specific suppressor cell defect exists in patients who are genetically predisposed to have AITD, or in most patients who have recovered from the illness, although observations on Treg and other recently defined T cell subsets appear to indicate defects that are likely to be causal.

 

Anti-Idiotype Antibodies

Whereas anti-idiotypic antibodies are thought to play a physiological role in immunoregulation, there is little evidence for participation in, or abnormality of, this function in AITD.   Immunoglobulins from some patients with Graves' disease bind TSH (317). This suggests that anti-idiotypes to TSH antibodies are present and might theoretically function as thyroid stimulating immunoglobulins; or conversely that anti-idiotypes to thyroid stimulating antibody exist and can bind TSH. Either possibility remains to be confirmed. Sikorska (318) demonstrated the presence of antibodies in sera of AITD patients which inhibit binding of TG to monoclonal TG antibodies, and interpreted these as anti-idiotypes. We have looked for anti-TG anti-idiotypes in patients with autoimmune thyroid disease and failed to find them (319). On the other hand, weak anti-idiotypes of the IgM class have been found which bind to TPO antibodies and are present in pooled normal immunoglobulins as well as certain patient sera (320). Although one could postulate that a failure to produce anti-idiotype antibodies could be a feature of AITD, a more likely hypothesis is that anti-idiotypic antibodies are simply rarely produced at a detectable level. Since anti-idiotype antibodies raised in animals will suppress in vitro TG antibody production, the theory that lack of anti-idiotype control is causal in AITD remains attractive, but data to support it are scant.

 

De Novo Expression Of Class Ii Antigens On Thyroid Cells

De novo expression of HLA-DR on thyroid epithelial cells, from patients with Graves' disease, was first reported by Hanafusa et al (321) and was proposed as the cause of autoimmunity by Bottazzo et al. (322) who suggested that de novo expression of MHC class II molecules on these cells, which are normally negative, allows them to function as APCs. Lymphocyte-produced IFN-g augments the expression of HLA-DR (also DP and DQ) on thyroid epithelial cells, and that TNF-a further increases the induction caused by IFN-g (323, 324). HLA-DR+ TECs definitely can stimulate T cells (325, 326) but this is critically dependent on the requirements of the T cell for a costimulatory signal, as Graves’ TECs do not express B7-1 or B7-2 (327, 328). In contrast B7.1 expression on Hashimoto TEC has been recorded, but how this is differentially regulated, compared to Graves’ disease, is unknown (329).   We have shown that TECs can present antigen to T cell clones which no longer require costimulation through B7, yet not only fail to stimulate B7-dependent T cells but also induce anergy in these cells by at least two mechanisms, one of which is Fas-dependent (330, 331). Perhaps the most conclusive proof that class II expression by thyroid cells cannot induce thyroiditis comes from the creation of transgenic mice expressing such molecules on TECs – such animals have no thyroiditis and have normal thyroid functionm (332). For reasons which remain unclear, thyroid follicular and papillary cancers may express B7.1 and B7.2, and B7.2 expression is associated with an unfavourable prognosis (333).

HLA-DR is also expressed on TECs in multinodular goiter and in many benign and malignant thyroid tumors, and this does not appear to induce thyroid autoimmunity (334). Aberrant DR expression has not been shown to develop before autoimmunity. Normal animal thyroids not expressing class II molecules can become the focus of induced thyroiditis, and then express class II molecules (335). Furthermore, HLA-DR expression on Graves' disease thyroid tissue is lost when tissue is transplanted to nude mice (336). Thus a consensus position is that class II expression could be important, but is a secondary phenomenon in AITD, dependent on the T cell-derived cytokine, g-IFN, and only allowing TECs to become APCs for T cells which have already received B7-dependent costimulation elsewhere. This could clearly exacerbate AITD once initiated, but teleologically the role of class II expression seems to be as a peripheral tolerance mechanism, allowing the induction of anergy in potentially autoreactive but still naive (ie. B7-dependent) T cells (Fig. 7-13). The recent description of hyperinducibility of HLA class II expression by TECs from Graves’ disease suggests that such patients may be genetically predisposed to display a more vigorous local class II response and this would increase the likelihood of disease progression (337). The genes controlling this response are therefore worthwhile candidates for future studies of genetic susceptibility.

 

Alternative outcomes of MHC class II expression by thyroid follicular cells.

Figure 7-13: Alternative outcomes of MHC class II expression by thyroid follicular cells. Reproduced from Weetman (1997) New Aspects of Thyroid Autoimmunity. Hormone Research 48 (Suppl 4), 5154 with permission.

ENVIRONMENTAL FACTORS

Environmental factors include viral and other infections, discussed above. Strong evidence for an important role for environmental factors is provided by the incomplete concordance seen in the monozygotic twins or other siblings of individuals with AITD. Also, there are temporal changes in disease incidence that can only be the result of environmental influences, such as the rise in Graves’ disease in children in Hong Kong, the steady rise in autoimmune thyroid disease in Calabria, Italy, the more than two-fold increase in lymphocytic thyroiditis over 31 years in Austria, and the changes in the rates of histologically diagnosed Hashimoto’s thyroiditis over a 124 year period (338, 339, 340, 341).

 

Such studies also show that environmental factors may change rapidly, making their ascertainment difficult and challenging. Epidemiological studies have also shown that there is a higher prevalence of thyroid autoimmunity in children raised in environments that have higher prosperity and standards of hygiene (342). This falls in line with the so-called hygiene hypothesis, that is, the idea that early exposure to infections may skew the immune system away from Th2 responses like allergy and also away from autoimmunity. IL-2 administration for treatment of cancer leads to the production of antithyroid antibodies, and hypothyroidism (and possibly a better tumor response) (343). IFN-a administration and other cytokines (91), as well as highly active antiretroviral therapy for HIV infection (344), have a similar effect, although interferon-b1b treatment has no significant adverse effect on AITD (345). However long-term follow up studies have shown that around a quarter of multiple sclerosis patients treated with this latter cytokine may develop autoimmune thyroid disease within the first year of treatment (346). It remains unclear how relevant any lessons from these observations are for AITD pathogenesis, as of course the doses of cytokines and drugs used therapeutically are vast. However, it has been reported that thyrotoxicosis tends to recur following attacks of allergic rhinitis (347). Possibly this is due to a rise in endogenous cytokines and the recent association of raised IgE levels with newly diagnosed Graves’ disease indicates that this may be mediated by preferential Th2 activation (348).

 

Cigarette smoking is associated with Graves' disease, and with ophthalmopathy (reviewed in 349) although it seems to be that smoking is associated with a lower risk of autoimmune hypothyroidism (350). The mechanisms behind these complex changes uncertain and is doubtless more complex than a local irritative effect. Environmental tobacco smoke induces allergic sensitization in mice, associated with increased production of Th2 cytokines, but a reduction in Th1 cytokines, by the respiratory tract (351). It is therefore possible that modulation of cytokines contributes to the worsening of ophthalmopathy with smoking. On the other hand, as noted above, the opposite effect prevails in hypothyroidism and smoking exposure was associated with a lower prevalence of thyroid autoantibodies in a large population survey of over 15000 US citizens (352) and smoking cessation is known to induce a transient rise in AITD (353). To explain this, investigations have been undertaken on anatabine, an alkaloid found in tobacco; this compound ameliorates EAT and reduces TG antibody levels in human subjects with Hashimoto’s thyroiditis (354).

 

More general environmental pollutants have not been thoroughly explored for their possible effects (although there is some evidence from older experiments that methylcholanthracene can induce thyroiditis) but a recent study has demonstrated that polychlorinated biphenyls can induce the formation of TPO antibodies and lymphocytic thyroiditis in rats (355). A cross-sectional survey in Brazil has fond that Hashimoto’s thyroiditis and thyroid autoantibodies are more frequent in individuals living near to a petrochemical complex than in controls (356). In addition pesticide use, especially of the fungicides benomyl and maneb/mancozeb, has been associated with an increased odds of developing thyroid dysfunction although the mechanism of action is unclear (357). it clear that this aspect of the environment warrants further study in human thyroid disease.

 

The role of dietary iodine is clearly established in animal models of AITD and circumstantial evidence exists for a similar role in man (358-360). The response is complex and recently it has been shown that iodide may exacerbate thyroiditis in NOD mice but not affect the production of TSH-R antibodies in the same strain (361). Such findings are intriguing as they raise the possibility that the thyroiditis which accompanies Graves’ disease may not be due to the immune response to the TSH-R. Iodine may affect several aspects of the autoimmune response, as detailed in the section on experimental thyroiditis above. In addition, iodide stimulates thyroid follicular cells to produce the chemokines CCL2, CXCL8, and CXCL14 (362). These observations suggest that iodide at high concentrations could induce AITD through chemokine upregulation thus attracting lymphocytes into thyroid gland.

 

Dietary selenium has also been proposed as a contributor. A recent large epidemiological survey of two counties of Shaanxi Province, China, one with adequate and the other with low selenium intake, showed that higher serum selenium was associated with lower odds ratio of autoimmune thyroiditis (0.47) and hypothyroidism (0.75) (362a). However a recent Cochrane Systematic Review of trials of selenium supplementation has shown no clear beneficial clinical effect in HT, although TPO autoantibody levels do fall over a 3 month period of supplementation (363). Vitamin D may be important in autoimmunity and many other disorders, as it is now recognized that individuals living in northerly latitudes may have suboptimal levels based on a fresh understanding of what normal levels of this vitamin should be. A significant inverse correlation has been observed between 25(OH)D levels and TPO antibody levels in Indian subjects, although the overall impact of this effect in terms of causality was low (364), and another prospective study has found no evidence for a role of vitamin D (250). A more recent large scale survey has found that for every 5 nmol/L increase in serum 25(OH)D there was an associated 1.5 to 1.6-fold reduction in the risk for developing Graves’ disease, Hashimoto’s thyroiditis or postpartum thyroiditis, but vitamin D was not strong associated with the level of thyroid autoantibodies (364a). These new results are also supported by a meta-analysis of all studies prior to this, indicating that low vitamin D levels, as well as frank deficiency, are indeed risk factors for AITD (364b).

 

A variety of lifestyle factors that are difficult to investigate may also be involved. It is otherwise difficult to account for the increase in AITD seen in same-sex marriages (365). Stress is likely to be important in the etiology of Graves’ disease, although studies to date have had to rely on retrospective measures of this (reviewed in 366). Moreover stress does not appear to be asociated with the development of TPO antibodies in euthyroid women (369). Presumably stress acts on the immune system via pertubations in the neuroendocrine network, including alterations in glucocorticoids, but the complex interaction between the nervous, endocrine and immune systems includes the actions of neurotransmitters, CRF, leptin and melanocyte stimulating hormone as well and so unravelling the pathways whereby stress may alter the course of autoimmunity is difficult in the extreme (370). Indirect support for such a mechanism, mediated through norepinephrine, comes from experiments showing dramatic enhancement of delayed-type hypersensitivity by acute stress, the result of sympathetic nervous system activation on the migration of dendritic cells and subsequent enhanced T cell stimulation (371). Moderate consumption of alcohol appears to have a protective effect with regard to AIITD (371, 372). Given the diversity of these environmental factors, presumably operating on different genetic backgrounds, it will be difficult (if not impossible with current tools) to establish the relative importance of each in AITD.

 

NORMAL AUTOIMMUNITY

"Normal" people express antithyroid immunity, as previously described, and this must be important in understanding the overall mechanism of AITD. Antibodies to TG and TPO are present in both Graves’ disease and Hashimoto’s thyroiditis up to 7 years prior to diagnosis, increasing over time in the former and consistently elevated in the alter (374). Many people with low levels of antibodies but without clinical disease can be shown to have lymphocyte infiltrates in the thyroid at autopsy. B cells from normal individuals can be induced to secrete anti‑TG antibody in vitro. These observations clearly show that incomplete deletion of clonal self‑reactive T cells is indeed the normal (and indeed perhaps necessary) circumstance, and provide strong support for the idea that disordered control of this low level immunity may be important in the etiology of AITD.

 

Effect of Antithyroid Drugs on the Immune Response

Antithyroid drugs are used in Graves' disease to decrease production of thyroid hormone, and also lead to diminution in TSI and other antibody levels. Clinical studies show that antithyroid drug administration also leads to a diminution in antibody production in thyroxine replaced Hashimoto's thyroiditis patients (375), proving that their effect is not simply due to control of hyperthyroidism in Graves' disease. Somewhat surprisingly (376), administration of KClO4 to patients with Graves' disease leads to diminished serum antibodies, suggesting that the effect of treatment is not specific for thionamide drugs, but could be mimicked by this compound. Antithyroid drugs inhibit macrophage function, interfering with oxygen metabolite production (377).

 

Following antithyroid drug treatment of active Graves' disease, there is a prompt short‑term increase of DR+CD8+ T cells in the bloodstream as described above.   Antithyroid drugs inhibit the production of cytokines, reactive oxygen metabolites and prostaglandin E2 by TECs and the reduction in these inflammatory mediators may explain the site-specificity of the immunomodulation produced by antithyroid drugs (125). Another pathway for an immunomodulatory action of these drugs is via the upregulation of Fas ligand expression, which may then attenuate the autoimmune response of Fas-expressing T cells (378). Only approximately 50% of patients enter remission after treatment with antithyroid drugs, a fact which must be accommodated in any hypothesis concerning an immunomodulatory action of these agents. Those patients with Graves’ disease who have the highest IgE and IL-13 levels in the circulation are the most likely to relapse (379). In turn, this suggests that antithyroid drugs only effect remission in individuals who do not have a strong Th2 response; those with the strongest such responses seem unlikely to be affected by the relatively weak action of such drugs.

AITD AS A CONSEQUENCE OF A MULTIFACTORAL PROCESS
(TABLE 4) (FIG. 7-14)

TABLE 4

DEVELOPMENT OF AUTOIMMUNE THYROID DISEASE

Stage 1 --Basal State
Normal exposure to antigen such as TG and normal low levels of antibody response
Inherited susceptibility via HLA-DR, DQ, or other genes
Stage 2a --Initial Thyroid Damage and Low Level Immune Response
Viral or other damage with release of normal or altered TG, TPO, or TSH-R
Increased antibody levels in genetically susceptible host with high efficiency HLA-DR, DQ, TCR molecules
Infection induced elevation of IL-2 or IFN-γIL-2 stimulation of antigen specific or nonspecific ThIFN-γ stimulation of DR expression and NK activation
Glucocorticoid-induced alterations in lymphocyte function during stress
Stage 2b --Spontaneous Regression of Immune Response
Diminished antigen exposure
Anti-idiotype feedback
Antigen specific Ts induction
Stage 3 --Antigen Driven Thyroid Cell Damage (or Stimulation)
Complement dependent antibody mediated cytotoxicity
Fc receptor+ cell ADCC by T, NK, or macrophage cells
NK cell attack
Direct CD4+ or CD8+ T cell cytotoxicity
Antibody-mediated thyroid cell stimulation
Stage 4 --Secondary Disease Augmenting Factors
Thyroid cell DR, DQ expression --APC function
Other molecules (cytokines, CD40, adhesion molecules) expressed by thyroid cell
Immune complex binding and removal of Ts
Stage 5 --Antigen Independent Disease Progression
Recruitment of nonspecific Th or autoreactive Th
Autoreactive Th bind DR+ TEC or B cells
IL-2 activation of bystander Th
Stage 6 --Clonal Expansion with Development of Associated Diseases
Antigen release and new Th and B recruitment
Cross reactivity with orbital antigen
IL-2, IFN-γ augmentation of normal immune response to intrinsic factor, acetylcholine receptor, DNA, melanocytes, hair follicles, etc.

 

Theoretical Sequence of Development of AITD.

Figure 7-14: Theoretical Sequence of Development of AITD.

 

Thus one is led to the uncomfortable position that AITD is probably not caused by a single factor, but rather due to very many factors which interact. In terms of genetic and environmental factors, as well as factors that may be termed existential (such as age, being female and parity), these may all have to coincide in a favorable way for AITD to occur, in keeping with the Swiss-cheese model for accidents (Fig 7-15). We have divided the roles of these potential disease activity factors into a series of stages, emphasizing the predisposing events, antigen driven responses, and then the secondary and nonspecific amplification which ensues.

A Swiss cheese model for the causation of autoimmune thyroid disease, showing the effect of cumulative environmental, genetic and existential weaknesses lining up to allow AITD to occur, like the holes in the slices of cheese.

Figure 7-15: A Swiss cheese model for the causation of autoimmune thyroid disease, showing the effect of cumulative environmental, genetic and existential weaknesses lining up to allow AITD to occur, like the holes in the slices of cheese. In reality each of the slices depicted is composed of many individual components. The Swiss cheese model for accident causation, for instance an airplane crashing, incorporates active failures (e.g. pilot error) and latent failures (e.g. maintenance deficiency). Some factors contributing to the initiation of AITD are latent (e.g. ageing, growing up in a hygienic environment) and others are active (e.g. possession of an HLA allele which permits presentation of a thyroid autoantigen). Reproduced with permission from Weetman AP, Europ Thy J 2013 in press.

Stage 1 -- In the basal state, Stage 1, immune reactivity to autologous antigen occurs as a normal process. This probably exists at a physiologically insignificant level, since not all T or B cells reacting with TSH-R, TPO or TG are clonally deleted, and Ag is normally present in the circulation. If assays become sensitive enough, we probably will find some level of antibodies to TSH-R, TPO and TG present in most or even all healthy persons, increasing in prevalence and concentration with age, and especially in women, since being female somehow augments antithyroid immunity many-fold. Patients who have inherited certain susceptibility genes will be especially prone to develop AITD because their T and B cell repertoire includes cells recognizing self-antigen, or their immunocytes are especially good at collecting, presenting, and responding to antigen.

Stage 2 -- Possibly viral infection, or other causes of cell damage, or cross‑reacting antibodies present after Yersinia (or other) infection, leads to release of increased amounts of (possibly modified) thyroid antigens which, in genetically prone individuals, leads to an increased but still a low level immune response. Nonspecific production of TNF-a and IFN-g, in response to any infection or immune response, may augment MHC class expression on TECs, allow these cells to function as APCs, and increase production of the already established, normally occurring low levels of antibodies. The process may be affected by stress, although the mechanism remains quite uncertain. The process may go on over years, and wax and wane, as it has been shown that thyroiditis can be clinically apparent and then disappear. Factors involved in temporary or permanent suppression of the autoimmune response may include diminished thyroidal release of antigen, peripheral tolerance induction by DR-positive TECs which cannot provide a costimulatory signal, B cell anti-idiotype feedback, or the induction of T cells with a regulatory function, including those engendered by the mutual regulation of Th1 and Th2 subsets. In some individuals, thyroid cells may be less able to express DR, or may secrete TGF-b and suppress immune responses. Glucocorticoid administration and other immunosuppressives can also temporarily prevent the expression of nascent autoimmunity.

Stage 3 -- If suppressive factors do not control the developing immune response, the disease progresses to a new intensity, now driven by specific antigens, inducing cell hyperfunction (TSI), or hypofunction (TSH blocking or NIS antibodies), or cell destruction. Direct T cell cytolysis and apoptosis, ADCC, and K or NK cell attack play an important role at this stage, and now the disease becomes clinically evident.

Stage 4 -- As the disease develops, a variety of secondary factors come into play, and augments antithyroid immunoreactivity. Any stimulus which causes increased DR expression on thyroid cells, such as T cell release of IFN-g, combined with increased TSH stimulation, may allow TECs to function as APCs.   Although perhaps poor in this function, they are large in number and localized in one area. The TECs may also participate in the autoimmune process by several other pathways, including the expression of adhesion molecules, Fas, Fas ligand, CD40 and complement regulatory proteins, and the production of a number of inflammatory mediators such as cytokines, reactive oxygen metabolites, nitric oxide and prostaglandins. These events are, like class II expression, dependent on cytokines and other signals generated by the intrathyroidal lymphocytic infiltrate. Some patients may inherit diminished T regulatory cell function. The ongoing immune reaction itself, may lead to nonspecific suppressor dysfunction, further augmenting immunoreactivity.

Stage 5 -- T cell derived cytokines may non-specifically induce bystander antigen specific T and B cells to be activated and produce antibody. Autoreactive cells will now accumulate in thyroid tissue because of the many strongly DR+ positive lymphocytes and TECs, and augment the developing response by lymphokine secretion or cytolysis, in a manner independent of thyroid antigens. At this stage in the disease, non-specific autoreactive immune processes may dominate a disease process which no longer depends upon antigen for its continuation.

Stage 6 -- As the concentration of activated T and B cells builds in thyroid tissue, and autoreactive and antigen nonspecific T cells become progressively involved, cell destruction may lead to release of new antigens. Cross-reacting epitopes, and nonspecific stimulation of T cells in genetically prone individuals, may cause the addition of new immunologic syndromes (exophthalmos, pretibial myxedema, atrophic gastritis) typical of older patients with more long standing and florid disease.

 

THYROIDITIS, MYXEDEMA, AND GRAVES' DISEASE AS AUTOIMMUNE DISEASES

 

HASHIMOTO'S THYROIDITIS (FIG. 7-16)

Balance of Immune Reactions Favoring Graves' or Hashimoto's Disease.

Figure 7-16: Balance of Immune Reactions Favoring Graves' or Hashimoto's Disease.

How well do the changes of Hashimoto's thyroiditis fulfill the criteria of an immunologic reaction? Neither the presence of autoantibodies in the serum of patients with Hashimoto's thyroiditis nor the demonstration in vitro of cytotoxicity of the serum constitutes definitive evidence that autoimmunity is the cause of the disease. Rarely, if ever, is there a well-defined initial immunizing event, and accordingly a shortened latent period after a secondary stimulus has not been observed.   Further, experimental passive transfer of the immune state in normal recipients has not yet been attempted and has failed when human sera have been transfused into monkeys and other animals. This experiment is conducted by nature during pregnancy, since maternal antibodies cross the placenta. Transplacental passage of thyroid stimulating antibodies can produce neonatal thyrotoxicosis, and TSH blocking antibodies can produce transient neonatal hypothyroidism. Passage of TG antibody or TPO antibody has no detectable cytotoxic effect.

 

Assays for T cell reactivity in man, supplemented by data from animal models, provide compelling evidence of the autoimmune basis for Hashimoto’s thyroiditis, but this does not exclude an amplifying role for TG and TPO antibodies via ADCC, and, for TPO antibodies, via complement fixation. It may well be that T cell-mediated damage is required initially for all of these antibody-mediated events to take place, as this could be necessary for such access. Another striking feature of Hashimoto’s thyroiditis is the development of Hürthle cells, with granular eosinophilic cytoplasm. This appears to be the result of a chronic inflammatory milieu, resulting in overexpression of immunoproteasomes (3680

 

The evidence is now overwhelming that an immune reaction mediated by T lymphocytes is involved in the development of experimental thyroiditis in animals and several mechanisms may operate singly or together in man to injure TECs. Lymphocytes presensitized to antigens of the thyroid are present in the circulation of most if not all patients and are believed to localize to the thyroid itself.   Since T cell mediated immunity is frequently lethal to cells, it is logical to assume that the T cell mediated immune response in thyroiditis could cause first a goiter, with lymphocyte infiltration and compensatory thyroid cell hyperplasia, and then gradual cell death and gland atrophy. The circulating antibodies may also be a functional part of this reaction. We can accept the idea that T cell-mediated immunity is the major pathogenic factor in thyroiditis.

 

Idiopathic Myxedema

Even before the present era of immunologic study, the basic unity of Hashimoto's thyroiditis and myxedema was realized. To quote from Crile, writing in 1954 (381): "Struma lymphomatosa is responsible not only for large lymphadenoid goiters, but also for fibrosis and atrophy of the thyroid. The clinical spectrum of struma lymphomatosa extends from spontaneous myxedema with no palpable thyroid tissue to a rapidly growing goiter associated with no clinical evidence of thyroid failure."

 

Hubble (382) also drew attention to the occurrence of syndromes intermediate between those of myxedema and Hashimoto’s thyroiditis, in which a small, firm thyroid gland can be felt on careful palpation. The histologic studies of Bastenie (383) and Douglass and Jacobson (384) revealed a close similarity in appearance of the thyroid remnant in myxedema and the Hashimoto gland. The immunologic studies of Owen and Smart (385), and the experience in most thyroid laboratories, indicate a similar incidence and titer of antibodies in myxedema and Hashimoto's thyroiditis. The familial association of myxedema and thyroiditis was described earlier and so far no clear genetic susceptibility difference has been reported in the two diseases. Attempts to ascribe atrophy of the thyroid gland in myxedema to particular antibodies, such as those inhibiting growth or TSH (386), or which mediate ADCC have not been confirmed by other studies (reviewed in 234).

 

Thus, idiopathic myxedema is the end result of Hashimoto's thyroiditis, in which the phase of thyroid enlargement was minimal or was overlooked. We may assume that in idiopathic myxedema the cell‑destructive T cell-mediated immune response is an important pathogenic factor in the illness, and that cytotoxic antibodies and TSH blocking antibodies contribute to the development of hypothyroidism, but perhaps in only a proportion.

 

Graves' Disease

Graves' disease is associated with a similar type of thyroid autoimmunity, since most hyperthyroid patients have circulating TG and TPO antibodies. High antibody levels are found in a small group of hyperthyroid patients and histologic examination of their glands show changes of both cell stimulation and focal thyroiditis (387). Some patients with clinical Graves' disease have tissue changes in the thyroid that are typical of thyroiditis (388).   This type of patient with Graves' disease most often becomes hypothyroid after operation (389), or after 131I therapy (390). It is also well known that some patients fluctuate from hyper- to hypothyroidism over a period of months and others behave in the converse fashion, and of course the familial association of Graves’ disease with autoimmune hypothyroidism is well established.

 

The humoral response in Graves' disease leads to production of TG and microsomal TPO antibodies, but most importantly, as described in Chapter 10, B cells produce TSI, TBII and, in some, TSH blocking antibodies (35). TSI stimulate thyroid release of hormone primarily via cyclic AMP, although other pathways may also be activated by TSI in a proportion of patients. TSI are true cell stimulators and can even induce experimental goiter. However, the clinical picture in Graves’ disease will be a balance between the stimulation produced by TSI and the opposing effects of any TSH blocking antibodies which may be present.

 

Evidence also supports a role for T cell mediated immunity to thyroid antigens in Graves' disease, and against orbital antigens in patients with associated ophthalmopathy. We speculate that Graves' disease may be a condition representing a semistable balance between stimulatory, blocking, and cell‑lethal immune responses. Thus, TSI could cause thyroid hyperplasia and produce hyperthyroidism. Other antibodies might block the action of TSI either directly or, as in the case of NIS antibodies, indirectly, and prevent this hyperplastic response in some patients. Cytotoxic T cells will also gradually destroy cells and produce hypothyroidism either spontaneously or after therapy. It must be admitted that the etiology of ophthalmopathy still remains rather obscure, although the key role of cytokines in pathogenesis, causing fibroblast activation, seems firmly established.

 

RELATION TO OTHER DISEASES

Thyroid Cancer

Thyroid antibodies are present in increased prevalence (up to 32%) in patients with carcinoma of the thyroid, and usually are at low titer. Histologic evidence of thyroiditis is found in up to 26% of tumors. Histologic changes range from diffuse thyroiditis to focal collections of lymphocytes around the tumor or reactive lymphoid hyperplasia. Possibly release of antigens leads to increased thyroid autoimmunity. Some evidence suggests that patients who have thyroid antibodies have a better prognosis than antibody negative patients. Lymphoma and lymphosarcoma of the thyroid are associated with Hashimoto's thyroiditis (391), and there is compelling evidence that thyroiditis precedes development of the tumor. An increased frequency of carcinoma, especially of the papillary type, has been suggested in Hashimoto's thyroiditis but this relationship remains to be fully established (392).

 

Adolescent Goiter Enlargement of the thyroid during the second decade, accompanied by normal results of function tests, usually is labeled adolescent goiter. If the examination includes needle biopsy, an appreciable incidence of Hashimoto's thyroiditis is found (393) - up to 65%.   Eighty percent of these children with thyroiditis have a positive thyroid antibody test result. The parents of many of them have either overt thyroid disease or circulating thyroid antibodies. Hyperplasia, in response to an increased demand for thyroid hormone, and colloid involution are at the root of some of these goiters, but Hashimoto's thyroiditis is the most frequent explanation of adolescent goiter in iodine sufficient areas.

 

These illnesses, all similar, involve an acute exacerbation of thyroid autoimmunity occurring independent of, or following pregnancy in women, and in men. They are characterized by sequential inflammation-induced T4 and TG release, transient hypothyroidism, usually return to euthyroidism, and are discussed in Chapters 8 and 14. They are considered subtypes of Hashimoto's thyroiditis, and in the postpartum period, appear to result from release of the immunoregulatory effects of normal pregnancy (211).

 

Focal Thyroiditis

Focal lymphocytic infiltrations are frequently seen in Graves' disease, nodular goiter, nontoxic or colloid goiter, and thyroid carcinoma. The significance of these changes is not precisely known, but they correlate with positive antibody titers and may represent variations that do not differ qualitatively from thyroiditis.

 

Riedel’s Thyroiditis And Ig4 Disease

This rare thyroid disorder is associated with both Hashimoto’s thyroiditis and Graves’ disease and in addition many patients have evidence of fibrosis elsewhere, such as the retroperitoneum, lung, biliary tract and orbit. In one large series, 12 of 15 patients had positive TPO antibodies (389) It is now recognized that some of these patients with multifocal fibrosclerosis have IgG4-related sclerosing disease in which lymphocytes and IgG4-positive plasma cells infiltrate the affected tissues, especially the lacrimal gland, biliary tree and pancreas but the exact relationship of this entity to Riedels’ thyroiditis is unclear at present. There is a predominance of IgA rather than IgG4 in Riedel’s thyroiditis, but the effects of steroids may obscure the few analyses that have been undertaken (395, 396). However it does appear that Hashimoto’s thyroiditis can be divided into two discrete entities based on whether IgG4 plasma cells predominate in the thyroid infiltrate: in those individuals with IgG4 predominance, there is a greater male frequency, more rapid progression to hypothyroidism and more intense gland fibrosis (397). These studies have been predominantly undertaken in Japanese subjects. In a recent survey from Europe, only 12.5% of Hashimoto thyroid glands showed this feature: there was an association with younger age and male sex, and fibrosis was identified in 96 % of the IgG4-related cases but also in 18 % of the non-IgG4-related cases (397a). The authors note that unlike other forms of IgG4-related disease, the fibrosis is not accompanied by intense eosinophilia or obliterative phlebitis. IgG4 subclass thyroid autoantibodies display heritability in individuals with high levels of both TPO and TG antibodies; it is possible that more sophisticated analysis of autoantibody subtypes could lead to new methods to predict the natural history of disease (398).

 

Other Problems

An association between the occurrence of maternal antithyroid antibodies and recurrent abortion has been reported (399) and although this association has been disputed, a recent study showed clear evidence that the presence of TPO antibodies was associated with a 3-4-fold increased risk of miscarriage in women having in vitro fertilization (400). There is also an association between breast cancer and thyroid autoimmunity (401, 402) and between depression in middle-aged women and the presence of TPO antibodies (403). The nature of these associations is unclear; does thyroid autoimmunity predispose to such adverse events, or is the presence of thyroid autoimmunity simply a marker of a non-specific disturbance in the immune system due to whatever has caused miscarriage, cancer or depression? Having thyroid autoimmunity is not all bad news. Community-dwelling older women who have TG and TPO antibodies are less likely to be frail than those who are antibody-negative (404). Again the reason for this unexpected finding is unclear but it certainly warrants follow-up.

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Hypocalcemia: Diagnosis and Treatment

ABSTRACT

Hypocalcemia is an electrolyte derangement commonly encountered on surgical and medical services.  This derangement can result from a vast spectrum of disorders.  The condition may be transient, reversing with addressing the underlying cause expeditiously, or chronic and even lifelong, when due to a genetic disorder or the result of irreversible damage to the parathyroid glands after surgery or secondary to autoimmune destruction.  Adult and pediatric endocrinologists must carefully assess patients with hypocalcemia, factoring into that assessment clinical presentation and symptomatology, concomitant laboratory abnormalities, past medical and family histories, recent medications, and even genetic sequencing analysis on the patient or affected family members.  Critical initial laboratory testing involves measuring serum phosphate, magnesium, intact parathyroid hormone (PTH), 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D levels.  Further evaluation is directed by the clinical and laboratory profiles that emerge.  Significant fundamental insights into the molecular pathogenesis of several disorders that cause hypocalcemia have been made.  These insights involve the molecular etiologies for PTH resistance (i.e., the different subtypes of pseudohypoparathyroidism); the role of the AIRE (autoimmune regulator) protein in autoimmune hypoparathyroidism and in mediating central tolerance to self-antigens; and the molecular bases for different genetic forms of magnesium wasting (that in turn causes hypocalcemia) and hypoparathyroidism.  Genetic etiologies for hypoparathyroidism involve mutations in the calcium-sensing receptor, the G protein subunit alpha 11 that couples the receptor to downstream signaling molecules in parathyroid cells, transcription factors essential for parathyroid gland development, and the PTH molecule itself.  Treatment of hypocalcemia depends on severity and chronicity.  A calcium infusion is indicated for severe acute and or symptomatic hypocalcemia, while the standard mainstays of oral therapy are calcium supplements and activated vitamin D metabolites.  Finally, and importantly, despite the rarity of chronic hypoparathyroidism, there have been several clinical trials supporting the use of recombinant human PTH (1-84) in the management of patients not well controlled on standard treatment. These trials have led to the approval of PTH (1-84) by the US Food and Drug Administration for adults with this disorder not well regulated on the usual therapy.  Future research is being directed toward designing ideal treatment regimens with PTH (1-84) as well as developing a better understanding of the risks for post-surgical hypoparathyroidism, the most common etiology of hypoparathyroidism in adult patients. For complete coverage of this and all related ares of Endocrinology, please see our FREE web-book www.endotext.org.

CLINICAL PRESENTATION OF HYPOCALCEMIA

Hypocalcemia can present as an asymptomatic laboratory finding or as a severe, life-threatening condition (Table 1).  Distinguishing acute from chronic hypocalcemia and asymptomatic from severely symptomatic hypocalcemia is critical for determining appropriate therapy.  In the setting of acute hypocalcemia, rapid treatment may be necessary.  In contrast, chronic hypocalcemia may be well tolerated, but treatment is necessary to prevent long-term complications.

Table 1. Clinical Features Associated With Hypocalcemia
Neuromuscular irritability

  • Chvostek's sign
  • Trousseau's sign
  • Paresthesias
  • Tetany
  • Seizures (focal, petit mal, grand mal)
  • Muscle cramps
  • Muscle weakness
  • Laryngospasm
  • Bronchospasm
Neurological signs and symptoms

  • Extrapyramidal signs due to calcification of basal ganglia
  • Calcification of cerebral cortex or cerebellum
  • Personality disturbances
  • Irritability
  • Impaired intellectual ability
  • Nonspecific EEG changes
  • Increased intracranial pressure
  • Parkinsonism
  • Choreoathetosis
  • Dystonic spasms
Mental status

  • Confusion
  • Disorientation
  • Psychosis
  • Fatigue
  • Anxiety
  • Poor memory
  • Reduced concentration
Ectodermal changes

  • Dry skin
  • Coarse hair
  • Brittle nails
  • Alopecia
  • Enamel hypoplasia
  • Shortened premolar roots
  • Thickened lamina dura
  • Delayed tooth eruption
  • Increased dental caries
  • Atopic eczema
  • Exfoliative dermatitis
  • Psoriasis
  • Impetigo herpetiformis
Smooth muscle involvement

  • Dysphagia
  • Abdominal pain
  • Biliary colic
  • Dyspnea
  • Wheezing
Ophthalmologic manifestations

  • Subcapsular cataracts
  • Papilledema
Cardiac

  • Prolonged QT interval on EKG
  • Congestive heart failure
  • Cardiomyopathy
Adapted from Schafer AL and Shoback D:  Hypocalcemia:  definition, etiology, pathogenesis, diagnosis and management.  Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, C. J. Rosen (ed), John Wiley and Sons, Eighth Edition.  pp 572-578, 2013.

The hallmark of acute hypocalcemia is neuromuscular irritability.  Patients often complain of numbness and tingling in their fingertips, toes, and the perioral region.  Paresthesias of the extremities may occur, along with fatigue and anxiety.  Muscle cramps can be very painful and progress to carpal spasm or tetany.  In extreme cases of hypocalcemia, bronchospasm and laryngospasm with stridor may occur.  Muscle symptoms can be so severe as to present with a polymyositis-like picture with elevated muscle  isoenzymes.  These symptoms are corrected by calcium replacement.  Clinically, neuromuscular irritability can be demonstrated by eliciting Chvostek's or Trousseau's signs.  Chvostek's sign is elicited by tapping the skin over the facial nerve anterior to the external auditory meatus.  Ipsilateral contraction of the facial muscles occurs in individuals with hypocalcemia.  Chvostek's sign is also present in 10% of normal individuals.  Trousseau's sign is elicited by inflation of a blood pressure cuff to 20 mm Hg above the patient's systolic blood pressure for 3-5 minutes.  Carpal spasm presents as flexion of the wrist and of the metacarpal phalangeal joints, extension of the interphalangeal joints, and abduction of the thumb.  It can be very painful.

Acute hypocalcemia may have cardiac manifestations.  Prolongation of the QT-interval due to lengthening of the ST-segment on electrocardiogram is fairly common in hypocalcemic patients.  T-waves are abnormal in approximately 50% of patients (1).  A pattern of acute anteroseptal injury on EKG without infarction has been associated with hypocalcemia and other electrolyte abnormalities (2).  Hypomagnesemia in concert with hypocalcemia may magnify the EKG abnormalities.  Rarely, congestive heart failure may occur (1,3,4).  Reversible cardiomyopathy due to hypocalcemia has been reported (5).  In patients with mild, asymptomatic hypocalcemia, calcium replacement can result in improved cardiac output, and exercise tolerance (6).

Chronic hypocalcemia may have an entirely different presentation (7,8).  Patients with idiopathic hypoparathyroidism or pseudohypoparathyroidism may develop neurological complications, including calcifications of the basal ganglia and other areas of the brain (9,10), and extrapyramidal symptoms.  Grand mal, petit mal, or focal seizures have been described. Increased intracranial pressure and papilledema may be present.  If the patient has pre-existing subclinical epilepsy, hypocalcemia may lower the excitation threshold for seizures (11).  Electroencephalographic changes may be acute and nonspecific or present with distinct changes in the electroencephalogram (EEG).  EEG changes may be present with or without symptoms of hypocalcemia.  The relationship between calcification of basal ganglia (9,10-12), cerebral cortex, or cerebellum with pre-existing epileptic or convulsive disorders is not well understood.

Epidermal changes are frequently found in patients with chronic hypocalcemia.  These include dry skin, coarse hair, and brittle nails.  If hypocalcemia has occurred prior to the age of 5, dental abnormalities may be present.  Dental abnormalities include enamel hypoplasia, defects in dentin, shortened premolar roots, thickened lamina dura, delayed tooth eruption, and an increase in the number of dental caries.  Alopecia has been noted following surgically-induced hypoparathyroidism and is also associated with autoimmune hypoparathyroidism.  Other skin lesions reported in patients with hypoparathyroidism include atopic eczema, exfoliative dermatitis, impetigo herpetiformis, and psoriasis.  Restoration of normocalcemia is reported to improve these skin disorders.

Changes in smooth muscle function with low serum levels of calcium may cause irritability of the autonomic ganglia and can result in dysphagia, abdominal pain, biliary colic, wheezing, and dyspnea.  Subscapular cataracts occur in chronic, longstanding hypocalcemia (12) and with treatment, especially when the calcium x phosphate product is chronically elevated.  Paravertebral ligamentous ossification has been noted in 50% of cases with hypoparathyroidism, and antalgic gait may be noted. In some cases of chronic hypoparathyroidism, psychoses, organic brain syndrome, and subnormal intelligence have been noted.  Treatment of the hypocalcemia may improve mental functioning and personality, but amelioration of psychiatric symptoms is inconsistent.  Delayed development, subnormal IQ, and poor cognitive function could also be a component of a syndrome that includes hypoparathyroidism as one of its features (7,8).  This is critically important to consider in young patients being evaluated for the condition.  In the elderly population, disorientation or confusion may be manifestations of hypocalcemia.

ETIOLOGY OF HYPOCALCEMIA

Hypocalcemia can result from disorders of vitamin D metabolism and action, hypoparathyroidism, resistance to parathyroid hormone (PTH), or a number of other conditions (Table 2) (13,14).  These topics are discussed in separate sections below.

Table 2. Causes of Hypocalcemia
Inadequate vitamin D production and action

  • Nutritional deficiency
  • Lack of sunlight exposure
  • Malabsorption
  • Post-gastric bypass surgery
  • End-stage liver disease and cirrhosis
  • Chronic kidney disease
  • Vitamin D-dependent rickets type 1 and type 2
Inadequate PTH production/Hypoparathyroidism—see Table 3
Functional hypoparathyroidism

  • Magnesium depletion
  • Magnesium excess
PTH resistance - Pseudohypoparathyroidism
Miscellaneous etiologies

Neonatal hypocalcemia

Hyperphosphatemia

  • Phosphate retention in acute or chronic renal failure
  • Excess phosphate absorption caused by enemas, oral supplements
  • Massive phosphate release caused by tumor lysis or crush injury

Drugs

  • Intravenous bisphosphonate therapy or denosumab therapy – especially in patients with vitamin D insufficiency or deficiency
  • Foscarnet

Rapid transfusion of large volumes of citrate-containing blood

Acute critical illness

“Hungry bone syndrome”

  • Post-thyroidectomy for Grave’s disease
  • Post-parathyroidectomy

Osteoblastic metastases

Acute pancreatitis

Rhabdomyolysis

Mitochondrial gene defects
Adapted from:  Schafer AL, Shoback, D:  Hypocalcemia:  definition, etiology, pathogenesis, diagnosis and management.  Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, C. J. Rosen (ed), John Wiley and Sons, Eighth edition, pp 572-578, 2013.

VITAMIN D DISORDERS RESULTING IN HYPOCALCEMIA

Both inherited and acquired disorders of vitamin D and its metabolism may be associated with hypocalcemia (15,16).  Data from the 2005-2006 National Health and Nutrition Examination Survey (NHANES) estimates that the true prevalence of vitamin D deficiency (25-hydroxy vitamin D level ≤20 ng/mL [50 nmol/L]) is 41.5% (17).

Nutritional Vitamin D Deficiency

The fortification of milk, cereals, breads, and other foods with vitamin D and the use of supplements are why there are so few cases of vitamin D deficiency in children in the United States.  Vitamin D deficiency has been recognized in the United States in children who have restricted diets or specialized diets (18).   In countries that do not fortify foods, childhood vitamin D deficiency is more common.  Vitamin D deficiency is recognized as a worldwide problem in older adults as well (15,16).  Exclusively breastfed infants are at high risk for vitamin D deficiency, as there is little vitamin D in human milk.

Lack of Sunlight Absorption

Decreased synthesis of vitamin D in the skin is not uncommon and may be due to the lack of sun exposure due to excessive sunscreen usage, skin pigmentation, protective clothing, winter season, increased latitude or aging.  Patients who are unable to be exposed to solar ultraviolet B radiation are at risk for vitamin D deficiency.  In cultures where traditional dress includes long garments, hoods or veils, this may result in reduced sun exposure and vitamin D deficiency (19,20).

Malabsorption

Fat malabsorption accompanying hepatic dysfunction, sprue, Whipple's disease, Crohn's disease, and gastric bypass surgery may result in intestinal malabsorption of vitamin D and result in lower concentrations of circulating 25-hydroxy (25[OH]) vitamin D (21).

Liver Disease

Liver disease is not a common cause of inadequate 25(OH) vitamin D levels, as over 90% of the liver has to be dysfunctional before the 25(OH) vitamin D drops to subnormal levels.  However, intestinal fat malabsorption occurs in both parenchymal and cholestatic liver disease, and this may cause vitamin D deficiency.  Certain anticonvulsant drugs can alter the kinetics and hepatic metabolism of 25(OH) vitamin D.  Vitamin D deficiency is usually easily corrected by additional vitamin D administration.

Renal Disease

Nephrotic syndrome with excretion of large amounts of protein has also been associated with lower levels of 25(OH) vitamin D and may be due to excretion of vitamin D binding protein.  Chronic renal failure with a reduction in glomerular filtration rate to <30% of normal may present with decreased production of 1,25-(OH)2 vitamin D.  In the setting of chronic renal failure, hyperphosphatemia and secondary hyperparathyroidism occur.  Serum calcium tends to be in the low normal range.  Hypocalcemia is usually not observed in the presence of low levels of 25(OH) vitamin D due to the compensatory rise of PTH, which will mobilize the calcium from skeletal stores.  Hypocalcemia only occurs when these stores are severely depleted.

Inherited Disorders of Vitamin D Metabolism and Action

Inherited disorders can result from the deficiency in the renal production of 1,25-(OH)2  vitamin D (vitamin D-dependent rickets type 1) or a defect in the vitamin D receptor (VDR) (vitamin D-dependent rickets type 2).  Both disorders are exceedingly rare.  Patients with vitamin D-dependent rickets (VDDR) type 1 usually present with rickets, hypocalcemia, hypophosphatemia, elevated alkaline phosphatase, and as a result of their hypocalcemia, secondary hyperparathyroidism.  Because type 1 VDDR results from a defect in the renal production of 1-alpha-hydroxylase (15,16), 1,25-(OH)2 vitamin D levels are decreased or undetectable.  In contrast, patients with VDDR type 2 have disrupted production or impaired function of the VDR, resulting in end-organ resistance to 1,25-(OH)2 vitamin D. The clinical presentation includes severe hypocalcemia, hypophosphatemia, and resultant secondary hyperparathyroidism with elevated alkaline phosphatase and rickets.  In this disorder, however, the constant stimulation of renal 1-alpha-hydroxylase from the chronic hypocalcemia, hypophosphatemia, and increased PTH levels results in elevated serum levels of 1,25-(OH)2 vitamin D (15,16).

HYPOPARATHYROIDISM

The causes of hypoparathyroidism are summarized in Table 3 (7,8,22).

Postsurgical Hypoparathyroidism and Hypocalcemia

One of the most common causes of hypocalcemia is inadvertent removal of, damage to, or inadvertent devascularization of the parathyroid glands during surgery for parathyroid or thyroid disease. This may be short-term, in which case it is parathyroid gland “stunning.”  If persistent (beyond 6 months), postoperative permanent hypoparathyroidism is the diagnosis.  Other causes of postoperative hypocalcemia include the “hungry bone syndrome” with low serum calcium levels resulting from remineralization of the bone, as the stimulus for high bone turnover (e.g., high PTH or thyroid hormone levels) is removed are discussed below.  There may be edema in the surgical field resulting in hypocalcemia due to the surgery itself, which may remit as the swelling subsides. The vascular supply to the remaining parathyroid glands may be compromised resulting in hypocalcemia.  In chronic hyperparathyroidism, the dominant hyperactive parathyroid adenoma may have suppressed the remaining normal parathyroid glands.  After removal of the adenoma, the remaining suppressed parathyroid glands will eventually regain their functional capacity, although this may take time.

After surgery for primary hyperparathyroidism, hypocalcemia can be a significant source of morbidity.  Risk factors for the development of early post-operative hypocalcemia have been assessed by several groups (23-27).  In one study, patients with hypocalcemia within the 4 days after surgery were more likely to have had higher pre-operative levels of serum osteocalcin, bilateral (as opposed to unilateral) surgery, and a history of cardiovascular disease (23).  Other studies have identified pre-operative vitamin D deficiency [25 (OH) vitamin D level <20 ng/mL] (24) and a drop in intraoperative PTH level of >80% (25) as significant risk factors for post-operative hypocalcemia.  Employing more specific case definitions, one group classified cases of post-operative hypocalcemia as either “hungry bone syndrome” (hypocalcemia and hypophosphatemia) or hypoparathyroidism (hypocalcemia and hyperphosphatemia).  Independent risk factors for the development of hungry bone syndrome were pre-operative alkaline phosphatase level, blood urea nitrogen level, age, and parathyroid adenoma volume (26,27).  Additional research is needed to understand better the risks for post-surgical hypoparathyroidism.

While the gold standard for the surgical treatment of primary hyperparathyroidism was once  bilateral neck exploration, advances in surgical technique, pre-operative localization capabilities, and intra-operative PTH monitoring have allowed for more limited parathyroid surgical approaches, including unilateral neck exploration and minimally invasive parathyroidectomy with a high degree of success.  Unilateral neck exploration was shown to cause less early severe symptomatic hypocalcemia than bilateral neck exploration in a randomized controlled trial (28).  Intraoperative PTH monitoring has also been shown to reduce the rate of complications (including hypoparathyroidism) in patients undergoing reoperations for primary hyperparathyroidism (29).

Neck explorations for reasons other than hyperparathyroidism are associated with hypocalcemia. Thyroid surgery, for example, is associated with hypocalcemia, presumably due to surgical disruption or vascular compromise of the parathyroid glands.  Transient hypocalcemia is observed in 16-55% of total thyroidectomy cases (30,31).  One group recently reported that of the 50% of patients who developed post-operative hypocalcemia, hypoparathyroidism persisted beyond one month in 38% (30).  In another retrospective study, transient hypocalcemia was observed in 35% of patients undergoing total thyroidectomy, 3% had chronic hypocalcemia 6 months post-operatively, and 1.4% had permanent hypoparathyroidism 2 years post-operatively (31).  The type of surgery performed is associated with the risk of developing hypocalcemia.  For example, risk of hypocalcemia is higher after completion thyroidectomy or total thyroidectomy with node dissection (30).  Transient hypocalcemia was observed more frequently after thyroidectomy for Graves’ disease than for nontoxic multinodular goiter, although incidence of permanent hypoparathyroidism was not different between groups (32).

With hospital stays after surgery typically short, it is important to know how long to monitor a postoperative thyroid or parathyroid surgical patient for hypocalcemia.  Bentrem et al. performed a chart review of 120 patients undergoing total/near-total thyroidectomy and/or parathyroidectomy, and they found that a low ionized calcium level 16 hours post-operatively was sufficient to identify patients at risk for post-operative hypoparathyroidism (33).  Another review of outcomes following thyroidectomy reported that a low intact PTH level 1 hour post-operatively predicted symptomatic hypocalcemia with an 80-87% diagnostic accuracy, and none of the patients studied experienced symptomatic hypocalcemia when the 1-hour post-op PTH level was greater than 10 pg/mL.  In the same study, a low ionized calcium level the morning after surgery predicted biochemical hypocalcemia with 78-95% diagnostic accuracy (34).

Another study attempted to use both perioperative PTH levels and serum calcium levels to predict hypocalcemia after total or near-total thyroidectomy (31).  PTH levels after surgical resection of the second thyroid lobe, age, and number of parathyroid glands identified intraoperatively were independently associated with decreased serum calcium levels measured at the nadir on postoperative day 1 or 2.  Low levels of intraoperative PTH and serum calcium less than 2.00 mmol/L predicted biochemical hypocalcemia with a similar sensitivity (90% vs 90%) and specificity (75% vs 82%).

Developmental Disorders of the Parathyroid Gland

Developmental abnormalities in the third and fourth pharyngeal pouches result in the DiGeorge syndrome (35,36). Aplasia or hyperplasia of the thymus, aplasia or hypoplasia of the parathyroid glands and associated conotruncal cardiac malformation are hallmarks of this disorder. Severe hypocalcemia resulting in seizures and tetany can occur. Immunodeficiency due to thymic defects and hypoplasia can lead to  recurrent infections. Other common associated cardiac defects include tetralogy of Fallot and truncus arteriosus. Abnormal patterns of aortic arch arteries, improper alignment of the aortic pulmonary outflow vessels, and defects in septation of the ventricles can occur. Occasionally, partial forms of the DiGeorge syndrome occur, and an EDTA challenge test may be necessary to confirm the diagnosis and unmask the hypoparathyroidism.  Fluorescence in situ hybridization is one test that can be used to make the diagnosis.

DiGeorge syndrome is the most frequent contiguous gene deletion syndrome in humans and occurs in 1 in 4000 live births.  The microdeletion is found in chromosome 22q11.2. The phenotypic presentation is variable.  In a small cohort of 16 patients who met clinical criteria for DiGeorge syndrome, correlation was made with a microdeletion on chromosome 22q11.2 and immunodeficiency characterized by recurrent respiratory infections and absent thymus gland.  Cell-mediated immunodeficiency and  infections characterize this disorder (37).  Other studies suggest that newborns with DiGeorge syndrome have preserved T-cell function, but the numbers of T cells are decreased.  There is variable improvement in peripheral blood T-cell counts as the patients increase in age.  A gene involved in the the features of the DiGeorge syndrome or sequence is the transcription factor TBX1  (35,38-40).  Transgenic mice  haploinsufficient for TBX1 demonstate some  components of the DiGeorge syndrome (41). Complete knockout of the gene is lethal in mice and results in a high incidence of cardiac outflow tract abnormalities (42).

Table 3. Differential Diagnosis of Hypoparathyroidism
 Iatrogenic

  • Post I-131 radiation
  • Surgically induced
Infiltrative/Destructive Diseases

  • Hemochromatosis
  • Iron overload due to transfusion dependence in thalassemia
  • Wilson's disease
  • Metastatic carcinoma
Neonatal

  • Maternal hyperparathyroidism
  • Maternal FHH
Autoimmune

  • Isolated
  • Autoimmune polyendocrine syndrome type 1 (APS-1)
Genetic or developmental disorders

  • DiGeorge Syndrome
  • Activating calcium-sensing receptor mutation
  • Hypoparathyroidism, deafness, and renal anomalies (HDR) syndrome
  • Hypoparathyroidism-retardation-dysmorphism (HRD) syndrome
  • Mitochondrial gene defects
Adapted from Schafer AL and Shoback D:  Hypocalcemia:  definition, etiology, pathogenesis, diagnosis and management.  Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, C. J. Rosen (ed), John Wiley and Sons, Eighth Edition, pp 572-578, 2013.

Genetic Etiologies of Hypoparathyroidism

Hypoparathyroidism in Association with Syndromes 

Hypoparathyroidism has been described in association with specific disorders such as the Kearns-Sayre syndrome, which presents with heart block, retinitis pigmentosa and ophthalmoplegia. The Kenny-Caffey syndrome has also been associated with hypoparathyroidism and includes medullary stenosis of the long bones and growth retardation (43).

Familial Isolated Hypoparathyroidism

A few rare cases of familial isolated hypoparathyroidism have been described and are heterogeneous in their modes of inheritance:  X-linked recessive, autosomal recessive, and autosomal dominant (7,8,44-51).

X-linked recessive idiopathic hypoparathyroidism was reported in 2 related kindreds from Missouri, USA (47). Epilepsy and hypocalcemia were discovered in affected males during infancy. The disorder appears to be due to an isolated congenital defect of the parathyroid gland development, and linkage studies established this recessive idiopathic hypoparathyroidism was linked to a gene Xq26-q27 (44).

In one kindred of autosomal dominant isolated hypoparathyroidism, the primary molecular defect was a mutation in the prepro-PTH gene (48).  A highly charged arginine residue replaced cysteine within the hydrophobic core of the prepro-PTH signal peptide sequence. This sequence is critical for the movement of newly formed prepro-PTH through the endoplastic reticulum. The inability to progress through the cell secretory pathway could explain why these patients lacked expression of bioactive PTH. Other mutations in the signal peptide of prepro-PTH gene have also been reported (49). Another example leading to apoptosis in the affected cells was also described.

An autosomal recessive mode of inheritance has been described in isolated hypoparathyroidism in a Bangladesh-Asian kindred (50). The prepro-PTH gene mutation resulted in a substitution of G to C in the first nucleotide position of the prepro-PTH intron 2. This mutation resulted in an aberrant prepro-PTH mRNA so that the entire signal sequence would be absent preventing PTH secretion. The patients were homozygous for the mutant allele and were the product of a consanguineous marriage.

A recent case of hypoparathyroidism was described in which the affected patient was found to have a homozygous mutation in PTH at residue 25 (arginine substituted with cysteine).  The proband (from among 3 affected siblings in the same family) produced very high or frankly low levels of PTH, depending on the immunoassay used to measure the hormone (51).  This made the diagnosis confusing as the possibility of pseudohypoparathyroidism was raised because the patient had both hypocalcemia and hyperphosphatemia.  The circulating hormone displayed dramatically impaired ability to activate the PTH/PTH-related protein (PTHrP) type 1 receptor (PTH/PTHrP-R1) in transfected cells.

Ding and coworkers described a large kindred with hypoparathyroidism due to a homozygous mutation in the glial cells missing-2 (GCM2) transcription factor mapped to 6p23-24 (52).  GCM2 is expressed in parathyroid cells, and its expression is essential to the development of a PTH-secreting cell.  Additional kindreds with other loss of function mutations in GCM2 have been described (53-56), further supporting the importance of this molecule in parathyroid gland development and in considering it diagnostically in kindreds affected by autosomal recessive hypoparathyroidism.  There have been additional patients with GCM2 mutations in which the mutant transcription factor behaves as a dominant negative and the disease is inherited in an autosomal dominant manner (54).

Mutations Affecting the Extracellular Calcium-Sensing Receptor and Other Molecules in Its Signaling Pathway

The calcium-sensing receptor (CaSR) is critical to the regulation of PTH secretion and parathyroid cell function. This receptor is a member of the G-protein coupled receptor superfamily and is strongly expressed in the kidney and parathyroid gland as well as other tissues.  If hypercalcemia occurs, the CaSR activates the G-protein signaling pathway, resulting in increased intracellular calcium levels and the suppression of PTH gene transcription.  Heterozygous inactivating mutations of Casr result in familial benign hypocalciuric hypercalcemia.  Homozygous inactivating mutations cause neonatal severe hyperparathyroidism.  Autosomal dominant and sporadic hypoparathyroidism can result from gain of function mutations of the Casr (57).  The clinical presentation in these patients ranges from asymptomatic to life-threatening hypocalcemia.  PTH levels are frankly low to low normal, and hypercalciuria may be present out of proportion to the degree of hypocalcemia.  Activating mutations typically are in the amino-terminal extracellular domain of the receptor.  Hypercalciuria can be exacerbated resulting in nephrocalcinosis and impairment of renal function if patients are treated with vitamin D or its analogues.  Casr mutations are considered to be second to post-surgical hypoparathyroidism in frequency as a cause of hypoparathyroidism in the adult.

Recently mutations have been described in the gene encoding the G protein alpha subunit (Ga11 or GNA11) that couples the CaSR to downstream signaling molecules and specifically to activation of intracellular calcium mobilization (58,59).  The patients described have heterozygous point mutations in Ga11 with these substitutions (Arg181Gln; Phe341Leu, Arg60Cys, and Ser211Trp), and they demonstrate mild hypocalcemia clinically.  When these mutant G-protein alpha subunits are expressed in transfected cells, there is an apparent gain of function in extracellular calcium-activated signal transduction compatible with the suppression of PTH secretion in vivo.

Hypoparathyroidism, Sensory Neural Deafness, Renal Dysplasia Syndrome

The HDR (hypoparathyroidism, sensory neural deafness and renal dysplasia) syndrome is a rare autosomal dominant inherited syndrome which includes a variety of renal anomalies and varying degrees of hearing deficits as well as hypoparathyroidism (60-65).  Linkage and mutational analysis have identified the gene responsible for the syndrome as GATA3, which encodes a zinc finger transcription factor involved in embryonic development of the parathyroid glands, kidney and otic vesicle (60).  Patients are usually asymptomatic with inappropriately normal, given their level of hypocalcemia, or frankly low PTH levels.

Autoimmune Hypoparathyroidism

The classic association of autoimmune parathyroid disease, adrenal disease and mucocutaneous candidiasis is termed autoimmune polyendocrine syndrome type 1 (APS-1) (66-69) (Table 4).  The presence of 2 of the 3 classic endocrine features of the disease establishes the diagnosis.  APS-1 is typically inherited in an autosomal recessive pattern, and most cases are due to mutations in AIRE (autoimmune regulator of endocrine function), a gene located on chromosome 21q22.3.  This gene encodes a transcription factor which is involved in the mechanisms of central tolerance to self-antigens in the thymus.  APS-1 has also been reported in association with the presence of a thymoma (70).  APS-1 has the onset of at least one disease component typically in early childhood with almost 100% penetrance. There are other autoimmune disorders associated with this syndrome including gonadal failure, hepatitis, malabsorption, type 1 diabetes, alopecia (totalis or aerata) and vitiligo.

Table 4. Features of Autoimmune Polyendocrine Syndrome Type I
Classic Triad

  • Hypoparathyroidism
  • Candidiasis
  • Adrenal insufficiency (Addison's disease)
Associated with 2 or 3 of the following

  • Type 1 diabetes mellitus
  • Primary hypogonadism (especially ovarian failure)
  • Autoimmune thyroid disease
  • Chronic active hepatitis
  • Alopecia (totalis or aerata)
  • Vitiligo
Bilezikian J et al:  Hypoparathyroidism in the adult:  epidemiology, diagnosis, pathophysiology, target organ involvement, treatment and challenges for future research.  J Bone Min Res 26: 2317-37, 2011; and Betterle C, Garelli S, Presotto F:  Diagnosis and classification of autoimmune parathyroid disease.  Autoimm Rev 13: 417-22, 2014.

The most common age of onset of APS-1 is 8 years, and it occurs equally in males and females. There is a temporal sequence of development of APS-1 with chronic cutaneous candidiasis frequently the herald event. Hypoparathyroidism may occur next and may present as a seizure during an acute illness. Addison's disease (adrenal insufficiency) follows. These disorders occur at the average of 5, 9 and 14 years of age, respectively (71). There may be variation within individual families in the clinical presentation.

As many as 50% of patients with APS-1 experience keratoconjunctivitis (72). Keratoconjunctivitis can intermittently recur but also can be chronic and disabling. Some propose that this is a hypersensitivity response to the candidiasis as opposed to a component of APS-1 per se. Histopathological features have been evaluated in corneal buttons obtained at keratoplasties. Severe atrophy of the corneal epithelium was evident. The anterior corneal layers, epithelium, the Bowman's membrane and anterior corneal stoma only are affected. The anterior corneal stroma is replaced by scar tissue with features of chronic inflammation consisting of lymphocytes and plasma cells (73). There are reported cases of keratoconjunctivitis in the absence of a candida infection. Other ocular abnormalities include retinitis pigmentosa, exotropia, pseudo-optosis, cataracts, papilledema, strabismus, recurrent blepharitis, and loss of eyebrows and eyelashes (74).

In children with APS-1, other ectodermal disorders have been described, but they are also found in patients with non-autoimmune mediated hypocalcemia. These include alopecia totalis or areata, piebaldism, vitiligo, cataracts and papilledema. In APS-1, approximately 20 to 30% of individuals have some form of alopecia. Vitiligo has been reported in 10% of affected individuals (66-69).

Another immune problem associated with APS-1 is delayed sensitivity due to T-cell abnormalities.  Dental dysplasia, including enamel hypoplasia, may predate the onset of hypoparathyroidism.  Fifteen percent of affected individuals have gastric atrophy and pernicious anemia. Ten percent of APS-1 individuals develop chronic active hepatitis with cirrhosis which may be a significant cause of mortality.  Due to intestinal malabsorption, management may be difficult due to malabsorption of calcium and vitamin D.

Autoantibodies occur routinely in APS-1 and are its hallmark.  One group studied individuals with hypoparathyroidism and APS-1 and showed that >50% of these patients demonstrated antibodies to NALP5 (nacht leucine repeat protein 5), a molecule potentially involved in signal transduction in parathyroid cells (75,76).  One of the strongest markers for the presence of APS-1, in patients with one or more features of the disease and even prior to disease onset in high-risk individuals, is the presence of neutralizing anti-interferon alpha or omega antibodies (69,77,78).  These antibodies are thought to play a pathogenic role in the disease features including impaired mucosal immune responses and recognition of self by the thymus based on findings in mouse models (79,80).

Treatment of patients with hypoparathyroidism due to an autoimmune mechanism can be difficult. In children with autoimmune hypoparathyroidism, the symptoms of Addison's disease can be masked, and lack of glucocorticoid therapy can result in a fatal outcome.  In adrenocortical insufficiency, serum calcium concentrations may be elevated and decrease rapidly to hypocalcemic levels after the introduction of corticosteroid replacement therapy.  The introduction of hormone replacement therapy for premature ovarian failure can also lead to a diminution in serum calcium levels.  Vitamin D malabsorption can occur secondary to diarrhea due to intestinal malabsorption.  Patients with features of APS-1 should be screened regularly for associated autoimmune abnormalities.  It is recommended that healthy siblings be screened in the first decade of life biochemically or preferable with genetic testing or testing for the presence of autoantibodies.  Several forms of autoimmune hypoparathyroidism can occur either with or without other endocrinopathies (i.e., as isolated hypoparathyroidism) (69).

PSEUDOHYPOPARATHYROIDISM

Pathophysiology

Pseudohypoparathyroidism (PHP) was initially described by Dr. Fuller Albright and colleagues in 1942, and the disorders involved according to current classification are shown in Table 5. These patients have clinical and biochemical features consistent with hypoparathyroidism but have neither a hypercalcemic nor phosphaturic response to exogenous parathyroid extract.  PTH resistance is the biochemical hallmark of PHP, and in untreated patients, serum levels of PTH are elevated sometimes very markedly so.  Biological resistance to PTH causes inadequate flow of calcium into extracellular fluids and deficient phosphate excretion by the kidney.  Hypocalcemia is due to impaired mobilization of calcium from bone, reduced intestinal absorption of calcium, and increased urinary losses.

Table 5. Comparison of Features of Pseudohypoparathyroidism (PHP) and Pseudopseudohypoparathyroidism (PPHP)

PHP 1a PHP 1b PHP 2  PPHP
AHO + - - +
Serum calcium  ↓   ↓  ↓ NL
cAMP Response to PTH   ↓   ↓   ↓ NL
Urinary Phosphate   ↓   ↓ ( ↓ ) NL NL
Response to PTH
Hormone Resistance PTH, TSH and other Gs-alpha coupled  hormones PTH target tissues only PTH target tissues only None
Molecular defect Reduced functional Gs-alpha levels Abnormalities in Gs-alpha gene transcription Unknown Gs-alpha
AHO = Albright’s hereditary osteodystrophy

PTH = parathyroid hormone
NL = normal
R = receptor
Gs alpha = alpha subunit of the stimulatory guanine nucleotide binding protein
+ = present; = decreased
PHP = pseudohypoparathyroidism; PPHP = pseudopseudohypoparathyroidism

Adapted from Juppner H and Bastepe M:  Pseudohypoparathyroidism.  Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, C. J. Rosen (ed), John Wiley and Sons, Eighth Edition, pp 590-600, 2013.

In PHP 1, there is autosomal dominant inheritance of the mutation.  However, there is imprinting of the GNAS locus with silencing of the paternal allele, which influences the expression of the phenotype.  In PHP 1a, if the mutation is inherited from the mother, the disease in the offspring manifests as PHP 1a with low serum calcium, high serum phosphate and high PTH levels plus the presence of features of Albright’s hereditary osteodystrophy (AHO) (described below).  If the mutation is passed down from the father, that allele is silenced in the proximal tubules of the kidney (through imprinting). The normal sequence from the maternal allele is expressed, and the biochemical and mineral abnormalities are absent.  Only the AHO phenotype is present in such an individual.  This is called pseudopseudohypoparathyroidism (PPHP).

The hallmark of the diagnosis of PHP 1a is a markedly attenuated urinary cyclic AMP response to exogenous administration of PTH. The resistance to PTH is caused by a defect in the PTH hormone receptor-adenylate cyclase complex that produces cyclic AMP. Receptors communicate with the catalytic unit of adenylate cyclase through guanyl nucleotide binding regulatory proteins (G-proteins). There are many G-proteins, some of which are stimulatory (Gs) or inhibitory (Gi) to receptor dependent activation of adenylate cyclase.

The original description by Albright of PHP focused on PTH resistance.  However, patients with PHP 1a may display partial resistance to other hormones and present with short stature, hypothyroidism, hypogonadism, and mental retardation (81-84). Patients with PHP 1a also can have ovulatory, gustatory and auditory dysfunction. The defect is in Gs alpha, a ubiquitous protein required for functional cyclic AMP production and the amounts of G-protein present can be measured in plasma membrane of accessible cells. Patients with PHP 1a have an ~50% reduction in Gs alpha in all tissues studied. A variety of mutations in the Gs alpha gene (GNAS) have been identified by sequencing analysis.

Patients with PHP also have a constellation of developmental and somatic defects that are referred to as AHO.  Short stature, round facies, brachydactyly (Figure), obesity, and subcutaneous calcifications are classic features of AHO (81-84). Phenotypes can vary and presentations may be subtle.  A variant phenotype of patients with features of AHO but lacking hormone resistance is considered to have PPHP.  These patients have normal serum calcium levels.

 
Figure. X-rays demonstrate congenital shortening of the third, fourth and fifth metacarpals on the right hand. This patient was a 40-year-old female with normal serum calcium, phosphate and alkaline phosphatase. Her findings are consistent with pseudopseudohypoparathyroidism.

Individuals classified as PHP 1b typically lack features of AHO. Typically patients with PHP1b have the same biochemical presentation with hypocalcemia and hyperphosphatemia with elevated levels of PTH. They lack the appropriate increase in urinary cyclic AMP in response to PTH infusion. These patients have been studied extensively and in affected kindreds the disorder has been mapped to the GNAS1 locus (81-84).  The disease is thought to be due to abnormalities in DNA methylation of the GNAS1 promoter, altering levels of Gs alpha expression in key target tissues.  There is also a group with specific promoter DNA methylation defects which lead to a reduction in GNAS1 transcription due to the loss of methylation.

In PHP 2, there is reduced phosphaturic response to administration of exogenous PTH, but a normal increase in urinary cyclic AMP excretion.  PHP 2 is a clinically heterogenous syndrome. The molecular pathophysiology is poorly understood. These patients may demonstrate normal cyclic AMP but absent phosphaturic responses to PTH. Hypocalcemia, decreased calcium mobilization from bone in response to PTH, and decreased serum 1,25-(OH)2 vitamin D are the prominent features of PTH resistance.

Signs and Symptoms

Patients with PHP can present with signs and symptoms of hypocalcemia.  Neuromuscular irritability, with an onset in mid childhood (average age ~8 years), has been described.  Other patients may remain asymptomatic and not be diagnosed with PHP until adulthood. Symptoms of hypocalcemia such as carpopedal spasms, paresthesias, convulsions, muscle cramps and stridor can all be found in PHP.  In some severe cases, life-threatening laryngeal spasm has been reported in children.  Posterior subscapular cataracts can occur in patients with longstanding untreated hypocalcemia.  Basal ganglia calcifications have been found in 50% of patients with PHP and may produce extrapyramidal movement disorders. In some patients with PHP, normal serum calcium levels may be present.  Hypocalcemia may develop insidiously and be preceded by increasing levels of serum PTH.  Psychiatric problems such as depression, paranoia, psychosis and delusions have been described in the presence of hypocalcemia.  Cognitive defects such as mental retardation and memory impairment have been encountered.  Dental defects include dental and enamel hypoplasia, blunted tooth root development, and delay of tooth eruption.

In patients with radiographic evidence of osteitis fibrosis cystica, there is clearly skeletal sensitivity to PTH.  In such individuals, serum alkaline phosphatase levels and biochemical markers of bone turnover, especially of bone resorption, may be increased.  Bone mineral density (BMD) may be normal or decreased.  Especially in those with skeletal sensitivity to PTH, there may be decreased BMD, especially at cortical sites.

Reproductive abnormalities have been described. In patients with AHO and PHP, 76% of patients were oligomenorrheic or amenorrheic, had delayed sexual development, and only 2 of 17 patients had a history of pregnancy. All of these patients had typical PTH resistance and a 50% reduction in Gs alpha activity. These patients were mildly hypoestrogenemic with normal to slightly elevated serum gonadotropin levels.  It was proposed that the reproductive dysfunction was due to partial resistance to gonadotropins since administration of the synthetic GnRH analogue produced normal FSH and LH responses (85).

Phenotypic variability is another problem with the diagnosis of AHO even among affected individuals in a kindred.  Nonspecific physical characteristics include short stature and obesity.  Brachydactyly is a more specific criterion and can be diagnosed by physical or radiographic examination.  In AHO, the most commonly shortened bones are the distal phalanx of the thumb and the fourth metacarpal.  On a radiograph of a normal hand, a line drawn tangential to the distal ends of the fourth and fifth metacarpals passes distal to the head of the third metacarpal.  Archibald’s sign is present if the line instead runs through the distal end of the third metacarpal (86).   Heterotopic ossification can also occur, but little is known about the cause.

Diagnosis and Management of Pseudohypoparathyroidism

Patients who present with hypoparathyroidism and an elevated serum level of PTH should be suspected for possible PHP. The typical phenotype of AHO (short stature, brachydactyly of the hands and feet, subcutaneous calcifications) should suggest PHP 1a or PPHP. Hypocalcemia, hyperphosphatemia, elevated levels of PTH and normal renal function lead one to be highly suspicious of the diagnosis. A positive family history lends further support.

The biochemical hallmark is failure of bone and kidney to respond adequately to PTH.  The classic tests of Ellsworth and Howard and of Chase, Melson, and Aurbach (87) involved administration of 200 to 300 USP units of bovine parathyroid extract with measurements of urinary cyclic AMP and phosphate.  The test hormone is now synthetic human PTH (1-34).  After administration of human PTH (1-34), normal subjects and patients with hypoparathyroidism display 10- to 20-fold increases in urinary cyclic AMP.  Patients with PHP 1a and 1b have markedly blunted responses.  The definitive test for the diagnosis of PHP 1 is the analysis of the Gs-alpha protein levels or sequencing of GNAS1, which can be done in reference laboratories.

Treatment of hypocalcemia in patients with PHP is the same as for other types of hypoparathyroidism.  However, patients with AHO may require specific therapies related to skeletal abnormalities.  Approximately 30% of patients with AHO have ectopic calcification and occasionally, large extraskeletal osteomas require surgical removal to relieve symptoms (88).  Ossification of ligaments may also require surgery to relieve neurological problems (89).

Pseudopseudohypoparathyroidism

Patients who express only the AHO phenotype are described as having PPHP (Table 5). These subjects have normal serum calcium levels and have no other evidence of hormone resistance.  These patients have the same mutant allele that can cause Gs alpha deficiency in other affected family members who have hormone resistance (81-84).  Tissue-specific imprinting of GNAS1 in the proximal tubules of the kidney and in the thyroid gland occurs, for example in this disease, such that the paternal allele (if it is non-mutant) is silenced and allows for the expression of only one mutant maternal allele of GNAS1 in those tissues, producing the phenotype of hormonal resistance.

OTHER CAUSES OF HYPOCALCEMIA

Neonatal Hypocalcemia

Skeletal mineralization of the fetus is due to active calcium transport from the mother across the placenta.  At term, the fetus is hypercalcemic relative to the mother and may have suppressed PTH levels.  Over the first 4 days of life, PTH levels fall first and then rise to normal adult levels by 2 weeks after birth (90).

In the first 24-48 days of life, “early” neonatal hypocalcemia may occur.  It is more common in premature infants, infants of diabetic mothers, and infants who have suffered asphyxia.  The proportional drop in ionized calcium may be less than the drop in total calcium, so those symptoms may not be manifest.  Hypocalcemia in premature infants is not unusual, but the reason is not understood.  One proposal is that an exaggerated rise in calcitonin occurs that provokes hypercalcemia.  Other hypotheses include the fact that PTH secretion may be impaired in the premature infant.  Infants of diabetic mothers have an exaggerated postnatal drop in circulating calcium levels, and strict maternal glycemic control during pregnancy reduces the incidence of hypocalcemia in these infants.

Between 5 and 10 days of life, "late" neonatal hypocalcemia may result in tetany and seizures.  This disorder is more common in full-term infants than in premature infants.  One risk factor is hyperphosphatemia due to administration of cow's milk, which may reflect an inability of the immature kidney to secrete phosphate.  Magnesium deficiency may also masquerade as hypocalcemia in an infant.  Congenital defects of intestinal magnesium absorption or renal tubular absorption can occur resulting in severe hypocalcemia.

Hyperparathyroidism during pregnancy is unusual but can result in hypocalcemia in the newborn (91).  Atrophy of the fetal parathyroid glands can occur during intrauterine life due to the increased calcium delivery to the fetus.  The infant's parathyroid glands are not able to respond to the hypocalcemic stimulus after birth and maintain normal serum calcium levels.

A typically benign autosomal dominant disorder, familial hypocalciuric hypercalcemia (FHH), can paradoxically produce neonatal hypocalcemia.  FHH is usually due to heterozygous inactivating mutations in the Casr.  There is a report of an infant with late onset life-threatening hypocalcemia secondary to relative hyperparathyroidism. The hypoparathyroidism was thought to be due to fetal parathyroid suppression secondary to high maternal calcium levels in a mother with FHH due to a heterozygous Casr mutation (92).

Factitious Hypocalcemia Due to Hypoalbuminemia

In patients with chronic illness, malnutrition, cirrhosis, or volume over-expansion, serum albumin may fall with a reduction in the total, but generally not the ionized, fraction of serum calcium. This is referred to as "factitious" hypocalcemia. Patients do not have any of the signs or symptoms listed above of hypocalcemia. There is a correlation between the extent of hypoalbuminemia and hypocalcemia such that one can calculate the corrected total serum calcium. If the serum albumin levels fall to <4.0 g/dL, the usual correction is to add 0.8 mg/dL to the measured total serum calcium for every 1.0 g/dL by which the serum albumin is lowered. This is not a completely precise method, and serum ionized calcium measurements can confirm whether true hypocalcemia is present.

Hypomagnesemia

Magnesium depletion is relatively common in the hospitalized patient, occurring in 10% of this population. Hypocalcemia is commonly associated with magnesium depletion. Hypomagnesemia may be caused by excessive losses through the gastrointestinal tract.  Chronic diarrhea of essentially any etiology can cause hypomagnesemia.  Non-tropical sprue, radiation therapy, bacterial and viral dysentery, and severe pancreatitis can cause hypomagnesemia. Bypass or resection of the small bowel may also result in intestinal magnesium loss. Mutations in several genes important in magnesium conservation may also cause intestinal magnesium loss (93,94).

Loss of magnesium through renal mechanisms may be due to osmotic diuresis or glycosuria, and it may also occur in hypoparathyroidism. Many drugs cause renal magnesium wasting such as diuretics, aminoglycosides, amphotericin B, cisplatinum, cyclosporin, and pentamidine (7,8).

A rare etiology is primary familial hypomagnesemia, which usually is diagnosed at a very young age.  Hypomagnesemia with secondary hypocalcemia, due to a mutation in TRPM6, was described simultaneously by two groups (95,96).  TRPM6 is a protein of the long transient receptor potential channel (TRPM) family.  It is highly similar to TRPM7, a bifunctional protein combining calcium- and magnesium-permeable cation channel activities with protein kinase activity. TRPM6 is present in both kidney tubules and intestinal epithelia and maps to chromosome 9q. This autosomal recessive disorder can sometimes present with the triad of hypomagnesemia, hypokalemia, and hypocalcemia.  It is treatable with life-long magnesium supplementation. Recognition and treatment can prevent long-term neurological defects.  Several other inherited disorders causing hypomagnesemia have been elucidated and are described in recent reviews (93,94).

Patients with severe hypocalcemia due to magnesium depletion should be treated with intravenous magnesium at a dose of 48 mEq over 24 hours.  Although magnesium can be administered intramuscularly, these injections are usually painful.  Even though intravenous magnesium administration may result in prompt normalization of magnesium levels, hypocalcemia may not be corrected for 3-7 days.  This may be because cellular uptake of magnesium is slow, and magnesium is predominantly an intracellular cation.  Repletion of magnesium thus requires sustained correction of the deficiency state.  Magnesium therapy may be continued until the biochemical signs of depletion (hypocalcemia and hypokalemia) resolve.

Hyperphosphatemia

Hyperphosphatemia can lower serum calcium.  There are many causes of hyperphosphatemia, including increased intake of phosphate, decreased excretion of phosphate or increased translocation of phosphate from tissue breakdown into the extracellular fluid. Renal insufficiency is probably the most common cause of hyperphosphatemia. The use of phosphate-containing enemas or zealous use of oral phosphate may also lead to hyperphosphatemia.  Vitamin D administration, especially of vitamin D metabolites like calcitriol, may also cause hyperphosphatemia.  The transcellular shift of phosphate from cells into the extracellular fluid compartment is seen in tissue destruction or increased metabolism.  Examples of transcellular shifts include changes in phosphate that accompany treatment of acute leukemias or lymphomas or large bulky solid tumors with effective chemotherapy. Rapid release of cellular phosphate may occur causing the tumor lysis syndrome (97).  In rhabdomyolysis due to crush injury, hypocalcemia and hyperphosphatemia may occur. Severe intravascular hemolysis may lead to a similar syndrome. In diabetic ketoacidosis, ketone-induced urinary losses of phosphate deplete total body stores, but patients may present with hyperphosphatemia. When the volume shifts during the correction of hyperglycemia and acidosis, the shift of phosphate back into cells can result in mild transient hypophosphatemia.

Hyperphosphatemia alters the calcium-phosphate product and the solubility of these ions.  This may lead to calcium salt deposition in soft tissues. Ectopic calcifications in tissues may form, including in blood vessels, heart valves, skin, periarticular tissues, and the cornea (band keratopathy). Hyperphosphatemia inhibits 1-alpha-hydroxylase activity in the kidney. The resulting lower circulating concentrations of 1,25(OH)2 vitamin D may further aggravate the hypocalcemia by impairing intestinal absorption of calcium. Hypocalcemia and tetany may occur if serum phosphate rises rapidly. Treatment should be directed towards reducing the hyperphosphatemia in order to correct the hypocalcemia.

Hyperphosphatemia-induced hypocalcemia inhibits vitamin D bioactivation in the kidney and the resulting low 1,25(OH)2 vitamin D levels may result in increased PTH secretion. Secondary hyperparathyroidism from long-term hyperphosphatemia has been well described and is usually associated with renal insufficiency. Ectopic calcifications in tissues may occur.

Medications and Toxins

There are many drugs associated with hypocalcemia.  One class of such agents is inhibitors of bone resorption. These drugs include bisphosphonates, calcitonin, and denosumab, the neutralizing monoclonal antibody to the receptor activator of nuclear factor kappa B ligand (RANK-L) (98-101). Calcitonin, given in multiple doses daily is a short-term treatment for hypercalcemia, so hypocalcemia is an expected therapeutic outcome with such a dosing regimen.  Hypocalcemia after administration of an intravenous bisphosphonate like pamidronate or zoledronic acid is uncommon but if it occurs can be prolonged  (99,100).  This is especially true in patients with underlying vitamin D deficiency.  Patients receiving denosumab for osteoporosis or metastatic cancer to bone are at risk for hypocalcemia, especially if there is underlying chronic kidney disease (101).  Oral or parental phosphate preparations can also lower serum calcium.  Hypocalcemia and osteomalacia have been described with prolonged therapy with anticonvulsants such as phenytoin (diphenylhydantoin) or phenobarbital.  Hypocalcemia has also been found in patients undergoing pheresis and plasmapheresis with citrated blood.  Fluid overdoses during dialysis, over-fluorinated public water supplies, and ingestion of fluoride-containing cleaning agents have all been associated with low serum calcium levels. In this case, hypocalcemia is thought to be due to excessive rates of skeletal mineralization secondary to formation of calcium difluoride complexes. Chemotherapeutic agents such as the combined use of 5-fluorouracil and leucovorin, may result in mild hypocalcemia. The hypomagnesemia caused by cisplatinum can induce hypocalcemia.

"Hungry Bone Syndrome"

Rapid remineralization of the bone  occurring postoperatively, after thyroidectomy for thyrotoxicosis or parathyroidectomy for hyperparathyroidism, is referred to as "hungry bone syndrome" (26,27,102).  It is due to a rapid increase in bone uptake of serum minerals after the removal of a stimulus of high rates of bone remodeling (thyroid hormone or PTH).  When the stimulus is removed, there is a dramatic increase in bone formation. Hypocalcemia can occur if the rate of skeletal mineralization exceeds the rate of osteoclast-mediated bone resorption. This syndrome can be associated with severe and diffuse bone pain and tetany.

A similar pathophysiology (net rapid uptake of calcium into bone) is the cause of hypocalcemia due to osteoblastic metastases.  This may occur in patients with prostate or breast cancer (7).  Acute leukemia or osteosarcoma can also result in hypocalcemia.  In patients with vitamin D deficiency and symptoms of osteomalacia, institution of vitamin D therapy can result in hypocalcemia. All these disease states result in hypocalcemia due to rapid mineralization of large amounts of unmineralized osteoid.

Pancreatitis

Pancreatitis can be associated with lipid abnormalities, hypocalcemia, and even tetany. With the development of animal models, the mechanism of hypocalcemia is known (103). When the pancreas is damaged, free fatty acids are generated by the action of pancreatic lipase. There are insoluble calcium salts present in the pancreas, and the free fatty acids avidly chelate the salts resulting in calcium deposition in the retroperitoneum. In addition, hypoalbuminemia may be part of the clinical picture so that there is a reduction in total serum calcium. If there is concomitant alcohol abuse, emesis or poor nutrition, hypomagnesemia may augment the problem. PTH levels can be normal, suppressed or elevated. If PTH levels are normal or suppressed, hypomagnesemia may be present. If PTH levels are elevated, this is a reflection of the hypocalcemia. In the treatment of these patients, parenteral calcium and magnesium replacements are indicated. Vitamin D status should be assessed to rule out malabsorption or nutritional deficiencies.

Hypocalcemia Associated With Critical Illness

There are multiple reasons why a patient with acute illness may experience hypocalcemia. Acute or chronic renal failure, hypomagnesemia, hypoalbuminemia ("factitious hypocalcemia"), medications, or transfusions with citrated blood may all alter levels of serum calcium. Pancreatitis, as stated above, may also result in hypocalcemia. Another setting in which hypocalcemia can occur is sepsis and usually confirms a grave prognosis (104). In gram negative sepsis or in the "toxic shock syndrome", there is a reduction in both total and ionized serum calcium. The mechanism of action remains unknown, but elevated levels of the cytokines IL-6 or TNF-alpha may be mediators of hypocalcemia.

In a study of patients with acute illnesses, the 3 most common factors identified with low calcium levels were hypomagnesemia, presence of acute renal failure, and transfusions. The level of hypocalcemia correlated with patient mortality (105).

To assess the incidence of hypocalcemia in critically ill patients, Zivin and colleagues (105) compared the frequency and degree of hypocalcemia in nonseptic critically ill patients.  Three groups of hospitalized patients were studied: critically ill patients admitted to medical, surgical, trauma, neurosurgical, burn, respiratory and coronary intensive care units (ICUs) (n=99); non-critically ill ICU patients discharged from an ICU within 48 hours (n=50) and non-ICU patients (n=50).  Incidences of levels of low ionized calcium were 88%, 66% and 26% for the 3 groups, respectively.  The occurrence of hypocalcemia correlated with mortality/hazard ratio for death, 1.65 for calcium decrements of 0.1 mmol/L, (p<0.002). No specific illness (renal failure, blood transfusions) was associated with hypocalcemia.

During surgical procedures, hypocalcemia may occur with the rapid infusion of citrated blood, with physiologic increases in serum PTH levels.  Symptoms are variable in this setting, and it is thought that the phenomenon is due to acute hemodilution by physiological saline and complexation of calcium by the large amounts of citrate infused.  This is noted also during hepatic transplantation when the liver’s capacity for clearance of citrate is interrupted.  Hypocalcemia due to hypoparathyroidism is well recognized in transfusion-dependent patients with beta-thalassemia (106-108).  It is thought that hypoparathyroidism and the other endocrinopathies seen in patients with thalassemia are due to iron overload.  Their presence correlates with disease duration and extent of transfusions.

TREATMENT OF HYPOCALCEMIA

The decision to treat is dependent on presenting symptoms, and the severity and rapidity with which hypocalcemia develops.  All treatment requires close monitoring.  If intravenous infusions are contemplated, hospitalization in an intensive care unit or specialized unit with access to cardiac monitoring and rapid ionized calcium determinations is ideal for optimal management and safety.

Acute Hypocalcemia

Acute hypocalcemia can be life-threatening, as patients may present with tetany, seizures, cardiac arrhythmias, laryngeal spasm, or altered mental status.  Calcium gluconate is the preferred intravenous calcium salt as calcium chloride often causes local irritation.  Calcium gluconate contains 90 mg of elemental calcium per 10 mL ampule, and usually 1 to 2 ampules (180 mg of elemental calcium) diluted in 50 to 100 mL of 5% dextrose is infused over 10 minutes.  This can be repeated until the patient's symptoms have cleared.  With persistent hypocalcemia, administration of a calcium gluconate drip over longer periods of time may be necessary.  The goal should be to raise the serum ionized calcium concentration into the low normal range (~1.0 mM), maintain it there, and control the patient’s symptoms.  Drip rates of 0.5-2.0 mg/kg/hour are recommended.  As soon as possible, oral calcium supplementation should be initiated and, if warranted, therapy with vitamin D or its analogues.

Intravenous administration of calcium is not without problems.  Rapid administration could result in arrhythmias so intravenous administration should be carefully monitored. Local vein irritation can occur with solutions >200 mg/100 mL of elemental calcium.  If local extravasation into soft tissues occurs, calcifications due to the precipitation of calcium phosphate crystals can occur (109).  Calcium phosphate deposition can occur in any organ and is more likely to occur if the calcium-phosphate product exceeds 55.  Calcium phosphate deposition in the lungs, kidney or other soft tissue may occur in patients receiving intravenous calcium especially in the presence of high serum phosphate levels.

It is essential to measure serum magnesium in any patient who is hypocalcemic, as correction of hypomagnesemia must occur to overcome PTH resistance before serum calcium will return to normal.

Chronic Hypocalcemia

In chronic hypocalcemia, patients can often tolerate remarkably severe hypocalcemia and remain asymptomatic.  For patients who are asymptomatic or with mildly symptomatic hypocalcemia, calcium homeostasis can be restored with oral calcium and vitamin D or an activated vitamin D metabolite such as calcitriol (7,8).

Oral calcium carbonate is often the most commonly administered salt, although many different calcium salts exist.  Oral doses calcium should be in the amount of 1 to 3 grams of elemental calcium in 3 to 4 divided doses with meals to ensure optimal absorption.  Calcium carbonate contains 40% elemental calcium by weight and is relatively inexpensive.  Lower amounts of elemental calcium are present in other types of calcium such as calcium lactate (13%), calcium citrate (21%) and calcium gluconate (9%), requiring a larger number of tablets.  There are expensive forms of calcium supplements that have relatively few additional advantages.  Liquid calcium supplements are available such as calcium glubionate that contains 230 mg of calcium per 10 mL or liquid forms of calcium carbonate.  In patients with achlorhydria, a solution of 10% calcium chloride (1- to 30 ml) every 8 hours can also effectively raise calcium levels.  Calcium phosphate salts should be avoided.

The overall goal of therapy is to maintain serum calcium in the low normal range, especially in patients with hypoparathyroidism (7,8,110).  Serum calcium should be tested every 3 to 6 months or when any changes in the medical regimen are made.  One potential side effect of therapy in patients with hypoparathyroidism is hypercalciuria which can be complicated by nephrocalcinosis, nephrolithiasis, and or renal insufficiency.  A 24-hour urine calcium along with creatinine determination should be done at least annually, once stable doses of supplements are established.  The target for urinary calcium excretion is <4 mg/kg/24 hr.  Serum levels of calcium are poor indicators of the presence of hypercalciuria and nephrocalcinosis (110).  The patient should also regularly see an ophthalmologist to screen for cataracts.  When treating hypocalcemia in the presence of hyperphosphatemia, special care must be taken (sometimes with the use of a phosphate binder) to avoid soft tissue calcium phosphate precipitation.  Soft tissue calcification can occur in any tissue, but involvement of vital organs such as the lungs, kidney, heart, blood vessels, or brain can result in substantial morbidity or mortality (10).

For patients with hypoparathyroidism, vitamin D2 or D3 (ergocalciferol or cholecalciferol, respectively) or vitamin D metabolites [calcitriol or 1,25-(OH)2 vitamin D or 1 alpha-OH vitamin D (not available in the US)] are often required.  Calcitriol, the active metabolite of vitamin D, is rapid-acting and physiologic and is often used for initial therapy.  Where rapid dose adjustment is necessary, such as growing children, this may be the most convenient approach (111).  Most patients require 0.25 mcg twice daily and may require up to 0.5 mcg 4 times a day of calcitriol.  Among other options, ergocalciferol is a less expensive choice and has a long duration of action. The usual dose is 50,000 to 100,000 IU/day. When therapy needs to be administered acutely, calcitriol should be given for the first 3 weeks but then tapered off as the dose of ergocalciferol becomes effective.  If calcitriol is the vitamin D metabolite administered, then the serum 25 (OH) vitamin D level should be checked periodically to assure that vitamin D sufficiency is maintained.  Serum 25 (OH) vitamin D levels should be kept stable at >20 ng/mL.

Thiazide diuretics can increase renal calcium reabsorption in patients with hypoparathyroidism. This approach may be needed to achieve a urinary calcium of <4 mg/kg/day. Furosemide and other loop diuretics can depress serum calcium levels and should be avoided. Other factors that may precipitate hypocalcemia are glucocorticoids since they can antagonize the action of vitamin D and its analogues.

Administration or withdrawal of exogenous estrogen can also influence calcium and vitamin D replacement therapy. Estrogen increases calcium absorption at the level of the intestine and indirectly through stimulation of renal 1-alpha-hydroxylase activity. Dose adjustment may be required after changes in estrogen therapy due to alteration in calcium homeostasis. During the pre- and postpartum period in pregnant patients with hypoparathyroidism, doses of vitamin D often need frequent adjustments.  This is due to placental production of 1,25-(OH)2 vitamin D in pregnancy, the increasing levels of PTH-rP from placental, maternal and fetal tissues later in pregnancy, and the high levels of PTH-rP in conjunction with the estrogen-deficient state of lactation (110).

How well current treatment strategies (calcium salts, vitamin D and its metabolites) maintain quality of life in patients with hypoparathyroidism has been assessed to a limited extent (112).   In a cross-sectional, controlled study, 25 women with postsurgical hypoparathyroidism on stable calcium and vitamin D treatment were compared to 25 control subjects with a history of thyroid surgery.  Quality of life, urinary calcium excretion and renal calcifications, serum creatinine, and the presence of cataracts by slit lamp examination were assessed.  Serum calcium was in the therapeutic target range in 18 of 25 hypoparathyroid patients.  Urinary calcium was elevated (>8 mmol/day) in 5 of 23 patients.  Eleven of 25 hypoparathyroid patients had cataracts, and 2 of 25 had renal stones (112).  Compared to the control group, those with hypoparathyroidism had higher global complaint scores with predominant increases in anxiety and phobic anxiety subscores and their physical equivalents using validated questionnaires.  Thus, by both physical and psychologic assessments, there were several parameters that were reduced compared to control subjects.

The long-term complications of standard treatment of hypoparathyroidism were recently examined in a cohort of 120 patients (73% women, average age 52 years, 66% post-surgical) followed at a tertiary medical center (10).  The time-weighted average for serum calcium (maintained between 7.5 and 9.5 mg/dL) occurred in 88% of patients.  Patients were estimated to be in this range ~86% of the time.  Just 53 of 120 patients had any 24-hour urine calcium level determined.  Of those patients, 38% had at least one elevated measurement (>300 mg/24 hours).  Among the 54 of 120 with renal imaging, intrarenal calcifications were detected in 31%.  Of those with brain imaging (31/120 patients), 52% had basal ganglia calcifications.  Analysis of renal function showed rates of chronic kidney disease (stage 3 or greater) of 2- to 17-fold higher than age-adjusted normal subjects (10).  Overall, it was concluded that patients with hypoparathyroidism suffer excess morbidity, especially with regard to renal outcomes.

Underbjerg et al (113,114) examined clinical outcomes in a case-control study of 688 Danish patients with hypoparathyroidism compared to age- and gender-matched controls.  Patients with postsurgical hypoparathyroidism demonstrated increased risk of renal complications (hazard ratio [HR], 3.67; 95% confidence interval [CI], 2.41-5.59) and hospitalizations due to seizures (HR, 3.82; 95% CI, 2.15-6.79), compared to controls (113).  No increased cardiovascular complications or deaths were seen.  Using the same cohort, Underbjerg et al (114) further reported greater risks of hospitalization for infections (HR, 1.42; 95% CI, 1.20-1.67) and of depression/bipolar affective disorder (HR, 1.99; 95% CI, 1.14-3.46).  Risks of cataracts, cancers, spinal stenosis, and fractures were not increased.

The same investigators assessed the epidemiologic features of nonsurgical hypoparathyroidism in Danish patients (115).  Based on data from 180 patients (collected from 1977 to 2012), these investigators found a marked increased in renal insufficiency (HR, 6.01; 95% CI, 2.45-14.75), as well as almost 2-fold increased risk of cardiovascular disease (HR, 1.91; 95% CI, 1.29-7.81).  Neuropsychiatric complications (HR, 2.45; 95% CI, 1.78-3.35) and risks of infections (HR, 1.94; 95% CI, 1.55-2.44), seizures (HR, 10.05; 95% CI, 5.39-18.72), cataracts (HR, 4.21; 95% CI, 2.13-8.34), and upper extremity fractures (HR, 1.93; 95% CI, 1.31-2.85) were also increased.  This was thought to be due to the longer duration (lifetime) of the genetic condition responsible for the nonsurgical hypoparathyroidism in these patients.

 

Replacement with PTH for Hypoparathyroidism

In hypoparathyroidism, ideal treatment would theoretically be to replace the hormone itself in a physiologic manner.  Several clinical studies have evaluated PTH (1-34) and PTH (1-84) as replacement therapy for hypoparathyroidism.  In a trial of hypoparathyroid patients, once daily administration of PTH (1-34) normalized serum and urine calcium levels, but the action lasted only 12 hours (116).  With twice-daily administration of PTH (1-34) compared to twice-daily calcitriol for 3 years in 27 patients, Winer et al (117) reported stabilization of serum calcium levels just below the lower limit of normal and a normalization of urinary calcium excretion (at the target level of 1.25-6.25 mmol/24 hours).  Patients on calcitriol in this trial had urinary calcium levels above normal.  Serum creatinine levels were stable over time in both groups of patients, and biochemical markers of bone turnover increased with PTH (1-34) treatment compared to control levels at baseline.  BMD by dual energy x-ray absorptiometry (DXA) increased slightly but significantly at the lumbar spine and whole body in the calcitriol-treated patients and remained stable over 3 years in the PTH-treated group.  These studies in adults (116,117) included patients with a variety of different etiologies for their hypoparathyroidism including patients with activating Casr mutations.  Two other studies done in children (118,119) demonstrated stabilization of serum calcium levels with twice-daily treatment and normalization of urinary calcium excretion on both PTH(1-34) and calcitriol.  The most promising results for lowering urinary calcium into an acceptable range was seen during continuous PTH(1-34) infusion.  This approach achieved urinary calcium levels of ~4 mmol calcium/24 hours vs ~9.7 mmol/24 hours with twice-daily injections (normal range, 1.25-6.25 mmol/24 hours) in 8 adults with hypoparathyroidism (120).  Serum calcium, phosphorus, and magnesium concentrations were comparable with the two modes of PTH(1-34) delivery.  These findings suggest that renal PTH receptors may require more continuous exposure to the hormone to reabsorb calcium adequately.

Three recent trials have tested the ability of PTH(1-84) therapy to permit lowering of calcium and calcitriol supplements safely while maintaining serum calcium homeostasis in patients with chronic hypoparathyroidism (121-124).  Rubin et al (121) gave PTH(1-84) (100 mcg every other day) to hypoparathyroid patients and was able to lower both calcium and calcitriol supplements substantially (30-40%), while maintaining serum calcium within the target range and mildly lowering urinary calcium excretion.  Since there was no placebo control group in this study, reports of improved quality of life parameters (122) must be interpreted cautiously.

Two other clinical trials, which included placebo treatment arms, further assessed the safety and efficacy of PTH(1-84) therapy in hypoparathyroid subjects (123,124).  In the first, using a randomized, placebo-controlled trial design, Sikjaer et al (123) added PTH(1-84) (fixed dose of 100 mcg/day) or placebo injections to 62 patients on a chronic regimen of calcium and active vitamin D supplements for 24 weeks.  As serum calcium levels rose, supplements were reduced.  A substantial percentage of serum calcium measurements in patients receiving PTH(1-84) were elevated during the trial (~20%), and ~96% of those episodes in the PTH-treated subjects versus the placebo-treated group.  This outcome may have affected the quality of life during the study because no differences were noted between the PTH(1-84)- and placebo-treated groups in those assessments.

In the second randomized, placebo-controlled trial, PTH(1-84) or placebo was administered for 24 weeks to 134 patients with chronic hypoparathyroidism as calcium supplements and activated vitamin D (calcitriol or alphacalcidol) were actively down-titrated (124).  Dose escalations of PTH(1-84) were made starting at 50 mcg/day and then up to 75 and 100 mcg/day as calcium and activated vitamin D metabolites were dose-reduced.  This study met its primary end-point, defined as a 50% or greater reduction in calcium supplements and in active vitamin D metabolites while maintaining a serum calcium concentration within the optimized range of 2.0-2.5 mM.  Urinary calcium levels did not differ substantially between PTH(1-84)- compared to placebo-treated groups.  Extension studies are in progress from two of these trials (121,124) to determine long-term safety and efficacy of PTH(1-84) in this patient population.  Based on these findings, the Food and Drug Administration of the US approved recombinant human PTH(1-84) in 2015 for the treatment of hypoparathyroidism in patients not well controlled on conventional therapy with calcium supplements and activated vitamin D analogues.  Future research is being directed toward designing ideal treatment regimens with PTH (1-84).

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ACKNOWLEDGEMENTS

Dr. Schafer is supported by the US Department of Veterans Affairs, Veterans Health Administration, Clinical Science Research and Development Service, Career Development Award-2 (5 IK2 CX000549).

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Surgery of the Thyroid

ABSTRACT

Incidence rates of thyroid cancer have increased substantially worldwide in the past several decades. Thus, diseases of the thyroid gland and their treatment remain one of the most interesting and dynamic areas of study in medicine. This chapter presents a clear and concise description of current thought and practice concerning the surgical treatment of thyroid diseases. Sections within this chapter include 1) normal and abnormal anatomy and embryology of the thyroid and surrounding neck structures; 2) indications for operation of benign lesions of this gland; 3) diagnosis of thyroid nodules, stressing the use of fine needle aspiration with cytologic analysis; 4) preparation for operation and care of patients with Graves’ disease; 5) surgical approaches for treatment of the different types of thyroid cancer; 6) operative techniques for thyroidectomy including descriptions of standard open, minimally invasive, endoscopic, robotic and transoral approaches; 7) complications of thyroidectomy and their treatment; and 8) developmental abnormalities of the thyroid and their treatment. This chapter offers information for physicians and endocrinologists, as well as for surgeons. For complete coverage of this and related areas in Endocrinology, visit our FREE web-book, www,thyroidmanager.org

HISTORY

The extirpation of the thyroid gland…typifies, perhaps better than any operation, the supreme triumph of the surgeon’s art…. A feat which today can be accomplished by any competent operator without danger of mishap and which was conceived more than one thousand years ago…. There are operations today more delicate and perhaps more difficult…. But is there any operative problem propounded so long ago and attacked by so many…which has yielded results as bountiful and so adequate? Dr. William S. Halsted, 1920

Modern thyroid surgery, as we know it today, began in the 1860s in Vienna with the school of Billroth (1). The mortality associated with thyroidectomy was high, recurrent laryngeal nerve injuries were common, and tetany was thought to be caused by “hysteria.” The parathyroid glands in humans were not discovered until 1880 by Sandstrom (2), and the fact that hypocalcemia was the definitive cause of tetany was not wholly accepted until several decades into the twentieth century. Kocher, a master thyroid surgeon who operated in the late nineteenth and early twentieth centuries in Bern, practiced meticulous surgical technique and greatly reduced the mortality and operative morbidity of thyroidectomy for goiter (3). He described “cachexia strumipriva” in patients years after thyroidectomy (Fig. 1). Kocher recognized that this dreaded syndrome developed only in patients who had total thyroidectomy. As a result, he stopped performing total resection of the thyroid. We now know that cachexia strumipriva was surgical hypothyroidism. Kocher received the Nobel Prize for his contributions to thyroid surgery and for this very important contribution, which proved beyond a doubt the physiologic importance of the thyroid gland.

Figure 1. The dramatic case of Maria Richsel, the first patient with postoperative myxedema to have come to Kocher’s attention. A , The child and her younger sister before the operation. B , Changes 9 years after the operation. The younger sister, now fully grown, contrasts vividly with the dwarfed and stunted patient. Also note Maria’s thickened face and fingers, which are typical of myxedema. (From Kocher T: Uber Kropfextirpation und ihre Folgen. Arch Klin Chir 29:254, 1883.)

Figure 1. The dramatic case of Maria Richsel, the first patient with postoperative myxedema to have come to Kocher’s attention. A , The child and her younger sister before the operation. B , Changes 9 years after the operation. The younger sister, now fully grown, contrasts vividly with the dwarfed and stunted patient. Also note Maria’s thickened face and fingers, which are typical of myxedema. (From Kocher T: Uber Kropfextirpation und ihre Folgen. Arch Klin Chir 29:254, 1883.)

By 1920, advances in thyroid surgery had reached the point that Halsted referred to this operation as a “feat which today can be accomplished by any competent operator without danger of mishap” (1). Unfortunately, decades later, complications still occur. In the best of hands, however, thyroid surgery can be performed today with a mortality that varies little from the risk of general anesthesia alone, as well as with low morbidity. To obtain such enviable results, however, surgeons must have a thorough understanding of the pathophysiology of thyroid disorders; be versed in the preoperative and postoperative care of patients; have a clear knowledge of the anatomy of the neck region; and, finally, use an unhurried, careful, and meticulous operative technique.

IMPORTANT SURGICAL ANATOMY

The thyroid (which means “shield”) gland is composed of two lobes connected by an isthmus that lies on the trachea approximately at the level of the second tracheal ring (Figs. 2 and 3). The gland is enveloped by the deep cervical fascia and is attached firmly to the trachea by the ligament of Berry. Each lobe resides in a bed between the trachea and larynx medially and the carotid sheath and sternocleidomastoid muscles laterally. The strap muscles are anterior to the thyroid lobes, and the parathyroid glands and recurrent laryngeal nerves are associated with the posterior surface of each lobe. A pyramidal lobe is often present. This structure is a long, narrow projection of thyroid tissue extending upward from the isthmus and lying on the surface of the thyroid cartilage. It represents a vestige of the embryonic thyroglossal duct, and it often becomes palpable in cases of thyroiditis or Graves’ disease. The normal thyroid varies in size in different parts of the world, depending on the iodine content in the diet. In the United States it weighs approximately 15 grams.

Figure 2. The normal anatomy of the neck in the region of the thyroid gland. (From Halsted WS, The operative story of goiter. Johns Hopkins Hospital Rep 19:71, 1920.)

Figure 2. The normal anatomy of the neck in the region of the thyroid gland. (From Halsted WS, The operative story of goiter. Johns Hopkins Hospital Rep 19:71, 1920.)

Figure 3. Anatomy of the thyroid and parathyroid glands. A , Anterior view. B , Lateral view with the thyroid retracted anteriorly and medially to show the surgical landmarks (the head of the patient is to the left). (From Kaplan EL: Thyroid and parathyroid. In Schwartz SI [ed: Principles of Surgery, 5th ed. New York, McGraw-Hill, 1989, pp 1613–1685. Copyright © by McGraw-Hill, Inc. Used by permission of McGraw-Hill Book Company.)”]

Figure 3. Anatomy of the thyroid and parathyroid glands. A , Anterior view. B , Lateral view with the thyroid retracted anteriorly and medially to show the surgical landmarks (the head of the patient is to the left). (From Kaplan EL: Thyroid and parathyroid. In Schwartz SI [ed: Principles of Surgery, 5th ed. New York, McGraw-Hill, 1989, pp 1613–1685. Copyright © by McGraw-Hill, Inc. Used by permission of McGraw-Hill Book Company.)”]

VASCULAR SUPPLY

The thyroid has an abundant blood supply (Figs. 2 and 3). The arterial supply to each thyroid lobe is two-fold. The superior thyroid artery arises from the external carotid artery on each side and descends several centimeters in the neck to reach the upper pole of each thyroid lobe, where it branches. The inferior thyroid artery, each of which arises from the thyrocervical trunk of the subclavian artery, crosses beneath the carotid sheath and enters the lower or midpart of each thyroid lobe. The thyroidea ima is sometimes present; it arises from the arch of the aorta and enters the thyroid in the midline. A venous plexus forms under the thyroid capsule. Each lobe is drained by the superior thyroid vein at the upper pole, which flows into the internal jugular vein; and by the middle thyroid vein at the middle part of the lobe, which enters either the internal ­jugular or the innominate vein. Arising from each lower pole is the inferior thyroid vein, which drains directly into the innominate vein.

NERVES

The relationship of the thyroid gland to the recurrent laryngeal nerve and to the external branch of the superior laryngeal nerve is of major surgical significance because damage to these nerves leads to disability in phonation and/or to difficulty breathing (4). Both nerves are branches of the vagus nerve.

Injury to the external branch of the superior laryngeal nerve leads to difficulty in singing and projection of the voice. Injury to one recurrent laryngeal nerve may lead to hoarseness of the voice, aspiration, and difficulty breathing. Bilateral recurrent laryngeal nerve injury is much more serious and often leads to the need for a tracheostomy. These injuries will be discussed in greater detail later in this chapter under “Postoperative Complications.”

Recurrent Laryngeal Nerve

The right recurrent laryngeal nerve arises from the vagus nerve, loops posteriorly around the subclavian artery, and ascends behind the right lobe of the thyroid (Fig. 4a). It enters the larynx behind the cricothyroid muscle and the inferior cornu of the thyroid cartilage and innervates all the intrinsic laryngeal muscles except the cricothyroid. The left recurrent laryngeal nerve comes from the left vagus nerve, loops posteriorly around the arch of the aorta, and ascends in the tracheoesophageal groove posterior to the left lobe of the thyroid, where it enters the larynx and innervates the musculature in a similar fashion as the right nerve. Several factors make the recurrent laryngeal nerve vulnerable to injury, ­especially in the hands of inexperienced surgeons (4,6)

Figure 4a. Anatomy of the recurrent laryngeal nerves. (From Thompson NW, Demers M: Exposure is not necessary to avoid the recurrent laryngeal nerve during thyroid operations. In Simmons RL, Udekwu AO [eds, Debates in Clinical Surgery, Chicago, Year Book Publishers, 1990.)

Figure 4a. Anatomy of the recurrent laryngeal nerves. (From Thompson NW, Demers M: Exposure is not necessary to avoid the recurrent laryngeal nerve during thyroid operations. In Simmons RL, Udekwu AO [eds, Debates in Clinical Surgery, Chicago, Year Book Publishers, 1990.)

  1. The presence of a nonrecurrent laryngeal nerve (Fig. 4b). Nonrecurrent nerves occur more often on the right side (0.6%) than on the left (0.04%) (5). They are associated with vascular anomalies such as an aberrant takeoff of the right subclavian artery from the descending aorta (on the right) or a right-sided aortic arch (on the left). In these abnormal positions, each nerve is at greater risk of being divided.
Figure 4b. “Nonrecurrent” right laryngeal nerves coursing ( A ) near the superior pole vessels or ( B ) around the inferior thyroid artery. Because of the abnormal location of “nonrecurrent” nerves, they are much more likely to be damaged during surgery. (From Skandalakis JE, Droulis C, Harlaftis N, et al: The recurrent laryngeal nerve. Am Surg 42:629–634, 1976.)

Figure 4b. “Nonrecurrent” right laryngeal nerves coursing ( A ) near the superior pole vessels or ( B ) around the inferior thyroid artery. Because of the abnormal location of “nonrecurrent” nerves, they are much more likely to be damaged during surgery. (From Skandalakis JE, Droulis C, Harlaftis N, et al: The recurrent laryngeal nerve. Am Surg 42:629–634, 1976.)

  1. Proximity of the recurrent nerve to the thyroid gland. The recurrent nerve is not always in the tracheoesophageal groove where it is expected to be. It can often be posterior or anterior to this position or may even be surrounded by thyroid parenchyma. Thus, the nerve is vulnerable to injury if it is not visualized and traced up to the larynx during thyroidectomy.
  2. Relationship of the recurrent nerve to the inferior thyroid artery. The nerve often passes anterior, posterior, or through the branches of the inferior thyroid artery. Medial traction of the lobe often lifts the nerve ante­riorly, thereby making it more vulnerable. Likewise, ­ligation of the inferior thyroid artery, practiced by many surgeons, may be dangerous if the nerve is not identified first.
  3. Deformities from large thyroid nodules (6). In the presence of large nodules the laryngeal nerves may not be in their “correct” anatomic location but may be found even anterior to the thyroid (Fig. 5). Once more, there is no substitute for identification of the nerve in a gentle and careful manner.
Figure 5. Recurrent laryngeal nerve displacements by cervical and substernal goiters. Such nerves are at risk during lobectomy unless the surgeon anticipates the unusual locations and is very careful. Rarely, the nerves are so stretched by the goiter that spontaneous palsy results. After careful dissection and preservation, functional recovery may occur postoperatively. (From Thompson NW, Demers M: Exposure is not necessary to avoid the recurrent laryngeal nerve during thyroid operations. In Simmons RL, Udekwu AO, eds, Debates in Clinical Surgery. Chicago, year Book, 1990.)

Figure 5. Recurrent laryngeal nerve displacements by cervical and substernal goiters. Such nerves are at risk during lobectomy unless the surgeon anticipates the unusual locations and is very careful. Rarely, the nerves are so stretched by the goiter that spontaneous palsy results. After careful dissection and preservation, functional recovery may occur postoperatively. (From Thompson NW, Demers M: Exposure is not necessary to avoid the recurrent laryngeal nerve during thyroid operations. In Simmons RL, Udekwu AO, eds, Debates in Clinical Surgery. Chicago, year Book, 1990.)

External Branch of the Superior Laryngeal Nerve

On each side, the external branch of the superior laryngeal nerve innervates the cricothyroid muscle. In most cases, this nerve lies close to the vascular pedicle of the superior poles of the thyroid lobe which requires that the vessels be ligated with care to avoid injury to it (Fig. 6) (7). In 21% of cases, the nerve is intimately associated with the superior thyroid vessels. In some patients the external branch of the superior laryngeal nerve lies on the anterior surface of the thyroid lobe, making the possibility of damage during thyroidectomy even greater (8). In only 15% of patients is the superior laryngeal nerve sufficiently distant from the superior pole vessels to be protected from manipulation by the surgeon. Unfortunately, many surgeons do not attempt to identify this nerve before ligation of the upper pole vessels of the thyroid (9, 9a).

Figure 6. Proximity of the external branch of the superior laryngeal nerve to the superior thyroid vessels. (From Moosman DA, DeWeese MS: The external laryngeal nerve as related to thyroidectomy. Surg Gynecol Obstet 127:1101, 1968.)

Figure 6. Proximity of the external branch of the superior laryngeal nerve to the superior thyroid vessels. (From Moosman DA, DeWeese MS: The external laryngeal nerve as related to thyroidectomy. Surg Gynecol Obstet 127:1101, 1968.)

PARATHYROID GLANDS

The parathyroids are small glands that secrete parathyroid hormone, the major hormone that controls serum calcium homeostasis in humans. Usually four glands are present, two on each side, but three to six glands have been found. Each gland normally weighs 30 to 40 mg, but they may be heavier if more fat is present. Because of their small size, their delicate blood supply, and their usual anatomic position adjacent to the thyroid gland, these structures are at risk of being ­accidentally removed, traumatized, or devascularized during thyroidectomy (100.

The upper parathyroid glands arise embryologically from the fourth pharyngeal pouch (Figs. 7 and 8). They descend only slightly during embryologic development, and their position in adult life remains quite constant. This gland is usually found adjacent to the posterior surface of the middle part of the thyroid lobe, often just anterior to the recurrent laryngeal nerve as it enters the larynx.

Figure 7. A and B, Shifts in location of the thyroid, parafollicular, and parathyroid tissues. C, approximates the adult location. Note that what has been called the lateral thyroid is now commonly referred to as the ultimobranchial body, which contains both C cells and follicular elements. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Gland, 2nd ed. Philadelphia, WB Saunders, 1980; adapted from Norris EH: Parathyroid glands and lateral thyroid in man: Their morphogenesis, histogenesis, topographic anatomy and prenatal growth. Contrib Embryol Carnegie Inst Wash 26:247–294, 1937.) Principles of Surgery, 5th ed. New York, McGraw-Hill, 1989, pp 1613–1685. Copyright © by McGraw-Hill, Inc. Used by permission of McGraw-Hill Book Company.)”]

Figure 7. A and B, Shifts in location of the thyroid, parafollicular, and parathyroid tissues. C, approximates the adult location. Note that what has been called the lateral thyroid is now commonly referred to as the ultimobranchial body, which contains both C cells and follicular elements. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Gland, 2nd ed. Philadelphia, WB Saunders, 1980; adapted from Norris EH: Parathyroid glands and lateral thyroid in man: Their morphogenesis, histogenesis, topographic anatomy and prenatal growth. Contrib Embryol Carnegie Inst Wash 26:247–294, 1937.) Principles of Surgery, 5th ed. New York, McGraw-Hill, 1989, pp 1613–1685. Copyright © by McGraw-Hill, Inc. Used by permission of McGraw-Hill Book Company.)”]

Figure 8. Descent of the lower parathyroid. Whereas the upper parathyroid occupies a relatively constant position in relation to the middle or upper third of the lateral thyroid lobe, the lower parathyroid normally migrates in embryonic life and may end up anywhere along the course of the dotted line. When this gland is in the chest, it is nearly always in the anterior mediastinum. (From Kaplan EL: Thyroid and parathyroid. In: Principles of Surgery, Schwartz SI [ed], 6th edition, Chpt 36, McGaw-Hill, Inc., New York, NY, 1993, pp 1611-1680).

Figure 8. Descent of the lower parathyroid. Whereas the upper parathyroid occupies a relatively constant position in relation to the middle or upper third of the lateral thyroid lobe, the lower parathyroid normally migrates in embryonic life and may end up anywhere along the course of the dotted line. When this gland is in the chest, it is nearly always in the anterior mediastinum. (From Kaplan EL: Thyroid and parathyroid. In: Principles of Surgery, Schwartz SI [ed], 6th edition, Chpt 36, McGaw-Hill, Inc., New York, NY, 1993, pp 1611-1680).

The lower parathyroid glands arise from the third pharyngeal pouch, along with the thymus; hence, they often descend with the thymus. Because they travel so far in embryologic life, they have a wide range of distribution in adults, from just beneath the mandible to the anterior mediastinum (see Fig. 8) (11). Usually, however, these glands are found on the lateral or posterior surface of the lower part of the thyroid gland or within several centimeters of the lower thyroid pole within the thymic tongue.

Parathyroid glands can be recognized by their tan appearance; their small vascular pedicle; the fact that they bleed freely when a biopsy is performed, as opposed to fatty tissue; and their darkening color of hematoma formation when they are traumatized. With experience, one becomes much more adept at recognizing these very important structures and in differentiating them from either lymph nodes or fat. Frozen section examination during surgery can be helpful in their identification.

LYMPHATICS

A practical description of the lymphatic drainage of the thyroid gland for the thyroid surgeon has been proposed by Taylor (12). The results of his studies, which are clinically very relevant to the lymphatic spread of thyroid carcinoma, are summarized in the following.

Central Compartment of the Neck

  1. The most constant site to which dye goes when injected into the thyroid is the trachea, the wall of which contains a rich network of lymphatics. This fact probably accounts for the frequency with which the trachea is involved by thyroid carcinoma, especially when it is anaplastic.
  2. A chain of lymph nodes lies in the groove between the trachea and the esophagus (Level 6, Fig. 8).
  3. Lymph can always be shown to drain toward the mediastinum and to the nodes intimately associated with the thymus (Level 7, Fig. 8).
  4. One or more nodes lying above the isthmus, and therefore in front of the larynx, are sometimes involved. These nodes have been called the Delphian nodes (named for the oracle of Delphi) because it has been said that if palpable, they are diagnostic of carcinoma. However, this clinical sign is often misleading.
  5. A bilateral central lymph node dissection, called a level 6 dissection (See Fig. 8) clears out all these lymph nodes from one carotid artery to the other carotid artery and down into the superior mediastinum as far as possible (12a).
Figure 9. The lymph node regions of the neck are divided into levels I through VII: Level I nodes are the submental and submandibular nodes; Level II are the upper jugular nodes; Level III are the midjugular nodes; Level IV are the lower jugular nodes; Level V are the posterior triangle and supraclavicular nodes; Level VI or central compartment nodes incorporate the Delphian/prelaryngeal, pretracheal, and paratracheal lymph nodes; and Level VII nodes are those within the superior mediastinum.

Figure 9. The lymph node regions of the neck are divided into levels I through VII: Level I nodes are the submental and submandibular nodes; Level II are the upper jugular nodes; Level III are the midjugular nodes; Level IV are the lower jugular nodes; Level V are the posterior triangle and supraclavicular nodes; Level VI or central compartment nodes incorporate the Delphian/prelaryngeal, pretracheal, and paratracheal lymph nodes; and Level VII nodes are those within the superior mediastinum.

Lateral Compartment of the Neck

A constant group of nodes lies along the jugular vein on each side of the neck (Levels 2, 3, and 4). The lymph glands found in the supraclavicular fossae or more laterally in the neck (Level 5) may also be involved in more extensive spread of malignant disease from the thyroid gland (12a). It should not be forgotten that the thoracic duct on the left side of the neck, a lymph vessel of considerable size, arches up out of the mediastinum and passes forward and laterally to drain into the left subclavian vein or the internal jugular vein near their junction. If the thoracic duct is damaged, the wound is likely to fill with lymph; in such cases, the duct should always be sought and ligated. A wound that discharges lymph postoperatively should always raise suspicion of damage to the thoracic duct or a major tributary. A lateral lymph node dissection encompasses removal of these lateral lymph nodes (Fig. 9a). Rarely, the submental nodes (Level 1) are involved by metastatic thyroid cancer as well.

Figure 9a. Lateral neck dissection. Note that during this procedure lymph nodes from Levels 2, 3, 4, and 5 are removed. The vagus nerve, sympathetic ganglia, phrenic nerve, brachial plexus, and spinal accessory nerve are preserved. In a modified radical neck dissection the sternocleidomastoid muscle is not usually divided, and the jugular vein is not removed unless lymph nodes with tumor are adherent to it. (From Sedgwick CE, Cady B: In Surgery of the Thyroid and Parathyroid Glands. Philadelphia, WB Saunders, 1980, p 180.)

Figure 9a. Lateral neck dissection. Note that during this procedure lymph nodes from Levels 2, 3, 4, and 5 are removed. The vagus nerve, sympathetic ganglia, phrenic nerve, brachial plexus, and spinal accessory nerve are preserved. In a modified radical neck dissection the sternocleidomastoid muscle is not usually divided, and the jugular vein is not removed unless lymph nodes with tumor are adherent to it. (From Sedgwick CE, Cady B: In Surgery of the Thyroid and Parathyroid Glands. Philadelphia, WB Saunders, 1980, p 180.)

INDICATIONS FOR THYROIDECTOMY

Thyroidectomy is usually performed for the following ­reasons:
1. As therapy for some individuals with thyrotoxicosis, both those with Graves’ disease and others with hot nodules
2. To establish a definitive diagnosis of a mass within the thyroid gland, especially when cytologic analysis after fine-needle aspiration (FNA) is either nondiagnostic, equivocal, or indeterminate
3. To treat benign and malignant thyroid tumors
4. To alleviate pressure symptoms or respiratory difficulties associated with a benign or malignant process
5. To remove an unsightly goiter (Figs. 9b and 9c)
6. To remove large substernal goiters, especially when they cause respiratory difficulties

Figure 9b. Large goiters are prevalent in areas of iodine deficiency. A woman from Switzerland operated upon by Dr. Theodor Kocher (From Kocher (3)).

Figure 9b. Large goiters are prevalent in areas of iodine deficiency. A woman from Switzerland operated upon by Dr. Theodor Kocher (From Kocher (3)).

Figure 9c. Large goiters are prevalent in areas of iodine deficiency. Large goiters still occur in many parts of the world, as demonstrated in this woman from a mountainous region of Vietnam, 1970.

Figure 9c. Large goiters are prevalent in areas of iodine deficiency. Large goiters still occur in many parts of the world, as demonstrated in this woman from a mountainous region of Vietnam, 1970.

SOLITARY THYROID NODULES

Solitary thyroid nodules are found in 4% to 9% of patients by clinical examination, and in 22% or more of patients by ultrasound in the United States; most are benign (13). Therefore, rather than operating on every patient with a thyroid nodule, the physician or surgeon should select patients for surgery who are at high risk for thyroid cancer. Furthermore, each surgeon must know the complications of thyroidectomy and be able to either perform a proper operation for thyroid cancer in a safe and effective manner or refer the patient to a center where it can be done.

LOW-DOSE EXTERNAL IRRADIATION OF THE HEAD AND NECK

A history of low-dose external irradiation of the head or neck (less than 1500 to 2000 rads) is probably the most important historical fact that can be obtained in a patient with a thyroid nodule because it indicates that cancer of the thyroid, usually papillary cancer, is more likely (in up to 35% of cases), even if the gland is multinodular (14,15). Low-dose irradiation and its implications are discussed elsewhere (15a). Fortunately, treatments of low-dose radiation for benign conditions--thymic enlargement, tonsils, and acne-- have long been discontinued. However, patients who had this therapy in infancy or childhood are still seen and are still at a greater risk of cancer (15b).

HIGH-DOSE EXTERNAL IRRADIATION THERAPY

High-dose external irradiation therapy (more than 2000 rads), does not confer safety from thyroid carcinoma, as was previously thought (16). Rather, an increased prevalence of thyroid carcinoma, usually papillary cancer, has been found, ­particularly in patients with Hodgkin’s disease and other lymphomas who received upper mantle irradiation that included the thyroid gland (15b). Usually, a dose of approximately 4000 to 5000 rads was given. Both benign and malignant thyroid nodules have been recognized, now that these persons survive for longer periods (17). If a thyroid mass appears, it should be treated aggressively. These patients should also be observed carefully for the development of hypothyroidism.

RISK OF IONIZING RADIATION

Children exposed to ionizing radiation in the area of the Chernobyl nuclear accident have been shown to have at least a 30-fold increase in papillary thyroid cancer (18). This cancer may also be more aggressive than the usual papillary carcinoma and demonstrated more local invasion and lymph node metastases. It is thought to be the result of exposure to iodine isotopes that were inhaled or that entered the food chain. The mechanism of radiation-induced thyroid cancer is thought to be caused primarily by chromosomal rearrangements such as RET/PTC (19) and less commonly to BRAF mutations (19a, 19b).

DIAGNOSIS OF THYROID NODULES

Ultrasound examinations are used very commonly as screening procedures for thyroid nodules or as exams after a thyroid nodule has been palpated to look for multicentricity, invasion, etc. When suspicious thyroid nodules are detected, the sonographer should examine the central and lateral neck areas for suspicious lymph nodes which suggest metastatic disease.

Diagnostic modalities such as nuclear scans had been used widely in the past, but currently they have been superseded by a fine needle aspiration (FNA) of the mass with cytologic analysis (Fig. 10). In the hands of a good thyroid ­cytologist, more than 90% of nodules can be categorized histologically. Approximately 60% to 70% are found to be compatible with a colloid (benign) nodule. Fifteen percent to 30% demonstrate sheets of follicular cells with little or no colloid (an indeterminate lesion). An indeterminate lesion can be classified further as a follicular lesion of undetermined significance (FLUS) or as a possible follicular neoplasm (Table 1). Five percent to 10% of FNA’s are malignant, and less than 10% are nondiagnostic. In order to improve the diagnostic ability of FNA, researchers are adding biomarkers to the cytologic analyses (20, 21). A system for reporting thyroid cytopathology with the potential risk of malignancy, called the Bethesda system, is shown in Table 1 (20a).

TABLE 1. Implied Risk of Malignancy and Recommended Clinical Management
Diagnostic category Risk of malignancy (%) Usual management
Nondiagnostic or unsatisfactory 1-4 Repeat FNA with ultrasound guidance
Benign 0-3 Clinical follow-up
Atypia of undetermined significance or follicular lesion of undetermined significance ˜5-15 Repeat FNA (or operate)
Follicular neoplasm or suspicious for a follicular neoplasm 15-30 Surgical lobectomy
Suspicious for malignancy 60-75 Near-total thyroidectomy or surgical lobectomy
Malignant 97-99 Near-total or total thyroidectomy

Adapted from: Cibas ES and Ali SZ: The Bethesda System for Reporting Thyroid Cytology. Thyroid 19:1159-1165, 2009 (20a).

As shown in Table 1, all patients who have malignant cytologic results should be operated upon. False positive results are rare. Patients with a “suspicious of malignancy” cytologic diagnosis should also be operated upon and are also likely to have a malignant lesion. Patients with the cytologic diagnosis of a follicular neoplasm or suspicion of a follicular neoplasm should also be operated upon, for up to 30% of these tumors prove to be carcinoma. When atypia of undetermined significance or a follicular lesion of undetermined significance (FLUS) is reported, some clinicians recommend a repeat FNA several months later (Table 1). However, others recommend operation, since up to 15% of these FLUS lesions also prove at operation to be malignant. In studies of follicular lesions, experiments are being conducted to determine whether the use of molecular markers such as BRAF, RAS, RET/PTC, PAX8-PPAR, or Galectin 3 will aid in differentiating benign from malignant lesions (21a). In another recent study of 265 indeterminate nodules, classified by FNA and then operated upon, 85 (32%) proved to be carcinomas. Using a diagnostic test that measures the expression of 167 genes, investigators were able to identify 78 of the 85 carcinomas as suspicious and to recognize most of the other lesions as benign (21b). Thus, in the future, perhaps these or similar tests will become routine and will reduce the number of operations currently performed for these indeterminate lesions which are ultimately found to be benign. An excellent review of the value of molecular diagnosis of FNA specimens for indeterminate nodules was written by Keutgen and associates (21c).

When the diagnosis of colloid nodule is made cytologically, the patient should be observed and not operated on unless tracheal compression or a substernal goiter is present, or unless the patient desires the benign mass to be removed. Finally, if an inadequate specimen is obtained, FNA with cytologic examination should be repeated. Usually one waits several months between needle biopsies.

Especially with small, nonpalpable masses, and probably in all cases, it is wise to perform FNA under sonographic guidance to be certain that the needle is in the correct location. Furthermore, when performing an FNA on a suspicious lymph node in the neck, one should measure thyroglobulin in the specimen as well as perform a cytologic analysis. The presence of thyroglobulin is diagnostic of metastatic thyroid cancer even if the cytology is non-diagnostic. FNA with cytologic assessment is the most powerful tool in our armamentarium for the diagnosis of a thyroid nodule.

In summary, the algorithm for the diagnosis of a thyroid nodule with isotope scintigraphy and ultrasonography as initial steps has been replaced in most hospitals by emphasizing the importance of early cytologic examination using fine needle aspirate (FNA) (Fig. 10). Far fewer isotope scans are being done because carcinomas represent only 5% to 10% of all cold nodules. This test is usually reserved for diagnosis of a “hot” nodule.

 

Figure 10. Algorithm for the diagnosis of a thyroid nodule with fine-needle aspiration (FNA) and cytologic examination of each nodule. Greater accuracy is obtained by using this diagnosis scheme. (Courtesy of Dr. Jon van Heerden.)

Figure 10. Algorithm for the diagnosis of a thyroid nodule with fine-needle aspiration (FNA) and cytologic examination of each nodule. Greater accuracy is obtained by using this diagnosis scheme. (Courtesy of Dr. Jon van Heerden.)

 

PREPARATION FOR SURGERY

Most patients undergoing a thyroid operation are euthyroid and require no specific preoperative preparation related to their thyroid gland. Determination of serum calcium and parathyroid hormone (PTH) levels is often helpful, and endoscopic or indirect laryngoscopy should definitely be performed in those who are hoarse and in others who have had a prior thyroid, parathyroid, or cervical disc operation in order to detect the possibility of a recurrent laryngeal nerve injury. Evaluation of vocal cord movement by a transcutaneous ultrasound technique has recently been described (21d). An increasing number of surgeons propose that all patients should have their vocal cords examined prior to undergoing thyroidectomy in order to rule out a vocal cord paralysis that is not clinically apparent. Certainly, if a surgeon is planning to report rates of recurrent laryngeal nerve injury postoperatively, both preoperative and postoperative vocal cord examination is important.

HYPOTHYROIDISM

Modest hypothyroidism is of little concern when treating a surgical patient; however, severe hypothyroidism can be a significant risk factor. Severe hypothyroidism can be diagnosed clinically by myxedema, as well as by slowness of affect, speech, and reflexes (22). Circulating thyroxine and triiodothyronine values are low. The serum thyroid-stimulating hormone (TSH) level is high in all cases of hypothyroidism that are not caused by pituitary insufficiency, and it is the best test of thyroid function. In the presence of severe hypothyroidism, both the morbidity and the mortality of surgery are increased as a result of the effects of both the anesthesia and the operation. Such patients have a higher incidence of ­perioperative hypotension, cardiovascular problems, gastrointestinal hypomotility, prolonged anesthetic recovery, and neuropsychiatric disturbances. They metabolize drugs slowly and are very sensitive to all medications. Therefore, when severe myxedema is present, it is preferable to defer elective ­surgery until a euthyroid state is achieved.

If urgent surgery is necessary, it should not be postponed simply for repletion of thyroid hormone. Endocrine consultation is imperative, and an excellent anesthesiologist is mandatory for success. In most cases, intravenous thyroxine can be started preoperatively and continued thereafter. In general, small doses of thyroxine are initially given to patients who are severely hypothyroid, and then the dose is gradually increased.

HYPERTHYROIDISM

In the United States, most patients with thyrotoxicosis have Graves’ disease. Furthermore, in the U.S., about 90% of all patients with Graves’ disease are treated with radioiodine therapy. Young patients, those with very large goiters, some pregnant women, and those with thyroid nodules or severe ­ophthalmopathy are commonly operated upon. Radioiodine therapy can make the ophthalmopathy worse in some cases.

Persons with Graves’ disease or other thyrotoxic states should be treated preoperatively to restore a euthyroid state and to prevent thyroid storm, a severe accentuation of the symptoms and signs of hyperthyroidism that can occur during or after surgery. Thyroid storm results in severe tachycardia or cardiac arrhythmias, fever, disorientation, coma, and even death. In the early days of thyroid surgery, operations on the toxic gland were among the most dangerous surgical procedures because of the common occurrence of severe bleeding, as well as all the symptoms and signs of thyroid storm. Now, with proper preoperative preparation, operations on the thyroid gland in Graves’ disease can be performed with about the same degree of safety as operations for other thyroid conditions (23).

In mild cases of Graves’ disease with thyrotoxicosis, iodine therapy alone has been used for preoperative preparation, although we do not recommend this approach (22). Lugol’s solution or a saturated solution of potassium iodide is given for 8 to 10 days. Although only several drops per day are needed to block the release of thyroxine from the toxic thyroid gland, it is our practice to administer two drops two or three times daily. This medication is taken in milk or orange juice to make it more palatable. Iodine therapy suppresses thyroid hormone release only in Graves’ disease and should not be given to patients with toxic nodular goiter.

Most of our patients with Graves’ disease are treated initially with the antithyroid drugs propylthiouracil (PTU) or methimazole (Tapazole) until they approach a euthyroid state. Then iodine is added to the regimen for 8 to 10 days before surgery. The iodine decreases the vascularity and increases the firmness of the gland. Sometimes thyroxine is added to this regimen to prevent hypothyroidism and to decrease the size of the gland. Beta-adrenergic blockers such as propranolol (Inderal) have increased the safety of thyroidectomy for patients with Graves’ disease (23). We use them commonly with antithyroid drugs to block adrenergic receptors and ameliorate the major signs of Graves’ disease by decreasing the patient’s pulse rate and eliminating the tremor. Some surgeons recommend preoperative use of propranolol alone or with iodine (24). These regimens, they believe, shorten the preparation time of patients with Graves’ disease for surgery and make the operation easier because the thyroid gland is smaller and less friable than it would otherwise be. We do not favor these regimens for routine preparation because they do not appear to offer the same degree of safety as do preoperative programs that restore a euthyroid state before surgery. Instances of fever and tachycardia have been reported in persons with Graves’ disease who were taking only propranolol. We have used propranolol therapy alone or with iodine without difficulty in some patients who are allergic to antithyroid medications. In such patients it is essential to continue the propranolol for several weeks postoperatively. Remember that they are still in a thyrotoxic state immediately after surgery, although the peripheral manifestations of their disease have been blocked.

The major advantages and disadvantages of radioiodine vs. thyroidectomy as definitive treatment of Graves’ disease are listed in Table 2. In our patients we have never had a death from thyroidectomy for Graves’ disease in over 45 years. Surgical resection involves subtotal, near total thyroidectomy (Fig. 11), or lobectomy with contralateral subtotal or near total lobectomy (Dunhill procedure). Previously we left 2 to 2.5 grams of thyroid in the neck. However, this resulted in a recurrence rate of approximately 12% at about the 10 year followup (25). Hence, we leave very small thyroid remnants and treat the patients with thyroxine replacement. Especially in children and adolescents, one should consider a total thyroidectomy or leaving a very small amount of tissue because the incidence of recurrence of thyrotoxicosis appears to be greater in this young group. Finally, when operating for severe ophthalmopathy, we try to perform near-total or total thyroidectomy, for improvement in the eyes may occur after this procedure. When operating on the thyroid, and especially in young patients with a benign condition, the surgeon should be very careful to avoid permanent hypoparathyroidism and nerve injury. These complications will be discussed later in this chapter.

The major benefits of thyroidectomy appear to be the removal of nodules if they are present, the speed with which normalization of thyroid function is achieved, possible improvement in the eyes, and possibly a lower rate of hypothyroidism than is seen after radioiodine therapy.

(Basic Surgery, 4th ed. St. Louis, Quality Medical Publishing, 1993, pp 162–195)

Figure 11. Common operations on the thyroid. In near-total thyroidectomy, a small amount of thyroid tissue is left to protect the recurrent laryngeal nerve and upper parathyroid gland. (From Kaplan EL: Surgical endocrinology. In Polk HC, Stone HH, Gardner B, eds, Basic Surgery, 4th edition, St. Louis, Quality Medical Publishing, 1993, pp 162-195.)

Figure 11. Common operations on the thyroid. In near-total thyroidectomy, a small amount of thyroid tissue is left to protect the recurrent laryngeal nerve and upper parathyroid gland. (From Kaplan EL: Surgical endocrinology. In Polk HC, Stone HH, Gardner B, eds, Basic Surgery, 4th edition, St. Louis, Quality Medical Publishing, 1993, pp 162-195.)

TABLE 2. Ablative Treatment of Graves’ Disease with Thyrotoxicosis

 Method  Dose or extent of surgery  Onset of responses  Complications  Remarks
 Surgery  Subtotal excision
of gland
(leaving about 1-2 g remnant or less)		 Immediate

Mortality: <1%

Permanent hypothyroidism:
20-30% or greater

Recurrent hyperthyroidism: <15%

Vocal cord paralysis: ~1%

Hypoparathyroidism: ~1%

 Applicable in young patients and pregnant women
 Radioiodine 5-10 mCi   Several weeks to months Permanent hypoparathyroidism:
50%-70% or more, often with delayed onset; multiple treatments sometimes necessary; recurrence possible		 Avoid in children or pregnant women

 

 

SURGICAL APPROACH TO THYROID NODULES

Colloid Nodule(s)

If a colloid nodule is diagnosed on FNA, there is no urgency to operate in most cases. Patients often are rebiopsied in the future to reduce the chance of error and are followed in 6 to 12 months with repeat ultrasound. Respiratory compromise, substernal goiter, rapid growth, and pain are reasons for operation. Some patients desire a thyroidectomy to get over the problem of further evaluation or wish to have surgery to rid themselves of an unsightly mass. Thyroid lobectomy, subtotal thyroidectomy, or near total or total thyroidectomy can be done for a goiter and each approach has advocates.A “toxic nodule” can occur and can be cured by enucleation since it is rarely a carcinoma and since normal thyroid function might follow this approach. Otherwise, a thyroid lobectomy is appropriate.

Follicular (indeterminate) Nodules

The surgical treatment of a nodule which is diagnosed on FNA as either a follicular lesion of undetermined significance (FLUS) or a follicular neoplasm is more controversial. The problem is that the pathologist rarely can tell which nodules are benign and which are malignant on frozen section. He/She cannot tell which are follicular adenomas and which are follicular carcinomas, for example, at the time of operation. This usually requires careful evaluation of many sections for capsular invasion or vascular invasion on permanent sections. Of course, at operation, lymph nodes can be biopsied, but in most small masses of this type they are negative.

These difficulties should be discussed with the patient preoperatively and usually he or she will guide the surgeon. There are two possible operative courses: a lobectomy or a near-total or total thyroidectomy. The American Thyroid Association guidelines recommend a thyroid lobectomy in such an instance and to await the final diagnosis (21a). This is the choice of most patients. However, if the lesion turns out to be a carcinoma on permanent pathologic analysis, a second operation is necessary, with a second anesthesia, etc., and may be more difficult because of adhesions. Furthermore, many patients are already on thyroid hormone or require thyroid hormone replacement therapy after only a thyroid lobectomy. Thus, some patients choose to have a near-total or total thyroidectomy at the first operation, especially if they have bilateral nodules. But the wise surgeon will have this discussion preoperatively and do what the patient wants, since there is no correct answer.

Irradiated Patients

Patients who received low-dose (less than 2000 rads) or high-dose external irradiation or who were exposed to excessive ionizing radiation are at increased risk of developing single or multiple nodules of the thyroid, both benign and malignant (15b). There is a greater chance of malignancy than in the non-irradiated gland. For single nodules, FNA analysis is performed and the decision as to whether or not to operate is determined by the result of the cytology. Multiple nodules in such a patient present more of a diagnostic problem since it is difficult to evaluate each nodule preoperatively and benign and malignant nodules often are found in the same gland. In such a patient, a decision to operate is likely. When an operation is performed in a patient with a radiation history and a suspicious nodule, we are more inclined to perform a near-total or total thyroidectomy rather than a lobectomy. This procedure removes all of the nodules and also removes all potentially damaged thyroid tissue.

SURGICAL APPROACH TO THYROID CANCER

PAPILLARY CARCINOMA

It is estimated that 62,450 new cases of thyroid cancer will be diagnosed in 2015, with 3 or 4 times the number of cases occurring in women when compared to men (26c). Thyroid cancer is the fastest increasing cancer in both men and women. Since 2004, incidence rates have been rising 6.6% per year in women and 5.5% per year in men. An estimated 1950 deaths from thyroid cancer are expected in 2015. The death rates of women and men have increased slightly from 2004 to present as well (25a).

Approximately 80% to 85% of all thyroid cancers are papillary cancer. The surgical treatment of papillary cancer is best divided into two groups based on the size, clinical characteristics, and aggressiveness of these lesions.

Treatment of Minimal or Micro-Papillary Carcinoma

The term minimal or micro papillary carcinoma refers to a small papillary cancer, less than 1 cm in diameter, that demonstrates no local invasiveness through the thyroid capsule, that is not associated with lymph node metastases, and that is often found in a young person as an occult lesion when thyroidectomy has been performed for another benign condition. In such instances, especially when the cancer is unicentric and smaller than 5 mm, lobectomy is sufficient and reoperations are unnecessary. Thyroid hormone is given to suppress serum TSH levels, and the patient is monitored at regular intervals (21a).

In recent studies from Japan (25b), between 1993 and 2011, 1235 patients with low risk papillary micro carcinomas were followed and not operated upon. Patients were operated upon if the lesions grew to 12 mm in size or if they developed lymph nodes suggestive of malignancy. One hundred ninety one of the 1235 patients underwent surgery after some period of observation. During this follow up period, none of the 1235 patients showed distant metastases or died of papillary cancer. Only a small number showed progression or developed clinical disease. This study demonstrates that many micro carcinomas (less than 10 mm in diameter) may be safely actively observed and not operated upon. They grow very slowly or not at all and were harmless. These studies add evidence for not performing FNA on most lesions of the thyroid that are less than 10 mm in diameter and suggest that a less aggressive treatment may be appropriate for these small papillary cancers.

Standard Treatment of Most Papillary Carcinomas

Most papillary carcinomas are neither minimal nor occult. These tumors are known to be microscopically multicentric in up to 80% of patients; they are also known occasionally to invade locally into the trachea or esophagus, to metastasize commonly to lymph nodes and later to the lungs and other tissues, and to recur clinically in the other thyroid lobe in 7% to 18% of patients if treated only by thyroid lobectomy (26a, 26b).

Most surgeons have treated papillary cancer that is not a micro-papillary lesion by near-total or total thyroidectomy (see Fig. 11), with appropriate central and lateral neck dissection when nodes are involved. The so-called cherry-picking operations, which remove only the enlarged lymph nodes, should not be performed. Rather, when lymph nodes with tumor are found in the lateral triangle, a modified radical neck dissection should be performed (Fig. 9a) (27). At the conclusion of a modified radical neck dissection, the lymph node–bearing tissue from the lateral neck is removed, whereas the carotid artery, jugular vein, phrenic nerve, sympathetic ganglia, brachial plexus, and spinal accessory nerve are spared and left in place. Sensory nerves--the posterior occipital, and greater auricular nerves--should be retained as well. On the left side, care should be exercised not to injure the thoracic duct. Prophylactic neck dissection of the lateral triangle should not be performed for papillary cancer; such dissections should be done only when enlarged nodes with tumor are found. For clarity and uniformity of reporting, the location of lymph nodes in the neck and upper mediastinum has been defined as shown in Fig. 8. Central lymph nodes (level VI) are frequently involved with metastases from ipsilateral thyroid cancers, as are levels III, IV, and V which are removed in most lateral neck dissections. Level II nodes may be involved as well and often require removal.

Should Prophylactic Central (Level 6) Lymph Node Dissections be Performed?

There is agreement that therapeutic central and lateral lymph node dissections should be performed at the time of total thyroidectomy when lymph nodes are suspicious or proved to harbor cancer by sonographic appearance or by FNA analyses preoperatively or when suspicious lymph nodes are found at operation. Prophylactic lateral lymph node dissections were common in the past, but have been abandoned for several decades or longer (12a). Delbridge and his group and others have proposed that unilateral or bilateral prophylactic central lymph node dissections (level 6 dissections) with parathyroid autotransplantation be performed in all cases of papillary thyroid cancer at the time of total thyroidectomy (12a, 27a). This, they state, might decrease mortality from thyroid cancer, would greatly decrease recurrence of cancer, and would further clarify who needs radioiodine therapy postoperatively. Some studies by very experienced surgeons demonstrate no increase in hypoparathyroidism or recurrent laryngeal nerve injuries after this procedure, while other equally competent surgeons have found an increase in permanent hypoparathyroidism (27b, 27c). We and others have not practiced routine bilateral central lymph node dissections prophylactically, but have generally reserved unilateral dissection for instances in which lymph nodes are clearly involved with tumor (27d). However, recently we have performed some prophylactic unilateral central lymph node dissections when a large carcinoma is present, as well as in some children.

Finally, surgeons with limited experience probably should not perform total or near-total thyroidectomy unless capable of doing so with a low incidence of recurrent laryngeal nerve injuries and permanent hypoparathyroidism, because these complications are serious. Otherwise, it may be advisable to refer such patients to a major medical center where such expertise is available.

Radioiodine Therapy

In the past, radioiodine therapy with 131I was commonly used in order to ablate any remaining normal thyroid remnant that was present in the thyroid bed after near-total or total thyroidectomy or to treat local or distant metastatic thyroid cancer (28, 28a). In order to prepare for RAI therapy, patients are placed on a low iodine diet for two to three weeks prior to treatment. Furthermore, in order to increase TSH levels to high values, either L-thyroxine is stopped for three weeks or injections of genetically engineered TSH (Thyrogen) are given for two days without stopping thyroxine. Then the radioiodine is given. More recently, there has been a trend to use radioiodine more sparingly in low risk patients with small tumors because this treatment has not been shown to definitively decrease mortality in such individuals. Radioiodine is commonly recommended postoperatively in patients with high risk papillary cancer and in all patients with metastatic disease, gross extrathyroidal extension, or when tumors are greater than 4 cm. It is recommended for selected patients with tumors 1 cm to 4 cm in diameter and others with lymph node metastases, but is optional in low risk patients with tumors less than 1 cm in diameter (21a).

Controversy remains in this area. Many reports indicate decreased recurrences after RAI is given to patients with tumors 1 cm or larger and without known metastases. Also, RAI ablation using low does (30 mCi) carries minimal risk, makes postoperative scans and the use of thyroglobulin (TG) determinations more effective and reliable, and simplifies follow-up. In higher doses, both short-term and long-term complications have been associated with radioactive iodine therapy. These are discussed elsewhere in these chapters.

If all or a substantial part of a lobe of normal thyroid remains after the first operation and radio-iodine therapy is to be given for treatment of metastases, this can be performed effectively after completion thyroidectomy has been performed. Usually, reoperative completion thyroidectomy is done and then the radioiodine is given.

Total Thyroidectomy versus thyroid Lobectomy? Controversies

Because randomized prospective studies have not been performed, controversy still exists over the proper treatment of papillary cancer in some patients. Most clinicians now accept that patients with this disease can be separated into different risk groups according to a set of prognostic factors. Using the AGES (29), AMES (30), or MACIS (31) criteria, which evaluate risk by age, distant metastases, extent of local involvement, and size (MACIS adds completeness of excision), approximately 80% of patients fall into a low-risk group. Treatment of this low-risk group is most controversial, perhaps because the cure rate is so good, certainly in the high 90% range. Should a lobectomy be done, or is bilateral thyroid resection more beneficial?

Low-risk Papillary Cancer

Hay and associates studied 1685 patients treated at the Mayo Clinic between 1940 and 1991; the mean follow-up period was 18 years (32). Of the total, 98% had complete tumor resection and 38% had initial nodal involvement. Twelve percent had unilateral lobectomy, whereas 88% had bilateral lobar resection; total thyroidectomy was done in 18%; while near-total thyroidectomy was performed in 60%. Cause-specific mortality at 30 years was 2%, and distant metastases occurred in 3%. These indices did not differ between the surgical groups; however, local recurrence and nodal metastases in the lobectomy group (14% and 19%, respectively) were significantly higher than the 2% and 6% rates seen after near-total or total thyroidectomy. This study is excellent. Although no differences in mortality were reported, a three-fold increase in tumor recurrence rates in the thyroid bed and lymph nodes was reported in the lobectomy group. In addition, this study recognizes patients’ anxiety about tumor recurrence, and their strong desire to face an operation only once and to be cured of their disease. If the operation can be done safely with low morbidity, this study supports the use of near-total or total thyroidectomy for patients with low-risk papillary cancer.

More recently, studies from Japan have shown excellent results (99% cause specific survival) for low-risk patients for papillary thyroid cancer treated by less than a total or near total thyroidectomy. This study and others like it are causing a reexamination of how low-risk papillary cancer is treated in the U.S.

High-risk Papillary Cancer

For high-risk patients, it is agreed that bilateral thyroid resection improves survival (29) and reduces recurrence rates (33) when compared with unilateral resection.

The Authors’ Series

In a retrospective study at the University of Chicago, total or near-total thyroidectomy has been practiced and most patients also received radioiodine ablation or treatment with radioiodine as indicated (34). In general, our studies (34, 35) and those of Mazzaferri and Jhiang (36) have demonstrated a decrease in mortality and in recurrence after near-total or total thyroidectomy followed by radioiodine ablation or therapy, when compared with lesser operations in papillary cancers 1 cm or greater.

Our series of patients have now been followed for a mean time of 27 years (36a). Predictors of death were increasing age, metastases, and advancing stages of disease. The mean time following diagnosis until recurrence of disease was 8.1 years and mean time of death was 9 years. However, 4% of recurrences and 17% of deaths were recognized only after a mean of 20 years, which emphasizes the importance of long-term followup of patients with papillary cancer.

FOLLICULAR CARCINOMA

True follicular carcinomas are far less common than papillary cancer. Remember that the “follicular variant” of papillary cancer should be classified and treated as a papillary carcinoma. Patients with follicular carcinoma are usually older than those with papillary cancer, and females predominate. Microscopically, the diagnosis of follicular cancer is made when vascular and/ or capsular invasion is present. Tumor multicentricity and lymph node metastases are far less common than in papillary carcinoma. Metastatic spread of tumor often occurs by hematogenous dissemination to the lungs, bones, and other peripheral tissues.

A follicular cancer that demonstrates only microinvasion of the capsule has a very good prognosis (37). In this situation, ipsilateral lobectomy is probably sufficient. However, for patients with follicular cancer that demonstrates gross capsular invasion or vascular invasion, the ideal operation is similar to that for papillary cancer, although the rationale for its performance differs. Near-total or total thyroidectomy should be performed not because of multicentricity but rather to facilitate later treatment of metastatic disease with radioiodine (36). Remnants of normal thyroid in the neck are ablated by radioiodine, and if peripheral metastases are detected (Fig. 12), they should be treated with high-dose radioiodine therapy. Although lymph node metastases in the lateral region of the neck are not ­commonly found, a modified radical neck dissection should be performed if metastatic nodes are identified.

Figure 12. Despite the fact that the chest radiograph was read as normal, a total body scan using radioiodine demonstrated uptake in both lung fields, thus signifying the presence of unknown metastatic thyroid cancer. Note that the thyroid has been removed surgically because no uptake of isotope is present in the neck.

Figure 12. Despite the fact that the chest radiograph was read as normal, a total body scan using radioiodine demonstrated uptake in both lung fields, thus signifying the presence of unknown metastatic thyroid cancer. Note that the thyroid has been removed surgically because no uptake of isotope is present in the neck.

Finally, regardless of the operation, patients with papillary or follicular cancer are usually treated for life with levothyroxine therapy in sufficient doses to suppress TSH to the appropriate level (36). Care should be taken to not cause cardiac or other problems from thyrotoxicosis, however. Recent studies have questioned whether TSH suppression is necessary in low-risk patients.

HÜRTHLE CELL TUMORS AND CANCER

Hürthle cell (oncocytic) tumors are thought to be variants of follicular neoplasms, but others regard them as a totally separate disease entity (38a). They are more difficult to treat than the usual follicular neoplasms, however, for several reasons (38): 1) the incidence of carcinoma varies from 5.3% to 62% in different clinical series; 2) benign-appearing tumors later metastasize in up to 2.5% of patients; and 3) Hürthle cell cancers are far less likely to concentrate radioiodine than are the usual follicular carcinomas, which makes treatment of metastatic disease particularly difficult.

The difficulty in diagnosing Hurtle cell cancers and differentiating them from benign lesions is shown in the following study. Of 54 patients with Hürthle cell tumors whom we treated, four had grossly malignant lesions (38). But during a mean follow-up period of 8.4 years, three additional Hürthle cell tumors were recognized as malignant after metastases were discovered. Thus, 7 of 54 (13%) of our patients who had a Hürthle cell tumor had Hürthle cell carcinoma. One of the seven patients with Hürthle cell cancer died of widespread metastases after 35 years, and the other six were currently free of disease.

In a more recent study, the size of the tumor was the major factor in determining whether or not a Hurthle cell neoplasm was malignant (38b). Overall, 20% of the tumors were malignant, but those less than 2 cm were always benign. Tumors 4 cm or larger had a greater than 50% chance of malignancy, and all tumors greater than 6 cm were universally cancers. Finally, overexpression of Cyclin D1 and D3 may help predict malignant behavior in fine needle aspirates suspicious for malignant behavior (38c).

We believe that treatment of these lesions should be individualized (38, 39). Total thyroid ablation is appropriate for frankly malignant Hürthle cell cancers, for all Hürthle cell tumors in patients who received low-dose childhood irradiation, for patients with associated papillary or follicular carcinomas, for all large tumors, certainly for those greater than 2 cm in diameter, and for patients whose tumors exhibit partial capsular invasion. On the other hand, single, well-encapsulated, benign-appearing Hürthle cell tumors that are small may be treated by lobectomy and careful follow-up because the chance that they will later exhibit malignant behavior is low (2.5% in our series and 1.5% among patients described in the literature) (38). Nuclear DNA analysis may aid the surgeon in recognizing tumors that are potentially aggressive, because such tumors usually demonstrate aneuploidy (40). Furthermore, increased genetic abnormalities have been shown in Hürthle cell carcinomas when compared with Hürthle cell adenomas (41).

In a review of follicular cancers at the University of Chicago, the overall mortality rate was 16%, twice that of papillary carcinomas (39). However, in non–Hürthle cell follicular cancers the mortality was 12%, whereas in Hürthle cell cancers it was 24%. This demonstrates the difficulty in treating metastatic disease which cannot be resected in the Hurthle group, because radioiodine therapy is almost always ineffective. These data are similar to those found in a recent large database study in which the overall death rate was approximately 18% for patients with Hurthle cell cancer and 11% for other well-differentiated thyroid cancers (41a).

ANAPLASTIC CARCINOMA

Anaplastic thyroid carcinoma remains one of the most aggressive of all cancers in humans. It makes up 1.3% to 9.8% of all thyroid cancers globally (1.7% of cancers in the U.S.) (40a, 40b, 40c). This tumor often arises from a differentiated thyroid cancer or from a prior goiter. It grows very rapidly, and systemic symptoms are common. Survival for most patients is measured in months. Median survival is 5 to 6 months, and one year survival is approximately 20%. The previously so-called small cell type is now known to be a lymphoma and is most often treated by a combination of external radiation and chemotherapy. The large cell type may be manifested as a solitary thyroid nodule early in its clinical course. If it is operated on at that time, near-total or total thyroidectomy should be performed, with appropriate central and lateral neck dissection. However, anaplastic cancer is almost always advanced when the patient is first evaluated. Widespread metastases to lymph nodes or to the lungs are common. Be sure to check vocal cord function preoperatively, for unilateral vocal cord paralysis is frequent.

With advanced disease, surgical cure is unlikely no matter how aggressively it is pursued. In particularly advanced cases, diagnosis by needle biopsy or by small open biopsy may be all that is appropriate. Sometimes the isthmus must be divided to relieve tracheal compression, or a tracheostomy might be beneficial. Most treatment, however, has been by external radiation therapy, chemotherapy, or both. Hyperfractionated external radiation therapy that uses several treatments per day has some enthusiasts, but complications may be high (42). Radioiodine treatment is almost always ineffective because tumor uptake is absent. Although some success has been observed with doxorubicin, prolonged remissions are rarely achieved, and multidrug regimens, especially with Paclitaxel or Cisplatin, and combinations of chemotherapy with radiation therapy are being tried (43). Although remissions do occur, cures have rarely been achieved in advanced cases. New experimental drugs (44) including monoclonal antibodies, kinase inhibitors, antiangiogenic drugs, and others are being tried because results of conventional therapy have been so dismal (40c). A phase 2 trial of efatutazone (alpha PPAR-gamma agonist) with paclitaxel versus efatutazone alone for patients with advanced anaplastic cancer is being conducted by the NIH (40d). American Thyroid Association guidelines for management of patients with anaplastic cancer including ethical considerations have recently been published (44a).

MEDULLARY THYROID CARCINOMA

Medullary thyroid carcinoma accounts for 5% to 8% of all thyroid cancers. It is a C-cell, calcitonin-producing tumor that contains amyloid or an amyloid-like substance. In addition to calcitonin, it may elaborate or secrete other peptides and amines such as carcinoembryonic antigen, serotonin, neurotensin, and a high-molecular-weight adrenocorticotropic hormone-like peptide. These substances may result in a carcinoid-like syndrome with diarrhea and Cushing’s syndrome, especially when widely metastatic tumor is present. Most medullary cancer of the thyroid is sporadic (about 70% to 80%), but it can also be transmitted in a familial pattern in 20% to 30% of cases. In the familial form, this tumor or its precursor, C-cell hyperplasia, occurs as a part of the multiple endocrine neoplasia type 2A (MEN2A) and type 2B (MEN2B) syndromes (45) (Table 3; Figs. 13 and 14); or, rarely, as part of the familial medullary thyroid cancer syndrome. MEN2A makes up approximately 95% of all hereditary cases and MEN2B approximately 5%. The MEN2 syndromes are transmitted as an autosomal-dominant trait, so 50% of the offspring would be expected to have this disease. Mutations of the RET oncogene on chromosome 10 (10 of 11.2) have been found to be the cause of the MEN2 syndromes (46). These defects are germ-line mutations and can therefore be found in blood samples. All patients with medullary thyroid carcinoma should probably be screened for hyperparathyroidism and pheochromocytoma (47). However, the risk of these two disease states accompanying the medullary cancer in MEN 2A is greatest in patients with a 630 or 634 RET mutation (50c). If a pheochromocytoma (or its precursor, adrenal medullary hyperplasia) is present, this should be operated on first because it has the greatest immediate risk to the patient. Family members, especially children, of a patient with medullary cancer of the thyroid should also be screened for medullary cancer of the thyroid if a patient has MEN2 with a RET oncogene mutation, but also if the tumor is bilateral or if C-cell hyperplasia is present. Genetic testing for RET mutations has largely replaced screening by calcitonin in family members. However, calcitonin and CEA measurements are still useful for evaluating patients with a thyroid mass when FNA analysis raises the possibility of medullary thyroid cancer.

The degree of clinical aggressiveness of a medullary cancer corresponds with the mutation of RET which is found (50d). A list of all known mutations with corresponding phenotype and risk profile can be found in the American Thyroid Association guidelines (50d). The most aggressive tumors called “highest risk, HST” are found in patients with the MEN2B and those with a RET codon M918T mutation. The “high risk, H” includes patients with RET codon C634 mutations, and the “moderate risk, MOD” category includes patients with RET codon mutations other than M918T and C634. In all of the hereditary cases (MEN2A, MEN2B, and familial MTC) germline mutations are found. However, about 50% of sporadic MTC have somatic RET mutations (only in the tumor). A RET codon M918T mutation found in a sporadic medullary cancer, for example, portends an aggressive clinical course and a poor prognosis, as well.

Figure 13. An 18-year-old female who demonstrates the appearance typically associated with multiple endocrine neoplasial type 2B (MEN2B) was found to have bilateral medullary carcinoma of the thyroid gland at surgery. The Marfan-like body habitus and facial features typically present in patients with MEN2B are clearly seen.

Figure 13. An 18-year-old female who demonstrates the appearance typically associated with multiple endocrine neoplasial type 2B (MEN2B) was found to have bilateral medullary carcinoma of the thyroid gland at surgery. The Marfan-like body habitus and facial features typically present in patients with MEN2B are clearly seen.

Figure 14. An 18-year-old female who demonstrates the appearance typically associated with multiple endocrine neoplasial type 2B (MEN2B) was found to have bilateral medullary carcinoma of the thyroid gland at surgery. Multiple neuromas of the tongue and lips are demonstrated. (Courtesy of Glen W. Sizemore.)

Figure 14. An 18-year-old female who demonstrates the appearance typically associated with multiple endocrine neoplasial type 2B (MEN2B) was found to have bilateral medullary carcinoma of the thyroid gland at surgery. Multiple neuromas of the tongue and lips are demonstrated. (Courtesy of Glen W. Sizemore.)

TABLE 3. Diseases Included in the MEN2 Syndromes
MEN2A MEN2B
Medullary carcinoma Medullary carcinoma
Pheochromocytoma

Pheochromocytoma

  • Hyperparathyroidism-unlikely
  • Ganglioneuroma phenotype
Hyperparathyroidism  
MEN = Multiple endocrine neoplasia

Medullary cancer spreads to the lymph nodes of the neck and mediastinum, and disseminates to the lungs, bone, liver, and elsewhere. The tumor is relatively radioresistant, does not take up radioiodine, and is not responsive to thyroid hormone suppression. Hence, an aggressive surgical approach is mandatory. The operation of choice for medullary cancer is total thyroidectomy coupled with aggressive resections of central and lateral lymph nodes, as well as mediastinal lymph nodes (46). Careful and extensive modified radical neck dissections are required. Reoperations for metastatic tumor were rarely considered to be rewarding until the work of Tisell and Jansson (48). Their work, and that of others (49), demonstrated that 25% to 35% of patients with ­elevated circulating calcitonin levels could be rendered eucalcitoninemic after extensive, meticulous, reoperative neck dissection under magnification to remove all the tiny lymph nodes. In other patients, computed tomography (CT) and magnetic resonance imaging (MRI) have localized some sites of tumor recurrence, whereas octreotide and meta-iodobenzylguanidine scanning have sometimes been helpful. Positron emission tomography combined with computerized tomography (PET-CT) have been successful in many patients (49a). Laparoscopic evaluation of the liver is helpful before a reoperation since small metastatic lesions on its surface can sometimes be identified.

Cure is best in young children who are found by genetic screening to have a mutated RET oncogene associated with the MEN2A syndrome. One hopes to operate on them when C-cell hyperplasia is present and before medullary cancer has started (46a). Patients with MEN2A syndrome have a better prognosis than do those with sporadic tumor (45). Patients with MEN2B syndrome have the most aggressive tumors and rarely survive to middle age. Thus, in recent years, children 5 years of age or younger who are found by genetic screening to have MEN2A with a 634 RET mutation have received prophylactic total thyroidectomy to prevent the development of medullary cancer. In children with MEN2B, screening for RET oncogene mutation should be done soon after birth because this cancer develops at a younger age than does MEN2A. With a mutated RET oncogene in MEN2B, total thyroidectomy should be done as early as possible within the first year of life in an experienced tertiary care setting. In MEN2B patients or infants and children older than one year, a prophylactic Level VI neck dissection should be considered as well since metastatic disease has been sometimes found in these very young children (50c). In all of these infants and children, preservation of parathyroid function and recurrent nerve integrity is of the highest priority (49). With these ­prophylactic operations, cures are possible. Careful genetic counseling is highly recommended.

Long-term studies of medullary cancer from the Mayo Clinic group have shown that in patients without initial distant metastatic involvement and with complete resection of their medullary cancer, the 20-year survival rate, free of distant metastatic lesions, was 81% (50). Overall 10- and 20-year survival rates were 63% and 44%, respectively. Thus, early diagnosis and complete initial resection of tumor are very important. A number of new therapies using tyrosine kinase inhibition are now being evaluated for metastatic disease (50a). Screening and treatment for pheochromocytoma and hyperparathyroidism in children with MEN syndromes are discussed elsewhere.

An excellent review of medullary cancer has been written by Pacini and colleagues (50b). In 2009, a very comprehensive set of guidelines for diagnosis, screening, surgical and medical treatment and followup of MEN2A and 2B, their pheochromocytomas and hyperparathyroidism as well as for sporadic medullary cancer has been published (50c).   A recent paper, “Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma” is excellent and is highly recommended (50d).

OPERATIVE TECHNIQUE FOR THYROIDECTOMY

While some groups have utilized local anesthesia with superficial cervical block, essentially all of our patients receive general anesthesia. The following description of thyroidectomy is used by many surgeons. The patient is placed in a supine position with the neck extended. A low collar ­incision is made and carried down through the subcutaneous tissue and platysma muscle (Fig. 15A). Currently, small incisions are the rule unless a large goiter is present. Superior and inferior subplatysmal flaps are developed, and the strap muscles are divided vertically in the midline and retracted laterally (Fig. 15B).

Figure 15. Upper left: Incision for thyroidectomy. The neck is extended and a symmetrical, gently curved incision is made 1 to 2 cm above the clavicle. In recent years, a much smaller incision is used except when a large goiter is present. Upper right: The sternohyoid and sternothyroid muscles are retracted to expose the surface of the thyroid lobe. Lower left: The surgeon’s hand retracts the gland anteriorly and medially to expose the posterior surfaces of the thyroid gland. The middle thyroid vein is identified, ligated and divided. Lower right: The superior thyroid vessels are ligated close to the thyroid capsule of the superior pole to avoid inadvertent injury to the external branch of the superior laryngeal nerve. This nerve can be seen in many cases.

Figure 15. Upper left: Incision for thyroidectomy. The neck is extended and a symmetrical, gently curved incision is made 1 to 2 cm above the clavicle. In recent years, a much smaller incision is used except when a large goiter is present. Upper right: The sternohyoid and sternothyroid muscles are retracted to expose the surface of the thyroid lobe. Lower left: The surgeon’s hand retracts the gland anteriorly and medially to expose the posterior surfaces of the thyroid gland. The middle thyroid vein is identified, ligated and divided. Lower right: The superior thyroid vessels are ligated close to the thyroid capsule of the superior pole to avoid inadvertent injury to the external branch of the superior laryngeal nerve. This nerve can be seen in many cases.

The thyroid isthmus is often divided early in the course of the operation. The thyroid lobe is rotated medially. The middle thyroid vein is ligated (Fig. 15C). The superior pole of the thyroid is dissected free, and care is taken to identify and preserve the external branch of the superior laryngeal nerve (see Fig. 6). The superior pole vessels are ligated adjacent to the thyroid lobe, rather than cephalad to it, to prevent damage to this nerve (Fig. 15D). This nerve can be visualized in over 90% of patients if it is carefully dissected (51). The inferior thyroid artery and recurrent laryngeal nerve are identified (Fig. 15E). To preserve blood supply to the parathyroid glands, the inferior thyroid artery should not be ligated laterally as a single trunk; rather, its branches should be ligated individually on the capsule of the lobe after they have supplied the parathyroid glands (Fig. 15F). The parathyroid glands are identified, and an attempt is made to leave each with an adequate blood supply while moving the gland off the thyroid lobe. Any parathyroid gland that appears to be devascularized can be placed in saline and later minced and implanted into the sternocleidomastoid muscle after a frozen section biopsy confirms that it is, in fact, a parathyroid gland. Care is taken to identify the recurrent laryngeal nerve and to gently follow along its course if a total lobectomy is to be done (Fig. 15G). The nerve is gently unroofed from surrounding tissue, with care taken to avoid trauma to it. The nerve is in greatest danger near the junction of the trachea with the larynx, where it is adjacent to the thyroid gland. Once the nerve and parathyroid glands have been identified and preserved, the thyroid lobe can be removed from its tracheal attachments by dividing the ligament of Berry (Fig. 15G). The contralateral thyroid lobe is removed in a similar manner when total thyroidectomy is performed. A near-total thyroidectomy means that a very small amount of thyroid tissue is left on the contralateral side to protect the parathyroid glands and recurrent nerve. Careful hemostasis and visualization of all important anatomic structures are mandatory for success. Some surgeons utilize the harmonic scalpel or electrothermal bipolar vessel sealing system and believe that they decrease the time of operation. However, one must be careful not to cause thermal damage (51a).

 

Figure 15. Top: With careful retraction of the lobe medially, the inferior thyroid artery is placed under tension. This facilitates exposure of the recurrent laryngeal nerve and the parathyroid glands. Lower left: The inferior thyroid artery is not ligated as a single trunk, but rather its tertiary branches are ligated and divided on the thyroid capsule. This preserves the blood supply to the parathyroid glands, which can be moved away from the thyroid lobe. Lower right: The ligament of Berry is then ligated and divided and the thyroid lobe is removed. (Courtesy of Drs. Alan P. B. Dackiw and Orlo H. Clark.)

Figure 15. Top: With careful retraction of the lobe medially, the inferior thyroid artery is placed under tension. This facilitates exposure of the recurrent laryngeal nerve and the parathyroid glands. Lower left: The inferior thyroid artery is not ligated as a single trunk, but rather its tertiary branches are ligated and divided on the thyroid capsule. This preserves the blood supply to the parathyroid glands, which can be moved away from the thyroid lobe. Lower right: The ligament of Berry is then ligated and divided and the thyroid lobe is removed. (Courtesy of Drs. Alan P. B. Dackiw and Orlo H. Clark.)

When closing, some surgeons do not tightly approximate the strap muscles in the midline; this allows drainage of blood superficially and thus prevents a hematoma in the closed deep space. Furthermore, one can obtain better cosmesis by not approximating the platysmal muscle. Rather, the dermis is approximated by interrupted 4-0 sutures, and the epithelial edges are approximated with a running subcuticular 5-0 absorbable suture. Sterile paper tapes (Steri-strips) are then applied and left in place for approximately one to two weeks. When it is needed, a small suction catheter is inserted through a small stab wound; it is generally removed within 12 hours.

SUBTOTAL THYROIDECTOMY

Bilateral subtotal or near total lobectomy is the operation which is often used for Graves’ disease. An alternative operation, which is equally good, is lobectomy on one side and subtotal or near total lobectomy on the other side (Dunhill procedure). Once more, the parathyroid glands and recurrent nerves should be identified and preserved. Great care should be taken to not damage the recurrent laryngeal nerve when cutting across or suturing the thyroid lobe. At the end of the operation, 1 or 2 grams or less of thyroid tissue is usually left in place. The aim is to achieve an euthyroid state without a high recurrence of hyperthyroidism. When the operation is performed for severe ophthalmopathy, however, near-total or total thyroidectomy is performed.

After thyroidectomy, even if a modified neck dissection is done for carcinoma, many patients can be safely discharged on the first postoperative day. Others are kept longer if the need arises. Some surgeons do not think that it is safe to discharge a patient on the day of surgery because of the risks of bleeding or hypocalcemia; however, same-day discharge is being practiced at some centers, usually after lobectomy (52).

ALTERNATIVE TECHNIQUE OF THYROIDECTOMY

An alternative technique of thyroidectomy is practiced by some excellent surgeons and is used by the authors in some operations (6, 53). In this technique, the dissection is begun on the thyroid lobe and the parathyroids are moved laterally, as described previously. However, the recurrent laryngeal nerve is not dissected along its length, but rather small bites of tissue are carefully divided along the thyroid capsule until the nerve is encountered near the ligament of Berry. Proponents of this technique believe that visualization of the recurrent laryngeal nerve by its early dissection may lead to greater nerve damage; however, most surgeons feel that seeing the nerve and knowing its pathway is safer and facilitates the dissection in many instances.

MINIMALLY INVASIVE OPTIONS FOR THYROIDECTOMY

Over recent years, the development of ultrasonic shears and other electrothermal bipolar vessel sealing devices for hemostasis and small size endoscopes has allowed surgeons to perform thyroidectomies through much smaller incisions than using traditional techniques. Two different approaches have been taken to minimally invasive thyroidectomies. One technique, largely popularized in areas of the Far East such as Japan, China, and Korea, involves making incisions away from the neck in hidden areas such as in the axillae, chest, or the areola of the breast. The surgeon then creates a tunnel up to the neck where the thyroidectomy is performed with endoscopic instruments utilizing the endoscope for visualization. Approaches such as this are generally performed with low pressure insufflation and can completely avoid any incisions on the neck itself. Thus, the major advantage is a thyroidectomy without a neck scar (54-57). Most reports suggest significantly longer operative times, especially during a learning period. Perhaps most concerning to many American surgeons with these approaches is that if bleeding problems are encountered in the course of the thyroid dissection, a separate neck incision may need to be made to solve the problem. Additionally, recent reports have suggested the possibility that recurrent thyroid cancer can develop in the subcutaneous tunnel after the performance of an endoscopic thyroidectomy (57a). Such complications will need to be carefully evaluated before wide acceptance of this technique can be recommended in cases of malignancy.

An alternative technique, developed by Dr. Paolo Miccoli, more widely utilized in Europe and to a less extent in the United States, utilizes a smaller incision than usual that is placed in the conventional location in the neck (58, 59). In general, a 1.5 to 2.0 cm incision is made in a conventional location in the neck and after the strap muscles are retracted from the thyroid gland, a 5 mm 30 degree endoscope is introduced into the incision. The scope is utilized to visualize the tissue along the lateral aspect of the thyroid gland and especially for the superior pole vessels. Usually after the superior and lateral aspects of the thyroid gland have been dissected free, the parathyroid glands and recurrent nerve are visualized and then the thyroid lobe is delivered through the neck incision. The remainder of the operation is performed in the conventional manner through the small cervical incision.

Several authors in the United States have reported good results in small series with this video-assisted approach (60, 61, 61a). A significant benefit of this approach is that the incision is in the usual location so that if any bleeding results in difficulty with visualization during the procedure, the incision can be enlarged and a conventional thyroidectomy can readily be completed. Most authors have found this approach to be similar to conventional thyroidectomy in operative time, although the small neck incision does limit the size of the thyroid gland that could be resected utilizing this technique (62-64).

Recently Miccoli and his group have shown that minimally invasive video-assisted thyroidectomy and conventional thyroidectomy have the same rate of hypoparathyroidism and recurrent laryngeal nerve injury following operations for papillary thyroid cancer (64a). Furthermore, no differences in outcome were noted at five years or in exposure to radioiodine therapy, suggesting the same degree of completeness of resection by both techniques. They believe that minimally invasive video-assisted thyroidectomy is a valid option to treat low and intermediate risk papillary thyroid cancer. In the United States, minimally invasive video-assisted thyroidectomy is offered in few specialized centers for selected patients with small thyroid nodules (usually less than 3 cm) and without evidence of thyroiditis. Except in the hands of surgeons very experienced in the technique, it should not be utilized for the treatment of most thyroid cancers. An excellent review of these minimally invasive techniques was written by Grogan and Duh (63a).

Robotic Transaxillary Thyroid Surgery

Using the da Vinci robot, several groups, largely from Korea, have developed a transaxillary approach to thyroidectomy (64b, 64c, 64d). An incision between 5 and 10 cm is made unilaterally or in both axillae and the dissection progresses from the axilla to the neck with performance of a lobectomy or total thyroidectomy. In malignant cases, a neck dissection can be performed as well (64c). In their hands, robotic surgery can be performed with low complications, but the procedure takes longer than open surgery. There is a prolonged learning curve. Furthermore, use of the robot is expensive. While reimbursement in Korea was higher for robotic surgery than for conventional thyroidectomy, in the U.S. reimbursement was the same for both procedures and the costs were higher for robotic thyroidectomy. The obvious advantage is cosmetic, since no scar is placed in the neck.

Initially there was considerable enthusiasm for robotic thyroidectomy in the U.S. However, because of increased costs and probably increased complications in American patients, who are generally larger than Korean counterparts, Intuitive Surgical, the company that makes the robots, decided it could no longer support the use of its robots for thyroid surgery. In 2010 to 2011 in the National Cancer Database, 224 patients with thyroid cancer underwent robotic thyroidectomy and close to 58,000 patients underwent open surgery (64e). More recently, fewer robotic thyroidectomy operations are being done in the U.S. and this procedure is limited to only a few institutions. In Korea, on the other hand, this procedure remains very popular.

Transoral Thyroidectomy

Experiments have been conducted to determine whether or not it is possible and safe to perform a thyroidectomy through the floor of the mouth (64f). Once more, the objective is to eliminate a scar in the neck. A number of investigators have moved this procedure to clinical practice. Both endoscopic and robotic techniques have been described. A summary of the experience in 2014 and the complications associated with these techniques are reviewed by Clark et al (64g).

POSTOPERATIVE COMPLICATIONS

Many authors have reported large series of thyroidectomies with no deaths. In other reports, mortality does not differ greatly from that from anesthesia alone. However, each patient should be evaluated for comorbidities, for it has been shown that elderly patients are more likely to suffer postoperative complications and longer hospitalizations than their younger counterparts following thyroidectomy (64h). Five major complications are associated with thyroid surgery: 1) thyroid storm, 2) wound hemorrhage, 3) wound infection, 4) recurrent laryngeal nerve injury, and 5) hypoparathyroidism.

Thyroid Storm

Thyroid storm reflects an exacerbation of a thyrotoxic state; it is seen most often in Graves’ disease, but it occurs less commonly in patients with toxic adenoma or toxic multinodular goiter. Clinical manifestations and management of thyroid storm are discussed elsewhere in this text.

Wound Hemorrhage

Wound hemorrhage with hematoma is an uncommon complication reported in 0.3% to 1.0% of patients in most large series. However, it is a well-recognized and potentially lethal complication (52). A small hematoma deep to the strap muscles can compress the trachea and cause respiratory distress. A small suction drain placed in the wound is not usually adequate for decompression, especially if bleeding occurs from an arterial vessel. Swelling of the neck and bulging of the wound can be quickly followed by respiratory impairment.

Wound hemorrhage with hematoma is an emergency situation, especially if any respiratory compromise is present. Treatment consists of immediately opening the wound and evacuating the clot, even at the bedside. Pressure should be applied with a sterile sponge and the patient returned to the operating room. Later, the bleeding vessel can be ligated in a careful and more leisurely manner under optimal sterile conditions with good lighting in the operating room. The urgency of treating this condition as soon as it is recognized cannot be overemphasized, especially if respiratory compromise is present.

Injury to the Recurrent Laryngeal Nerve

Injuries to the recurrent laryngeal nerve occur in 1% to 2% of thyroid operations when performed by experienced neck surgeons, and at a higher prevalence when thyroidectomy is done by a less experienced surgeon. They occur more commonly when thyroidectomy is performed for malignant disease, especially if a total thyroidectomy is done. Sometimes the nerve is purposely sacrificed if it runs into an aggressive thyroid cancer. Nerve injuries can be unilateral or bilateral and temporary or permanent, and they can be deliberate or accidental. Loss of function can be caused by transection, ligation, clamping, traction, or handling of the nerve. Tumor invasion can also involve the nerve. Occasionally, vocal cord impairment occurs as a result of pressure from the balloon of an endotracheal tube on the recurrent nerve as it enters the larynx. In unilateral recurrent nerve injuries, the voice becomes husky because the vocal cords do not approximate one another. Shortness of breath and aspiration of liquids sometimes occur as well. Most nerve injuries are temporary and vocal cord function returns within several months; it certainly returns within 9 to 12 months if it is to return at all. If no function returns by that time, the voice can be improved by operative means. The choice is insertion of a piece of Silastic to move the paralyzed cord to the midline; this procedure is called a laryngoplasty. Early in the course of management of a patient with hoarseness or aspiration, the affected vocal cord can be injected with various substances to move it to the midline and to alleviate or improve these symptoms.

Bilateral recurrent laryngeal nerve damage is much more serious, because both vocal cords may assume a medial or paramedian position and cause airway obstruction and difficulty with respiratory toilet. Most often, tracheostomy is required. In the authors’ experience, permanent injuries to the recurrent laryngeal nerve are best avoided by identifying and carefully tracing the path of the recurrent nerve. Accidental transection occurs most often at the level of the upper two tracheal rings, where the nerve closely approximates the thyroid lobe in the area of Berry’s ligament. If recognized, many believe that the transected nerve should be reapproximated by microsurgical techniques, although this is controversial. A number of procedures to later reinnervate the laryngeal muscles have been performed with improvement of the voice in unilateral nerve injuries, but with only limited success when a bilateral nerve injury has occurred (65).

Injury to the external branch of the superior laryngeal nerve may occur when the upper pole vessels are divided (Fig. 6) if the nerve is not visualized (9). This injury results in impairment of function of the ipsilateral cricothyroid muscle, a fine tuner of the vocal cord. This injury causes an inability to forcefully project one’s voice or to sing high notes because of loss of function of the cricothyroid muscle. Often, this disability improves during the first few months after surgery.

Recurrent Laryngeal Nerve Monitoring

Many surgeons have sought to try to further diminish the low incidence of recurrent laryngeal nerve (RLN) injury by use of nerve monitoring devices during surgery. Although several devices have been utilized, all have in common some means of detecting vocal cord movement when the RLN or the ipsilateral vagus nerve is stimulated. Many small series have been reported in the literature assessing the potential benefits of monitoring to decrease the incidence of nerve injury (65a, 65b). Given the low incidence of RLN injury, it is not surprising that no study has shown a statistically significant decrease in RLN injury when using a nerve monitor. The largest series in the literature by Dralle reported on a multi-institutional German study of 29,998 nerves at risk in thyroidectomy (65c). Even with a study this large, no statistically significant decrease in rates of RLN injury could be shown with nerve monitoring. Despite this, the use of nerve monitoring has become more popular.

Among the problems of nerve monitoring technology are that the devices can malfunction, often because of endotracheal tube misplacement, so that the surgeon cannot depend on the device to always identify the nerve. Proponents of nerve monitoring suggest that the technology is helpful even if a statistically significant decrease in the rate of RLN cannot be shown. Goretzki and his group, for example, have published data that when operating upon bilateral thyroid disease, if nerve monitoring suggests a nerve injury on the first side, they have modified or restricted the resection of the contralateral thyroid lobe (65d). In this way, they have decreased or eliminated the incidence of bilateral RLN injuries. This is very important.

Many authors have suggested that RLN monitors may be most helpful in difficult reoperative cases when significant scar tissue is encountered, and have limited their use to such cases. This has not generally been shown to be the case. Most authors have advocated routine use (67a). Nerve monitoring technology in thyroid surgery should never take the place of meticulous dissection. Surgeons may choose to use the technology, but the data do not support the suggestion that nerve monitors allow thyroid surgery to be performed in a safer fashion than that by a good surgeon utilizing careful technique. An excellent international guide to the use of electrophysiologic RLN monitoring during thyroid and parathyroid surgery has been published (65f). Furthermore, a review of the medical, legal, and ethical aspects of RLN monitoring has been written by Angelos (65e).

A recent advance is continuous intraoperative recurrent laryngeal monitoring, done by continuously stimulating the ipsilateral vagus nerve. It has been shown that a decrease in signal amplitude and an increase in signal latency often occur before some recurrent laryngeal nerves are permanently damaged. In a recent experimental study, it was shown that modification of the surgical maneuver by the operator (such as release of traction) led to recovery of the EMG changes and aversion of the impending recurrent nerve injury (65G). This technique shows great promise for helping the surgeon intraoperatively, for it signals that the nerve is in danger and that the surgeon should stop doing whatever is causing the problem. A note of caution is appropriate, however. Terris and associates recently described two serious adverse events in nine patients who underwent continuous vagal nerve monitoring—hemodynamic instability and reversible vagal neuropraxia—both attributable to the monitoring apparatus (65h). They feel that this technique may cause harm and they have stopped its routine use. Clearly, further clarification and study must be done, but surgeons should use caution until it’s safety has been proved.

HYPOPARATHYROIDISM

Postoperative hypoparathyroidism can be temporary or permanent. The incidence of permanent hypoparathyroidism has been reported to be as high as 20% when total thyroidectomy and radical neck dissection are performed, and as low as 0.9% for subtotal thyroidectomy. Other excellent neck surgeons have reported a lower incidence of permanent hypoparathyroidism, even about one percent following total thyroidectomy (66). Postoperative hypoparathyroidism is rarely the result of inadvertent removal of all of the parathyroid glands but is more commonly caused by disruption of their delicate blood supply. Devascularization can be minimized during thyroid lobectomy by dissecting close to the thyroid capsule, by carefully ligating the branches of the inferior thyroid artery on the thyroid capsule distal to their supply of the parathyroid glands (rather than ligating the inferior thyroid artery as a single trunk), and by treating the parathyroids with great care. If a parathyroid gland is recognized to be ischemic or nonviable during surgery, it can be autotransplanted often after identification by frozen section. The gland is minced into 1 to 2 mm cubes and placed into a pocket(s) in the sternocleidomastoid muscle.

Postoperative hypoparathyroidism results in hypocalcemia and hyperphosphatemia; it is manifested by circumoral numbness, tingling of the fingers and toes, and intense anxiety occurring soon after surgery. Chvostek’s sign appears early, and carpopedal spasm can occur. Symptoms develop in most patients when the serum calcium level is less than 7.5 to 8 mg/dL. Parathyroid hormone is low or absent in most cases of permanent hyopoparathyroidism.

Patients who have had a thyroid lobectomy rarely develop significant hypocalcemia postoperatively since two contralateral parathyroid glands are left intact. Many of these patients may be discharged on the day of operation if they are otherwise satisfactory. However, patients who have had a total or near total thyroidectomy for cancer or for Graves’ disease are at greater risk of a low calcium and are generally observed in the hospital postoperatively. We have found the one hour postoperative PTH level to be equivalent to the parathyroid hormone level drawn the following morning. A value of 15 pg/ml or greater at one hour is very reassuring and is rarely associated with symptomatic postoperative hypocalcemia or with permanent hypoparathyroidism. A central lymph node dissection makes transient hypocalcemia more likely.

As well as one hour PTH, we measure the serum calcium and parathyroid hormone levels approximately 12 hours after operation and thereafter. Most patients are able to leave the hospital on the morning after surgery if they are asymptomatic and their serum calcium level is 7.8 mg/dL or above. Oral calcium pills are used liberally. Patients with severe symptomatic hypocalcemia are treated in the hospital with 1 g (10 mL) of 10% calcium gluconate infused intravenously over several minutes. Then, if necessary, 5 g of this calcium solution is placed in each 500 mL intravenous bottle to run continuously, starting with approximately 30 mL/hour. Oral calcium, usually as calcium carbonate (1250 mg to 2500 mg four times per day), should be started. Each 1250 mg pill of calcium carbonate contains 500 mg of calcium. With this treatment regimen most patients become asymptomatic. The intravenous therapy is tapered and stopped as soon as possible, and the patient is sent home and told to take oral calcium pills. This condition is referred to as transient or temporary hypocalcemia or transient hypoparathyroidism. Serum phosphorus determinations are helpful to rule out bone hunger in which both calcium and phosphorus levels are low, while PTH may be normal.

Management of persistent severe hypocalcemia requires the addition of a vitamin D preparation to facilitate the absorption of oral calcium. We prefer the use of 1,25-­dihydroxyvitamin D (Calcitriol) because it is the active metabolite of vitamin D and has a more rapid action than regular vitamin D. Calcitriol 0.5 mcg with oral calcium carbonate therapy is given four times daily for the first several days, then this priming dose of vitamin D is reduced. The usual maintenance dose for most patients with permanent hypoparathyroidism is Calcitriol 0.25 to 0.5 mcg once daily, along with calcium carbonate, 500 mg Ca 2+ once or twice daily, although some patients require larger doses. When high doses of vitamins are used, serum calcium levels must be monitored carefully after discharge, and the dosage of the medications is adjusted promptly to prevent hypercalcemia as well as hypocalcemia. Finally, the serum parathyroid hormone level should be analyzed periodically to determine whether permanent hypoparathyroidism is truly present, because the authors and others have seen cases of postoperative tetany, perhaps caused by “bone hunger,” that later resolved completely. In such cases, circulating parathyroid hormone is normal and all therapy can be stopped. Remember that in bone hunger, both the serum calcium and phosphorus values are low, whereas in hypoparathyroidism, the serum calcium value is low but the phosphorus level is elevated. Permanent hypoparathyroidism is usually not diagnosed until at least six months have passed and parathyroid hormone remains low or absent.

McHenry has shown that the incidence of complications following thyroidectomy varies greatly (66a). In general, those surgeons with excellent training and a large experience with this operation have fewer complications, particularly following cancer procedures and reoperative surgery.

DEVELOPMENTAL ABNORMALITIES OF THE THYROID

To understand the different thyroid anomalies, it is important to briefly review normal development of this gland. The ­thyroid is embryologically an offshoot of the primitive alimentary tract, from which it later becomes separated (Figs. 16 and 17) (67-70). During the third to fourth week in utero, a median anlage of epithelium arises from the pharyngeal floor in the region of the foramen cecum of the tongue (i.e., at the junction of the anterior two thirds and the posterior third of the tongue). The main body of the thyroid, referred to as the median lobe or median thyroid component, follows the descent of the heart and great vessels and moves caudally into the neck from this origin. It divides into an isthmus and two lobes, and by 7 weeks it forms a “shield” over the front of the trachea and thyroid cartilage. It is joined by a pair of lateral thyroid lobes originating from the fourth and fifth branchial pouches (Fig. 17). From these lateral thyroid components, now commonly called the ultimobranchial bodies, C cells (parafollicular cells) enter the thyroid lobes. C cells contain and secrete calcitonin and are the cells that give rise to medullary carcinoma of the thyroid gland. Williams and associates have described cystic structures in the neck near the upper parathyroid glands in cases in which thyroid tissue was totally lingual in location (71). These cysts contained both cells staining for calcitonin and others staining for thyroglobulin. This study, they believe, offers evidence that the ultimobranchial body contributes both C cells and follicular cells to the thyroid gland of humans.

Figure 16. Early embryologic development of the pharyngeal anlage in a 4mm embryo. Note the beginning of thyroid development in the median thyroid diverticulum. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2d ed. Philadelphia, WB Saunders, 1980, p 7; adapted from Weller GL: Development of the thyroid, parathyroid and thymus glands in man. Contrib Embryol Carnegie Inst Wash 24:93–142, 1933.)

Figure 16. Early embryologic development of the pharyngeal anlage in a 4mm embryo. Note the beginning of thyroid development in the median thyroid diverticulum. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2d ed. Philadelphia, WB Saunders, 1980, p 7; adapted from Weller GL: Development of the thyroid, parathyroid and thymus glands in man. Contrib Embryol Carnegie Inst Wash 24:93–142, 1933.)

Figure 17. Stages in the development of the thyroid gland. A, 1, Thyroid primordium and pharyngeal epithelium of a 4.5mm human embryo; 2, section through the same structure showing a raised central portion. B, 1, Thyroid primordium of a 6.5mm embryo; 2, section through the same structure. C, 1, Thyroid primordium of an 8.2mm embryo beginning to descend; 2, lateral view of the same structure. D, Thyroid primordium of an 11mm embryo. The connection with the pharynx is broken, and the lobes are beginning to grow laterad. E, Thyroid gland of a 13.5mm embryo. The lobes are thin sheets curving around the carotid arteries. Several lacunae, which are not to be confused with follicles, are present in the sheets. (From Weller GL: Development of the thyroid, parathyroid and thymus glands in man. Contrib Embryol Carnegie Inst Wash 24:93–142, 1933.)

Figure 17. Stages in the development of the thyroid gland. A, 1, Thyroid primordium and pharyngeal epithelium of a 4.5mm human embryo; 2, section through the same structure showing a raised central portion. B, 1, Thyroid primordium of a 6.5mm embryo; 2, section through the same structure. C, 1, Thyroid primordium of an 8.2mm embryo beginning to descend; 2, lateral view of the same structure. D, Thyroid primordium of an 11mm embryo. The connection with the pharynx is broken, and the lobes are beginning to grow laterad. E, Thyroid gland of a 13.5mm embryo. The lobes are thin sheets curving around the carotid arteries. Several lacunae, which are not to be confused with follicles, are present in the sheets. (From Weller GL: Development of the thyroid, parathyroid and thymus glands in man. Contrib Embryol Carnegie Inst Wash 24:93–142, 1933.)

As the gland moves downward, it leaves behind a trace of epithelial cells known as the thyroglossal tract. From this structure both thyroglossal duct cysts and the pyramidal lobe of the thyroid develop. The mature thyroid gland may take on many different configurations depending on the embryologic development of the thyroid and its descent (Fig. 18).

Figure 18. Variations of normal adult thyroid anatomy resulting from embryologic descent and division of the thyroid gland. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2d ed. Philadelphia, WB Saunders, 1980; adapted from Gray SW, Skandalakis JE: Embryology for Surgeons. Philadelphia, WB Saunders, 1972.)

Figure 18. Variations of normal adult thyroid anatomy resulting from embryologic descent and division of the thyroid gland. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2d ed. Philadelphia, WB Saunders, 1980; adapted from Gray SW, Skandalakis JE: Embryology for Surgeons. Philadelphia, WB Saunders, 1972.)

THYROID ABNORMALITIES

The median thyroid anlage may, on rare occasions, fail to develop. The resultant athyrosis, or absence of the thyroid gland, is associated with cretinism. The anlage also may differentiate in locations other than the isthmus and lateral lobes. The most common developmental abnormality, if looked on as such, is the pyramidal lobe (Fig. 18), which has been reported to be present in as many as 80% of patients in whom the gland was surgically exposed. Usually, the pyramidal lobe is small; however, in Graves’ disease or in lymphocytic thyroiditis, it is often enlarged and is commonly clinically palpable. The pyramidal lobe generally lies in the midline but can arise from either lobe. Origin from the left lobe is more common than is origin from the right lobe (72).

THYROID HEMIAGENESIS

More than 100 cases have been reported in which only one lobe of the thyroid is present (73). The left lobe is absent in 80% of these patients. Often, the thyroid lobe that is present is enlarged, and both hyperthyroidism and hypothyroidism have been reported at times. Females are affected three times as often as males. Both benign and malignant nodules have been reported in this condition (74).

Other variations involving the median thyroid anlage represent an arrest in the usual descent of part or all of the thyroid-forming material along the normal pathway. Ectopic thyroid development can result in a lingual thyroid (Fig. 19) or in thyroid tissue in a suprahyoid, infrahyoid, or intratracheal location. Persistence of the thyroglossal duct as a sinus tract or as a cyst (called a thyroglossal duct cyst) is the most common of the clinically important anomalies of thyroid development (Fig. 20). Finally, the entire gland or part of it may descend more caudally; this results in thyroid tissue located in the superior mediastinum behind the sternum, adjacent to the aortic arch or between the aorta and pulmonary trunk, within the upper portion of the pericardium, and even within the interventricular septum of the heart. Most intrathoracic goiters, however, are not true anomalies, but rather are extensions of pathologic elements of a normally situated gland into the anterior or posterior mediastinum. Each of these abnormalities is discussed in greater depth.

ECTOPIC THYROID

Lingual Thyroid

A lingual thyroid is relatively rare and is estimated to occur in 1 in 3000 cases of thyroid disease. However, it represents the most common location for functioning ectopic thyroid tissue. Lingual thyroid tissue is associated with an absence of the normal cervical thyroid in 70% of cases. It occurs much more commonly in women than in men.

The diagnosis is usually made by the discovery of an incidental mass on the back of the tongue in an asymptomatic patient (Fig. 19). The mass may enlarge and cause dysphagia, dysphonia, dyspnea, or a sensation of choking (75). Hypothyroidism is often present and may cause the mass to enlarge and become symptomatic, but hyperthyroidism is very unusual. In women, symptomatic lingual thyroid glands develop during puberty or early adulthood in most cases. Buckman, in his review of 140 cases of symptomatic lingual thyroids in females, reported that 30% occurred in puberty, 55% between the ages of 18 and 40 years, 10% at menopause, and 5% in old age (76). He attributed this distribution to hormonal disturbances, which are more apparent in female subjects during puberty and may be precipitated by pregnancy. The incidence of malignancy in lingual thyroid glands is low (77). The diagnosis of a lingual thyroid should be suspected when a mass is detected in the region of the foramen cecum of the tongue, and it is definitively established by radioisotope scanning (see Fig. 19).

 

Figure 19. Left , The appearance of a large lingual thyroid. Right , A radioiodine scan demonstrating all activity to be above the hyoid bone, with no evidence of the presence of normally placed thyroid issue. (From Netter RA: Endocrine system and selected metabolic diseases. In Ciba Collection of Medical Illustrations. Summit, NJ, Ciba-Geigy, 1974, p 45.)

Figure 19. Left , The appearance of a large lingual thyroid. Right , A radioiodine scan demonstrating all activity to be above the hyoid bone, with no evidence of the presence of normally placed thyroid issue. (From Netter RA: Endocrine system and selected metabolic diseases. In Ciba Collection of Medical Illustrations. Summit, NJ, Ciba-Geigy, 1974, p 45.)

The usual treatment of this condition is thyroid hormone therapy to suppress the lingual thyroid and reduce its size. Only rarely is surgical excision necessary. Indications for extirpation include failure of suppressive therapy to reduce the size, ulceration, hemorrhage, and suspicion of malignancy (78). Autotransplantation of thyroid tissue has been tried on rare occasions when no other thyroid tissue is present, and it has apparently been successful. A lingual thyroid was reported in two brothers, which suggests that this condition may be inherited (79).

Suprahyoid and Infrahyoid Thyroid

In these cases, thyroid tissue is present in a midline position above or below the hyoid bone. Hypothyroidism with elevation of thyrotropin (TSH) secretion is commonly present because of the absence of a normal thyroid gland in most instances. An enlarging mass commonly occurs during infancy, childhood, or later life. Often, this mass is mistaken for a thyroglossal duct cyst, because it is usually located in the same anatomic position (80). If it is removed, all thyroid tissue may be ablated, a consequence that has definite physiologic as well as possible medicolegal implications. To prevent total thyroid ablation, it is recommended that an ultrasound examination be performed in all cases of thyroglossal duct cyst before its removal to be certain that a normal thyroid gland is present. Furthermore, before removing what appears to be a thyroglossal duct cyst, a prudent surgeon should be certain that no solid areas are present. If any doubt exists, the normal thyroid gland should be explored and ­palpated. Finally, if ectopic thyroid tissue rather than a thyroglossal duct cyst is encountered at surgery in an infant, its blood supply should be preserved; the ectopic gland divided vertically; and each half translocated laterally, deep to the strap muscles, where it is no longer manifested as a mass. If normal thyroid tissue is demonstrated to be present elsewhere or in the adult, it may be ­better to remove the ectopic tissue rather than transplant it, because carcinoma arising from these developmental abnormalities, although rare, has been reported.

THYROGLOSSAL DUCT CYSTS

Both cysts and fistulas can develop along the course of the thyroglossal duct (Fig. 20) (81). These cysts are the most common anomaly in thyroid development seen in clinical practice (82). Normally, the thyroglossal duct becomes obliterated early in embryonic life, but occasionally it persists as a cyst. Such lesions occur equally in males and females. They are seen at birth in about 25% of cases; most appear in early childhood; and the rest, about one third, become apparent only after age 30 years (83). Cysts usually appear in the midline or just off the midline between the isthmus of the thyroid and the hyoid bone. They commonly become repeatedly infected and may rupture spontaneously. When this complication occurs, a sinus tract or fistula persists. Removal of a thyroglossal cyst or fistula requires excision of the central part of the hyoid bone and dissection of the thyroglossal tract to the base of the tongue (the Sistrunk procedure) if recurrence is to be minimized. This procedure is necessary because the thyroglossal duct is intimately associated with the central part of the hyoid bone (Fig. 21). Recurrent cysts are very common if this operative procedure is not followed.

Figure 20. Location of thyroglossal cysts: (A) in front of the foramen cecum; (B) at the foramen cecum; (C) suprahyoid; (D) infrahyoid; (E) area of the thyroid gland; (F) suprasternal. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2nd ed. Philadelphia, WB Saunders, 1980.)

Figure 20. Location of thyroglossal cysts: (A) in front of the foramen cecum; (B) at the foramen cecum; (C) suprahyoid; (D) infrahyoid; (E) area of the thyroid gland; (F) suprasternal. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2nd ed. Philadelphia, WB Saunders, 1980.)

Figure 21. Diagram of the course of the thyroglossal tract. Note its proximity to the hyoid bone. (From Allard RHB: The thyroglossal cyst. Head Neck Surg 5:134–146, 1982.)

Figure 21. Diagram of the course of the thyroglossal tract. Note its proximity to the hyoid bone. (From Allard RHB: The thyroglossal cyst. Head Neck Surg 5:134–146, 1982.)

At least 115 cases of thyroid carcinoma have been reported to originate from the thyroglossal duct (82). Often, in such cases an association is noted with low-dose external irradiation of the head and neck in infancy or childhood. Almost all carcinomas have been papillary, and their prognosis is excellent. If a carcinoma is recognized, at the time of surgery the thyroid gland should be inspected for evidence of other tumor nodules, and the lateral lymph nodes should be sampled. Our practice and that of many others is to perform near-total or total thyroidectomy with appropriate nodal resection when a thyroglossal duct carcinoma is found and resected. In one series of 35 patients with papillary carcinoma arising in a thyroglossal duct cyst, the thyroid gland of 4 patients (11.4%) also contained papillary cancer (82). This operative ­procedure permits later radioiodine therapy as well.

In addition to papillary cancer, approximately 5% of all carcinomas arising from a thyroglossal duct cyst are squamous; rare cases of Hürthle cell and anaplastic cancer have also been reported. Finally, three families have been reported in which a total of 11 members had a thyroglossal duct cyst (84).

LATERAL ABERRANT THYROID

Small amounts of histologically normal thyroid tissue are occasionally found separate from the thyroid. If these tissue elements are near the thyroid, not in lymph nodes, and entirely normal histologically, it is possible that they represent developmental abnormalities. True lateral aberrant thyroid tissue or embryonic rests of thyroid tissue in the lymph nodes of the lateral neck region are very rare. Most agree that the overwhelming number of cases of what in the past was called “lateral aberrant thyroid” actually represented well-differentiated thyroid cancer metastatic to a cervical lymph node rather than an embryonic rest. In such cases, we favor near-total or total thyroidectomy with a modified radical neck dissection on the side of the lymph node, possibly followed by radioiodine therapy.

Several lateral thyroid masses have been reported that are said to be benign adenomas in lateral ectopic sites (85, 86). The authors of these studies suggest that they may develop ectopically because of failure of fusion of the lateral thyroid component with the median thyroid. However, before accepting this explanation, it is important to be certain that each of these lesions does not represent a well-differentiated metastasis that has totally replaced a lymph node and in which the primary thyroid carcinoma is small or even microscopic and was not recognized.

SUBSTERNAL GOITERS

Developmental abnormalities may lead to the finding of thyroid tissue in the mediastinum or, rarely, even within the tracheal or esophageal wall. However, most substernal goiters undoubtedly originate in the neck and then “fall” or are “swallowed” into the mediastinum and are not embryologically determined at all.

Substernal goiters have been reported to occur in 0.1% to 21% of patients in whom thyroidectomies were performed. This large variability is undoubtedly caused partly by a difference in classification among the authors, but it may also be caused by the incidence of endemic goiter. More recent series report an incidence of 2% or less (87).

Many substernal goiters are found on routine chest radiography in patients who are completely asymptomatic. Other patients may have dyspnea or dysphagia from tracheal or esophageal compression or displacement. Superior vena caval obstruction can occasionally occur with edema and cyanosis of the face, and venous engorgement of the arms and face (Fig. 22) (88). Most individuals with substernal goiters are euthyroid or hypothyroid; however, hyperthyroidism occurs as well. Although the goiters of Graves’ disease are rarely intrathoracic, single or multiple “hot” nodules may occur within an intrathoracic goiter and result in hyperthyroidism as part of a toxic nodular goiter.

Figure 22. Large substernal goiter resulting in superior vena caval syndrome. Left , A venogram demonstrated complete obstruction of the superior vena cava, displacement of the innominate veins, and marked collateral circulation. Right , Three weeks after thyroidectomy, patency of the vena cava was restored. Some displacement of the innominate veins remained at that time. (From Lesavoy MA, Norberg HP, Kaplan EL: Substernal goiter with superior vena caval obstruction. Surgery 77:325–329, 1975.)

Figure 22. Large substernal goiter resulting in superior vena caval syndrome. Left , A venogram demonstrated complete obstruction of the superior vena cava, displacement of the innominate veins, and marked collateral circulation. Right , Three weeks after thyroidectomy, patency of the vena cava was restored. Some displacement of the innominate veins remained at that time. (From Lesavoy MA, Norberg HP, Kaplan EL: Substernal goiter with superior vena caval obstruction. Surgery 77:325–329, 1975.)

Intrathoracic goiters are usually found in the anterior mediastinum and, less commonly, in the posterior mediastinum. In either instance the diagnosis is suggested if a goiter can be palpated in the neck and if it appears to continue below the sternum. Rarely, however, no thyroid enlargement in the cervical area is present, and instead of being in continuity, the intrathoracic component may be attached to the cervical thyroid only by a narrow bridge of thyroid or fibrous tissue. The diagnosis of an intrathoracic thyroid mass can be made by the use of a thyroid isotope scan; however, CT or MRI are usually more helpful. Regarding therapy, we generally agree with the recommendation made by Lahey and Swinton more than 50 years ago that goiters that are definitely intrathoracic should usually be removed if the patient is a good operative risk (89). Because of the cone-shaped anatomy of the upper thoracic outlet, once part of a thyroid goiter has passed into the superior mediastinum, it can increase its size only by descending further into the chest. Thus, delay in surgical management may lead to increased size of the lesion, a greater degree of symptoms, and perhaps a more difficult or hazardous operative procedure.

Substernal goiters should be operated on initially through a cervical incision, because the blood supply to the substernal thyroid almost always originates in the neck and can be readily controlled in this area. Only rarely does an intrathoracic goiter receive its blood supply from mediastinal vessels; however, such a finding favors a developmental cause. Thus, in most instances, good hemostasis can be obtained by control of the superior and inferior thyroid arteries in the neck. Thus, most substernal goiters can be removed through the neck, but in some cases a VATS procedure or a partial sternotomy may be necessary. The authors like to divide the isthmus and the upper pole vessels early in the dissection. The affected thyroid lobe is then carefully dissected along its capsule by blunt dissection into the superior mediastinum. While gentle traction is exerted from above, the mass is elevated by the surgeon’s ­fingers or blunt, curved clamps (Fig. 23). Usually these maneuvers suffice to permit extraction of a mass from the mediastinum and into the neck area. Any fluid-filled cysts may be aspirated to reduce the size of the mass and permit its egress through the thoracic outlet. However, piecemeal morcellation of the thyroid gland should not be practiced, because this occasionally has led to severe bleeding. Furthermore, rarely a substernal goiter has been found to contain carcinoma, and this technique violates all principles of cancer surgery.

Figure 23. Finger dissection of a substernal goiter. Note that the index finger is inserted into the mediastinum outside the thyroid capsule and is swept around until the gland is freed from the pleura and other tissue in the mediastinum. Occasionally, despite traction, a substernal goiter does not pass out through the superior thoracic outlet because of its size. In such cases, it may be necessary to evacuate some of the colloid material from within the goiter. Then, with gentle upward traction on the capsule, the mass can be elevated into the neck wound and resected. Occasionally a partial sternotomy is necessary. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2nd ed. Philadelphia, WB Saunders, 1980.)

Figure 23. Finger dissection of a substernal goiter. Note that the index finger is inserted into the mediastinum outside the thyroid capsule and is swept around until the gland is freed from the pleura and other tissue in the mediastinum. Occasionally, despite traction, a substernal goiter does not pass out through the superior thoracic outlet because of its size. In such cases, it may be necessary to evacuate some of the colloid material from within the goiter. Then, with gentle upward traction on the capsule, the mass can be elevated into the neck wound and resected. Occasionally a partial sternotomy is necessary. (From Sedgwick CE, Cady B: Surgery of the Thyroid and Parathyroid Glands, 2nd ed. Philadelphia, WB Saunders, 1980.)

With the use of this method, the great majority of substernal thyroid glands can be removed transcervically. If the thyroid gland cannot be easily extracted from the mediastinum, however, a partial or complete sternotomy should be performed. This procedure affords direct control of any mediastinal vessels and permits resection of the thyroid gland to be carried out safely.

As in all thyroid surgery, the recurrent laryngeal nerves must be preserved and treated with care. The parathyroid glands should be identified and preserved, and the inferior thyroid artery’s branches should be ligated close to the thyroid capsule to prevent ischemia of the parathyroid glands, which might result in hypoparathyroidism.

STRUMA OVARII

Ectopic development of thyroid tissue far from the neck area can also lead to difficulties in rare instances. Dermoid cysts or teratomas, which are uncommon ovarian germ cell tumors, occur in female subjects of all age groups. About 3% can be classified as an ovarian struma, because they contain functionally significant thyroid tissue or thyroid tissue occupying more than 50% of the volume of the tumor. Many more such tumors contain small amounts of thyroid tissue. Some strumae ovarii are associated with carcinoid-appearing tissue. These strumal-carcinoid tumors secrete or contain thyroid hormones as well as somatostatin, chromogranin, serotonin, glucagon, insulin, gastrin, or calcitonin (90). Some are associated with carcinoid syndromes.

Struma ovarii is sometimes manifested as an abdominal mass lesion, often with a peritoneal or pleural effusion that may be bloody. Most of these lesions synthesize and iodinate thyroglobulin poorly, and thus, despite growth of the mass, thyrotoxicosis does not develop. However, perhaps one fourth to one third of ovarian strumae are associated with thyrotoxicosis (91, 92). Many of these lesions may be contributing to autoimmune hyperthyroidism in response to a common stimulator such as thyroid-stimulating immunoglobulins. In other instances, the struma alone is clearly responsible for the thyrotoxicity. An elevated free thyroxine index or free T4, a suppressed TSH value, and uptake of radioiodine in a mass in the pelvis are the obvious prerequisites for making the diagnosis (93). Often, in ovarian struma, symptoms and findings of thyrotoxicosis are present in patients who have low uptake of radioiodine in their thyroid glands. Thus, a “high index of suspicion” is most important. Usually, operative resection of an ovarian tumor is indicated. After surgery, transient postoperative hypothyroidism and “thyroid storm” have occasionally been reported.

Benign thyroid adenomas in strumae are common, and about 5% manifest evidence of carcinoma (94). Usually, these lesions are resectable, but external radiation therapy and/or 131 I ablation has been advised after resection of the malignant tumors to avoid the tendency for late recurrence or metastatic disease, which has sometimes been fatal. Metastatic disease occurs in approximately 5% of these malignant tumors. It is best treated with 131 I therapy. Thyroidectomy is necessary in such cases before giving radioiodine therapy. Then, TSH should be suppressed with thyroxine as is done for thyroid cancer originating in the usual location.

STRUMA CORDIS

Functioning, apparently normal intracardiac thyroid tissue has been reported a few times and has been visualized by radioiodine imaging (95). The clinical finding is usually a right ventricular mass, and the diagnosis has typically been made after operative removal.

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Thyroid Hormone Synthesis and Secretion

ABSTRACT

The main function of the thyroid gland is to make hormones, T4 and T3, which are essential for the regulation of metabolic processes throughout the body. As at any factory, effective production depends on three key components - adequate raw material, efficient machinery, and appropriate controls. Iodine is the critical raw material, because 65% of T4 weight is iodine. Ingested iodine is absorbed and carried in the circulation as iodide. The thyroid actively concentrates the iodide across the basolateral plasma membrane of thyrocytes by the sodium/iodide symporter, NIS. Intracellular iodide is then transported in the lumen of thyroid follicles. Meanwhile, the thyrocyte endoplasmic reticulum synthesizes two key proteins, TPO and Tg. Tg is a 660kDa glycoprotein secreted into the lumen of follicles, whose tyrosyls serve as substrate for iodination and hormone formation. TPO sits at the apical plasma membrane, where it reduces H2O2, elevating the oxidation state of iodide to an iodinating species, and attaches the iodine to tyrosyls in Tg. H2O2 is generated at the apex of the thyrocyte by Duox, a NADPH oxydase. Initial iodination of Tg produces MIT and DIT. Further iodination couples two residues of DIT, both still in peptide linkage, to produce T4, principally at residues 5 in the Tg polypeptide chain. When thyroid hormone is needed, Tg is internalized at the apical pole of thyrocytes, conveyed to endosomes and lysosomes and digested by proteases, particularly the endopeptidases cathepsins B, L, D and exopeptidases. After Tg digestion, T4 and T3 are released into the circulation. Nonhormonal iodine, about 70% of Tg iodine, is retrieved intrathyroidally by DEHAL1, an iodotyrosine deiodinase and made available for recycling within the gland. TSH is the stimulator that affects virtually every stage of thyroid hormone synthesis and release. Early control involves the direct activation of the cellular and enzymatic machineries while delayed and chronic controls are on gene expression of key proteins. Iodine supply, either too much or too little, impairs adequate synthesis. Antithyroid drugs act by interfering with iodide oxidation. Genetic abnormalities in any of the key proteins, particularly NIS, TPO, Duox and Tg, can produce goiter and hypothyroidism. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org.

 

INTRODUCTION

The thyroid contains two hormones, L-thyroxine (tetraiodothyronine, T4) and L-triiodothyronine (T3) (Figure 2-1, below). Iodine is an indispensable component of the thyroid hormones, comprising 65% of T4's weight, and 58% of T3's. The thyroid hormones are the only iodine-containing compounds with established physiologic significance in vertebrates.

Fig. 2-1: Structural formula of thyroid hormones and precursor compounds

The term "iodine" occasionally causes confusion because it may refer to the iodine atom itself but also to molecular iodine (I2). In this chapter "iodine" refers to the element in general, and "molecular iodine" refers to I2. "Iodide" refers specifically to the ion I-.

Ingested iodine is absorbed through the small intestine and transported in the plasma to the thyroid, where it is concentrated, oxidized, and then incorporated into thyroglobulin (Tg) to form MIT and DIT and later T4 and T3 (Figure 2-2). After a variable period of storage in thyroid follicles, Tg is subjected to proteolysis and the released hormones are secreted into the circulation, where specific binding proteins carry them to target tissues. This chapter discusses these broad steps as: (a) iodine availability and absorption; (b) uptake of iodide by the thyroid; (c) oxidation of iodide, which involves the thyroperoxidase (TPO), H2O2, and H2O2 generation; (d) Tg, whose iodination leads to hormone formation; (e) storage of thyroid hormones in a Tg-bound form; (f) Tg breakdown and hormone release; (g) control of synthesis and secretion by iodine supply and TSH; and (h) effects of drugs and other external agents on the process.

Fig. 2-2: The iodide cycle. Ingested iodide is trapped in the thyroid, oxidized, and bound to tyrosine to form iodotyrosines in thyroglobulin (TG); coupling of iodotyrosyl residues forms T4 and T3. Hormone secreted by the gland is transported in serum. Some T4 is deiodinated to T3. The hormone exerts its metabolic effect on the cell and is ultimately deiodinated; the iodide is reused or excreted in the kidney. A second cycle goes on inside the thyroid gland, with deiodination of iodotyrosines generating iodide, some of which is reused without leaving the thyroid.

The production of thyroid hormones is based on the organization of thyroid epithelial cells in functional units, the thyroid follicles. A single layer of polarized cells (Fig. 2-4A) forms the enveloppe of a spherical structure with an internal compartment, the follicle lumen. Thyroid hormone synthesis is dependent on the cell polarity that conditions the targeting of specific membrane protein, either on the external side of the follicle (facing the blood capillaries) or on the internal side (at the cell-lumen boundary) and on the tightness of the follicle lumen that allows the gathering of substrates and the storage of products of the reactions.Thyroid hormone secretion relies on the existence of stores of pre-synthetized hormones in the follicle lumen and cell polarity-dependent transport and handling processes leading to the delivery of hormones into the blood stream.

IODINE AVAILABILITY AND TRANSPORT

The daily iodine intake of adult humans varies from less than 10 µg in areas of extreme deficiency to several hundred milligrams for some persons receiving medicinal iodine. Milk, meat, vitamin preparations, medicines, radiocontrast material, and skin antiseptics are important sources (Table 2-1) (1;2). In the United States, the average intake in 1960 was about 100-150 µg/day, then rose to 200-500 µg/day in the following decade. It is currently about 150 µg/day (3). The use of iodate as a bread conditioner in the baking industry greatly increased average iodine consumption; this additive has been replaced more recently by other conditioners that do not contain iodine. Iodophors as sterilizing agents in the milk industry also added much iodine to the food chain, but this source may also be diminishing. In the USA and elsewhere, most consumers are unaware of the amount of iodine they ingest. Commerce and manufacturing technology rather than health dictate the presence of iodine in most products. The amounts of iodine are usually unrevealed, and changes in them unannounced.
In the USA, where iodized salt use is optional, about 70% of the population consumes table salt containing approximately 76 ppm iodine (76 mg I/kg salt). Most prepared food in the USA and Europe uses uniodized salt (Switzerland and Macedonia are exceptions) and only about 15% of the daily salt intake is added at the table, so iodized salt in these areas makes only a modest contribution to daily iodine intake (4). The National Health and Nutrition Examination Surveys (NHANES) showed that the median national urinary iodine excretion in the USA in samples collected between 1988 and 1994 was 145 µg/L, a marked decrease from the 321 µg/L in a similar survey two decades before (5;6). Estimates from the NHANES (2001) are about 160 µg/L. The Total Diet Study of the U.S. Food and Drug Administration reported a parallel decrease in iodine consumption between 1970 and 1990 (7) . These fluctuations in iodine intake result from changes in societal and commercial practices that are largely unrecognized and unregulated. Canada mandates that all salt for human consumption contain KI at 100 ppm (76 ppm as iodine). Calculations of the representative Canadian diet in 1986 estimated slightly over 1 mg iodine/person/day, of which iodized salt contributed over half (8). Urinary iodine excretion in a group of men in Ottawa in 1990 was less than 50% of that in the Canadian national survey of 1975 (9), suggesting a decrease in dietary intake there as well as in the USA. Some countries have areas with very high iodine intake (10), from dietary custom (e.g., seaweeds in Japan) or from iodine-rich soil and water (e.g., a few places in China). But many countries have had some degree of iodine deficiency (11) in at least part of their territory. This has been corrected by the widespread programs of iodine prophylaxis promoted by ICCIDD (12).
Too much iodine increases the incidence of iodine-induced hyperthyroidism, autoimmune thyroid disease and perhaps thyroid cancer. Too little causes goiter, hypothyroidism and their consequences i.e.features of the so-called iodine deficiency disorders (5). The global push to eliminate iodine deficiency in the current decades has put both excess and deficiency of iodine in the spotlight. Some countries have already moved rapidly from severe iodine deficiency to iodine excess, while others are only now recognizing iodine deficiency as a problem (5;12). Their experience, as well as that in the USA and Canada, emphasizes the need for continued monitoring to assess trends in iodine intake.
Medicinal sources can provide iodine in amounts much larger than those consumed in an average diet (Table 2-1). For example, 200 mg of amiodarone contains 75 mg of iodine. Radiographic contrast materials typically contain grams of iodine in covalent linkage, and significant amounts (milligrams) may be liberated in the body. Skin disinfectants (e.g., povidone iodine) and iodine-based water purification systems can greatly augment iodine intake. At the other end, some individuals with little consumption of dairy products and of iodized salt have low iodine intakes.

 

Table 2-1. Some common sources of iodine in adults USA (1,2)
Dietary iodine Daily intake (µg)
Dairy products 52
Grains 78
Meat 31
Mixed dishes 26
Vegetables 20
Desserts 20
Eggs 10
Iodized salt 380
Other iodine sources (µg)
Vitamin/mineral prep (per tablet) 150
Amiodarone (per tablet) 75,000
Povidone iodine (per mL) 10,000
Ipodate (per capsule) 308,000

Most dietary iodine is reduced to iodide before absorption throughout the gut, principally in the small intestine. Absorption is virtually complete. Iodinated amino acids, including T4 and T3, are transported intact across the intestinal wall. Short-chain iodopeptides may also be absorbed without cleavage of peptide bonds (13). Iodinated dyes used in radiography are absorbed intact, but some deiodination occurs later. Except in the postabsorptive state, the concentration of iodide in the plasma is usually less than 10 µg/L. Absorbed iodide has a volume of distribution numerically equal to about 38% of body weight (in kilograms) (14), mostly extracellular, but small amounts are found in red cells and bones.

The thyroid and kidneys remove most iodide from the plasma. The renal clearance of iodide is 30-50 mL plasma/min (14-16) and appears largely independent of the load of iodide or other anions. In certain species, such as the rat, large chloride loads can depress iodide clearance. In humans, renal iodide clearance depends principally on glomerular filtration, without evidence of tubular secretion or of active transport with a transfer maximum (17). Reabsorption is partial, passive, and depressed by an extreme osmotic diuresis. Hypothyroidism may decrease and hyperthyroidism may increase renal iodide clearance, but the changes are not marked (14;18).

On iodine diets of about 150 µg/day, the thyroid clears iodide from 10-25 mL of serum (average, 17 mL) per minute (14). The total effective clearance rate in humans is thus 45-60 mL/min, corresponding to a decrease in plasma iodide of about 12%/hr. Thyroidal iodide clearance may reach over 100 mL/min in iodine deficiency, or as low as 3 or 4 mL/min after chronic iodine ingestion of 500-600 µg/day.

The salivary glands and the stomach also clear iodide and small but detectable amounts appear in sweat and in expired air. Breast milk contains large amounts of iodide, mainly during the first 24 hours after ingestion (19). Its content is directly proportional to dietary iodine. For example, in one part of the USA with community adult iodine intake of about 300 µg daily per person, breast milk contained about 18 µg iodine/dL, while in an area of Germany consuming 15 µg iodine per capita daily, the breast milk iodine concentration was only 1.2 µg/dL (20). Milk is the source of virtually all the newborn's iodine, so milk substitutes need to provide adequate amounts.

UPTAKE OF IODINE BY THE THYROID

Thyroid cells extract and concentrate iodide from plasma (21;22). As shown in Fig. 2-3, shortly after administration, radioiodide is taken up from the blood and accumulates within thyroid follicular cells. About 20% of the iodide perfusing the thyroid is removed at each passage through the gland (23). The normal thyroid maintains a concentration of free iodide 20 to 50 times higher than that of plasma, depending on the amount of available iodine and the activity of the gland (24). This concentration gradient may be more than 100:1 in the hyperactive thyroid of patients with Graves' disease. The thyroid can also concentrate other ions, including bromide, astatide, pertechnetate, rhenate, and chlorate, but not fluoride (25;26).

Fig. 2-3: Radioautographs of rat thyroid sections. Animals received iodide shortly before sacrifice, and radioautographs of thyroid sections were coated with emulsion after being stained by the usual methods. The radioautographs indicated the presence of iodide primarily over the cells at these early time intervals. (From Pitt-Rivers, R.J., S.F. Niven, and M.R. Young, in Biochemistry, 90:205, 1964, with permission of the author and publisher.)

The protein responsible for iodide transport, the so-called sodium/iodide symporter or NIS, is located at the basolateral plasma membrane of thyrocytes (Fig. 2-4.). NIS-mediated I- accumulation is a Na+-dependent active transport process that couples the energy released by the inward translocation of Na+ down to its electrochemical gradient to the simultaneous inward translocation of I- against its electrochemical gradient. The maintenance of the Na+ gradient acting as the driving force is insured by Na+-K+-ATPase. NIS belongs to the sodium/glucose cotransport family as the SLC5A5 member. Iodide transport is energy-dependent and requires O2. Ouabain, digitoxin, and other cardiac glycosides block transport in vitro (27;28). Iodide uptake by thyroid cells is dependent on membrane ATPase. During gland hyperplasia, iodide transport usually varies concordantly with plasma membrane Na+-K+-activated, ouabain-sensitive ATPase activity (29).

NIS cDNA was first cloned in rat FRTL-5 cells by Dai et al. (30). The rat NIS gene gives rise to a 3kb transcript with an open reading frame of 1,854 nucleotides encoding a polypeptide chain of 618 amino acids. The mature protein is a glycoprotein with an apparent molecular mass of 85kDa (31;32).It has 13 membrane spanning domains, with the carboxy terminus in the cytoplasm and the amino terminus located outside the cells (33). In the model of Levy et al. (34), a Na+ ion first binds to the transporter which, in the presence of iodide, forms a complex that then transfers iodide and two Na+ ions to the cell interior.

The human NIS gene located on chromosome 19 (35) codes for a protein of 643 amino acids that is 84% homologous with rat NIS (36). The mouse NIS polypeptide chain (37) has the same size (618 amino acids) as the rat NIS. At variance with other species, three different transcripts are generated from the porcine NIS gene by alternative splicing (38); the main form encodes a polypeptide of 643 amino acids as human NIS.

Functional studies clearly show that NIS is responsible for most of the events previously described for iodide concentration by the thyroid. TSH stimulates NIS expression (39;40) and iodide transport (31;32). TSH exerts its regulatory action at the level of transcription through a thyroid-specific far-upstream enhancer denominated NUE (NIS Upstream Enhancer) that contains binding sites for the transcription factor Pax8 and a cAMP response element-like sequence. This original demonstration made on the rat NIS gene (41) has now been extended to human (42) and mouse (43) NIS genes. It has been suggested that TSH could also regulate NIS expression at post-transcriptional level (44). Data from TSH receptor-null mice (44-46) clearly show that TSH is required for expression of NIS. Moderate doses of iodide in the TSH-stimulated dog thyroid inhibit expression of the mRNAs for NIS and TPO, while not affecting that for Tg and TSH receptor (40) . The decrease in thyroid iodide transport resulting from excess iodide administration (escape from the Wolff-Chaikoff effect, see further) is related to a decrease in NIS expression (40;47). Both NIS mRNA and NIS protein are suppressed by TGFb, which also inhibits iodide uptake (48;49). Reviews focus on NIS and its functional importance (50;51).

Several mutations in the NIS gene causing defective iodide transport have been reported in humans (52-59). The most commonly found mutation corresponds to a single base alteration T354P in the ninth putative transmembrane domain of NIS (55). Site directed mutagenesis of rat NIS cDNA to substitute for threonine at residue 354 and transfection into COS cells lead to loss of iodide transport activity (60). Other mutations lead to truncated NIS (58) or to alterations of membrane targeting of the NIS protein (59) . NIS expression is increased in Grave’s disease and hyperactive nodules (61-63) and decreased in adenomas and carcinomas (64;65) appearing as cold nodules at scintigraphy.In hypofuctioning benign or malignant tumors, the impairment of iodide transport would result from both transcriptional and post-transcriptional alterations of NIS expression (66).Other tissues that concentrate iodide also show NIS expression, including salivary glands (67) and mammary glands (68;69).

Iodide supply of follicular lumen involves a two-step transport process: the active transport across the basolateral plasma membrane of thyrocytes by NIS and a passive transport across the apical plasma membrane. The protein(s) insuring the second step is (are) not yet identified. A potential iodide transporter has been proposed: pendrin (70;71). Pendrin, encoded by the PDS gene (72) and composed of 780 amino acids, is expressed in different organs including kidney, inner ear and thyroid. In the thyroid, pendrin is a 110kDA membrane glycoprotein (73), selectively located at the apical plasma membrane (74). Its activity as transporter of anions including iodide has been demonstrated in different experimental systems (71;75-77). Pendrin belongs to the SLC family under the reference SLC26A4. However, the implication of pendrin in thyroid iodide transport remains uncertain for several reasons. First, there is still no direct demonstration of a pendrin-mediated efflux of iodide from thyrocytes to the follicular lumen. Second, the genetic alterations of the PDS gene found in patients with the Pendred syndrome, which lead to a loss of the anion transport activity of pendrin and to a constant and severe hearing loss, only have a moderate impact on the thyroid functioning, generally a euthyroid goiter (78). Third, PDS knock-out mice (79) do not show any thyroid dysfunction. In summary, contrary to NIS for which the anion selectivity (25) corresponds to what was expected, the ion selectivity of thyroid pendrin remains to be elucidated. In the thyroid as in the kidney, pendrin could act primarily as a chloride/bicarbonate anion exchanger. Rather than pendrin, anoctamin-1/TEM 16A, a calcium-activated chloride channel, seems to be responsible for most of the iodide efflux accross the apical membrane of the thyrocytes (80).

Fig. 2-4: NIS-mediated transport of iodide. A, immunolocalization of the human NIS protein at the basolateral plasma membrane of thyrocytes in their typical follicle organization. B, schematic representation of the membrane topology of the NIS polypeptide chain deduced from secondary structure prediction analyses (33). C, transport of iodide from the extracellular fluid (or plasma) to the thyroid follicle lumen. The uptake of iodide at the basolateral plasma membrane of thyrocytes must be active; it operates against an electrical gradient (0 - 50 mV) and a concentration gradient, [ I- ]c being higher than extracellular [ I- ]. The transport of iodide from the cytoplasm to the follicle lumen should be a passive process, the electrical and concentration gradients being favorable.

Iodide that enters the thyroid remains in the free state only briefly before it is further metabolized and bound to tyrosyl residues in Tg. A significant proportion of intrathyroidal iodide is free for about 10-20 minutes after administration of a radioactive tracer (81), but in the steady state, iodide contributes less than 1% of the thyroid total iodine. A major fraction of the intrathyroidal free iodide pool comes from deiodination of MIT and DIT; this iodide is either recycled within the thyroid or leaked into the circulation. Some data suggest that iodide entering the gland by active transport segregates from that generated by deiodination of Tg within the gland (82;83). Once in the thyroid, iodide is organically bound at a rate of 50 to 100% of the pool each minute (24;84). The proportion of an iodide load that is bound varies little, despite wide shifts in daily intake. In contrast, NIS activity is sensitive to both iodine availability and TSH stimulation, and transport rather than intrathyroidal binding is the controlling factor in making iodide available for hormonogenesis.

IODINE IN OTHER TISSUES

The thyroid is not the only organ to concentrate iodine; the others endowed with this capacity are salivary glands, gastric mucosa, mammary glands, and choroid plexus. Ductal cells of the salivary glands express NIS (67) . The plasma membrane of the mammary gland epithelium contains a NIS protein with a molecular mass different from that of thyroid NIS (~75 kDa vs ~90 kDa). In the mammary gland, NIS is processed differently after translation and subjected to regulation by lactogenic stimuli (68). It has been reported that over 80% of human breast cancer samples express this symporter. As it is absent in normal non-lactating tissue, NIS may represent a marker for breast malignancy and even a possible target for radioiodine therapy (69). The thyroid, salivary glands, and gastric mucosa share a common embryologic derivation from the primitive alimentary tract and, in each of these tissues; iodide transport is inhibited by thiocyanate, perchlorate, and cardiac glycosides. TSH stimulates transport only in the thyroid. An active transport for iodide in the gastric mucosa has an obvious value because it provides iodine to the circulation for use in the thyroid. Active concentration by the breast helps transfer iodide to milk. Iodide concentration by the choroid plexus and salivary glands does not have any obvious physiologic benefit, but needs to be remembered for possible insights into pathways as yet undiscovered.

Iodine, particularly in the form of I2, may enter additional metabolic pathways outside the thyroid. Rats administered I2 orally showed much less circulating free iodide and much more iodine bound to proteins and lipids than did animals given iodide (85). In another comparison of I2 versus iodide, administration of iodide to iodine-deficient rats eliminated thyroid hyperplasia much more efficiently than did I2. Additionally, I2 decreased lobular hyperplasia and periductal fibrosis in the mammary glands, while iodide increased the former and had no effect on the latter (86).

THYROPEROXIDASE (TPO)

After concentrating iodide, the thyroid rapidly oxidizes it and binds it to tyrosyl residues in Tg, followed by coupling of iodotyrosines to form T4 and T3. The process requires the presence of iodide, a peroxidase (TPO), a supply of H2O2, and an iodine acceptor protein (Tg).

Thyroperoxidase oxidizes iodide in the presence of H2O2. In crude thyroid homogenates, enzyme activity is associated to cell membranes. It can be solubilized using detergents such as deoxycholate or digitonin. The enzyme activity is dependent on the association with a heme, the ferriprotoporphyrin IX or a closely related porphyrin (87;88). Chemical removal of the prosthetic group inactivates the enzyme, and recombination with the heme protein restores activity (89). The apoprotein from human thyroid is not always fully saturated with its prosthetic group (90). Some congenitally goitrous children have poor peroxidase function because the apoprotein has weak binding for the heme group (90).

Antibodies directed against the thyroid "microsomal antigen," which are present in the serum of patients with autoimmune thyroid disease (AITD), led to identification of TPO. These antibodies were found to react with proteins of 101-107 kDa and to immunoprecipitate thyroid peroxidase (TPO), thus identifying microsomal antigen as TPO (91-95). A monoclonal antibody to purified microsomal antigen or antibodies directed againt thyroperoxidase were then used to clone human TPO (96-98). Different laboratories then cloned TPO from various species: pig (99), rat (100), and mouse (101). Kimura et al. (96) cloned two different cDNAs of humanTPO.TPO1 coded for a protein of 933 residues and TPO2 was identical to TPO1 except that it lacked exon 10 and was composed of 876 residues. Both forms occur in normal and abnormal human thyroid tissue. The C-terminal portion of the proteins exhibits a hydrophobic segment (residues 847-871), likely corresponding to a transmembrane domain; thus, TPO has a short intracellular domain and most of the polypeptide chain is extracellular (Fig. 2-5A). TPO1 is active, but TPO2 appears enzymatically inactive because it does not bind heme, degrades rapidly, and fails to reach the cell surface in transfected cell lines (102). Different degradative pathways exist for the two forms (103). Several other TPO variants resulting from exon skipping have been identified; they appear either active or inactive (104). Pig TPO contains 926 amino acids (99) ; mannose-rich oligosaccharide units occupy four of its five glycosylation sites (105).

Human TPO, which has 46% nucleotide and 44% amino acid sequence homology with human myeloperoxidase, clearly belongs to the same protein family. The TPO gene resides on chromosome 2p13, spans over 150 kbp, and has 17 exons (106). As NIS, Tg, and the TSH receptor (TSHr), TPO expression is controlled by the TSH cAMP pathway (107) through thyroid-specific transcription factors. These include TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 (108;109). Tg and TPO genes have the same binding sites for TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 in their promoters, and the genes for both have TTF-1/NKx2.1 sites in enhancer regions.

Inactivating mutations in the TPO gene are responsible for a subtype of congenital hypothyroidism characterized by thyroid dyshormonogenesis due to iodide organification defect. More than 60 annotated mutations have been reported; most of them result in total iodide organification defect with severe and permanent hypothyroidism (110;111).

TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes core glycosylation. TPO is then transported to the Golgi where it is subjected to terminal glycosylation and packaged into transport vesicles along with Tg (112) (Fig. 2-6). These vesicles fuse with the apical plasma membrane in a process stimulated by TSH. TPO delivered at the apical pole of thyrocytes exposes its catalytic site with the attached heme in the thyroid follicular lumen (113). TPO activity is restricted to the apical membrane, but most of the thyroid TPO is intracellular, being located in the perinuclear part of the endoplasmic reticulum (114;115). Most of this intracellular protein is incompletely or improperly folded; it contains only high mannose-type carbohydrate units, while the membrane TPO has complex carbohydrate units. Glycosylation is essential for enzymatic activity (115). Chronic TSH stimulation increases the amount of TPO and its targetting at the apical membrane (116).

Fig. 2-5: Schematic representation of the membrane topology of Thyroperoxidase, TPO (A) and NADPH thyroid oxidase, ThOX (Duox) (B) at the apical plasma membrane of thyrocytes. C, hypothetical reaction scheme for TPO. H2O2 is presumed to oxidize the free enzyme with a loss of two electrons leading to the formation of complex I. Iodide binds to complex I, is oxidized and form complex II, which then reacts with a tyrosyl residue of Tg, Tyr-Tg.The newly-formed I0 and Tyr0-Tg free radicals interact to form MIT-Tg and the enzyme returns to its free state. I2 may be generated from two cxidized iodine atoms

H 2 O 2 GENERATING SYSTEM

By definition, a peroxidase requires H2O2 for its oxidative function. A large body of older work (reviewed in (117)) investigated possible sources using various in vitro models (117-120). It was already suggested in 1971 that H2O2 would be produced at the apical plasma membrane of the thyrocyte by an enzyme that requires calcium and NADPH originating from the stimulation of the pentose phosphate pathway (121). Further biochemical studies showed that the enzymatic complex producing H2O2 for TPO is a membrane-bound NADPH-dependent flavoprotein (122-126). H2O2 produced by this NADPH-dependent protein is the limiting step of protein iodination and therefore of thyroid hormone synthesis when iodide supply is sufficient (127-129). In human thyroid, the H2O2 production and iodination process are stimulated by the calcium-phosphatidylinositol pathway (129). The quantity of H2O2 produced is important especially in stimulated thyrocytes; it is comparable to the ROS production of activated leukocytes. While the activated leukocyte lives a few hours, the life of an adult thyrocyte is 7 yr (130;131). Thus thyroid cells may be exposed to high doses of H2O2 and have to adapt to it by developing highly regulated generator and efficient protective systems.

More than twenty years passed between the initial biochemical studies and the cloning of Duox as the catalytic enzymatic core of the H2O2 thyroid generating system. By two independent molecular strategies Duox enzymes were uncovered from the thyroid. Starting from a purified fraction of pig thyroid membrane bound NADPH flavoprotein, the team of C. Dupuy isolated p138 Tox which turned out to be Duox2 lacking the first 338 residues (132). Simultaneously, De Deken et al cloned two cDNAs encoding NADPH oxidases using the strategy based on the functional similarities between H2O2 generation in the leukocytes and the thyroid according to the hypothesis that one of the components of the thyroid system would belong to the known gp91phox gene family and display sequence similarities with gp91phox, now called NOX2. Screenings of two cDNA libraries at low stringency with a NOX2 probe enabled the isolation of two sequences coding for two NADPH oxidases of 1551 and 1548 amino acids respectively initially named Thox1 and Thox2 (133). The encoded polypeptides display 83% sequence similarity and are clearly related to gp91phox (53 and 47% similarity over 569 amino acids of the C-terminal end) . The whole protein is composed of : a N-terminus ecto-sequence of 500 amino acids showing a similarity of 43% with thyroperoxidase (hence named Dual oxidase-Duox in the present terminology); a first transmembrane segment preceding a large cytosolic domain which contains two calcium binding EF-hand motifs; the C-terminal portion componed of six transmembrane segments, harbouring the four His and two Arg characteristic of the Nox family protein heme binding site and the conserved FAD- and NADPH-binding sites at the extreme C-terminal cytolic portion (Fig. 2-5B). Duox proteins are localized like TPO at the apical plasma membrane of the thyrocyte as fully glycosylated forms (~190kDa) and in the endoplasmic reticulum as high mannose glycosylated forms (~180kDa) (Fig. 2-6C).

Duox1 and Duox2 genes are co-localized on chromosome 15q15.3, span 75kb, have opposite transcriptional orientations and are separated by a ~16kb region (Fig. 2-6A). Duox1 gene is more telomeric, spans 36 kb and is composed of 35 exons; two first of them are non-coding. Duox2 spans 21.5 kb and is composed of 34 exons; the first being non-coding (134).

In addition to thyroid, Duox expression is reported in several tissues: Duox1 is expressed in lung epithelia, in oocytes (135-137) and Duox2 in gastrointestinal mucosa and salivary glands (138;139). Multiple functions are attributed to Duox enzymes: airway fuid acidification (140), mucin secretion (141), wound healing (142;143) and innate hoste defense (144-147) .

Most of the time Duox activity is associated to a peculiar peroxidase activity like in oocyte with the ovoperoxidase involved in the fertilization process or with the lactoperoxidase in lung epithelia or in the gut (144;145;148;149) . Beside these killing mechanisms, Duox and H2O2 are certainly also involved in the interaction between host mucosa and bacteria to maintain mucosal homeostasis e.g. in bronchi and intestine (146;150). In the thyroid, the specificity of the thyroid hormone machinery using Duox lays on TPO. Thus colocalization of Duox and TPO and their probable association at the apex of the thyrocyte would increase the efficiency of H2O2 producer-consumer system (151-153).

 

Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid differentiation marker. The proteins involved in the synthesis of thyroid hormones are expressed just after the thyroid precursor cells have completed their migration from the primitive pharynx and reached their final location around the trachea (154;155). This final morphological maturation begins in mouse with the expression of Tg at embryonic day 14 followed one day later (E15) by the expression of TPO, NIS, TSH receptor and Duox concomitant with the apparition of iodinared Tg (46;156).

 

Until 2006, the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell system for Duox correctly expressed at the plasma membrane in its active state. Several cell lines transfected with Duox1 and/or Duox2 showed Duox expression completely retained in the endoplasmic reticulum in their immature form without displaying any production of H2O2 (157). HEK293 cells transfected with Duox2 generate rather small quantities of superoxide anions in a calcium-depnedent manner (158). The reconstitution of a Duox-based functional H2O2 generating system requires a maturation factor called DuoxA. The two human DuoxA paralogs were initially identified as thyroid specific expressed genes by in silico screenings of multiple parallel signature sequencing data bases (159). The two genes are located on chromosome 15 in the Duox1/Duox2 intergenic region in a tail to tail orientation, DuoxA1 facing Duox1 and DuoxA2 facing Duox2 (Fig. 2-6A.). DuoxA2 ORF spans 6 exons and encodes a 320 amino acid protein predicted to compose five transmembrane segments, a large external loop presenting N-glycosylation sites between the second and third transmembrane helices and a C-terminal cytoplamic region (Fig. 2-6B). DuoxA1 gene was initially annotated “homolog of Drosophila Numb-interacting protein: NIP” (160). Four alternatively spliced DuoxA1 variants have been identified (161). One of the most expressed transcript, DuoxA1α, is the closest homolog of DuoxA2 and encodes a 343 amino acid protein (58% identity of sequence with DuoxA2) adopting the same predicted structure.

In heterologous systems DuoxA proteins in the absence of Duox are mainly retained in the endoplasmic reticulum. When co-transfected with Duox they cotransported with Duox to the plasma membrane where they probably form complexes. Only the Duox1/DuoxA1 and Duox2/DuoxA2 pairs produce the highest levels of H2O2 as they undergo the glycosylation steps through the Golgi. Duox2/DuoxA1 pair does not produce H2O2 but rather superoxide anions and Duox1/DuoxA2 is unable to produce any ROS. In addition it has been shown that the type of Duox-dependent ROS poduction is dictated by defined sequences in DuoxA (162). This means that the Duox activators promote Duox maturation but also are parts of the H2O2 generating complex (163;164). Mice deficient in DuoxA maturation factors present a maturation defect of Duox, lacking the N-glycan processing, and a loss of H2O2 production. These mice develop severe goitrous congenital hypothyroidism with undetectable serum T4 and high serum TSH levels (165).

The reconstitution of this functional H2O2 producing system has been useful to measure and compare the intrinsic enzymatic activities of Duox1 and Duox2 in relationship with their expression at the plasma membrane under stimulation of the major signalling pathways active in the thyroid. It has been shown that the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium binding motifs. However, the two oxidase enzymatic activities are differently regulated after activation of the two main signalling cascades in the thyroid. Duox1 but not Duox2 activity is stimulated by the cAMP dependent cascade triggered by forskolin (EC50=0.1µM) via protein kinase A-mediated phosphorylation on serine 955 of Duox1. In contrast, phorbol esters, at low concentrations, induce Duox2 phosphorylation via protein kinase C activation associated with high H2O2 generation (EC50= 0.8nM) (166). These results suggest that both Duox proteins could be involved in thyroid hormone synthesis by feeding H2O2 to TPO to oxidize iodide and couple iodotyrosines.

 

From in vitro and in vivo data it has been concluded that Duox-DuoxA constitutes the major if not the unique component of the hormonogenic thyroid H2O2 generating system. The bidirectional promoter allows the coexpression of Duox and DuoxA in the same tissue but the mechanisms regulating their transcription are not well and definitely characterized (167;168). It has been recently shown that Th2 cytokines, IL4 and IL13, up-regulate Duox2 and DuoxA2 genes in human thyrocytes through an activation of Jak-Stat pathway opening new perspectives for a better understanding of the eventual role of Duox in autoimmune diseases (169).

 

Defects in Duox and/or DuoxA were rapidly recognized possible causes of congenital hypothyroidism (CH) due to thyroid dyshormonogenesis in patients born with a hyperplastic thyroid or developing a goiter postnatally when T4 treatment is delayed after birth.

The first screening of mutations in Duox genes in 2002 was performed on 9 patients who had idiopathic congenital hypothyroidism with positive ClO4- discharge (>10%), one with permanent and 8 with transient hypothyroidism (170). They were identified in the Netherlands by neonatal screening and followed up to determine the evolution of CH with the time. One of the patients with total organification defect (TIOD) presented a permanent hypothyroidism and the 8 others presented a transient hypothyroidism with a partial organification defect (PIOD). Of these last 8 patients 3 harboured heterozygous nonsense or frameshift mutations (Q686X, R701X, S965fsX994) meaning that a single defective Duox2 allele can cause haploinsufficency resulting in mild transient CH. It is noteworthy that this hypothyroid status was limited to the neonatal period, when thyroid hormone requirement is the highest, and was not detectable in adulthood since adult heterozygotes in these families presented normal TSH serum levels. The only case with severe permanent CH was homozygous for a nonsense mutation (R434X= protein devoid of the catalytic core) leading to the conclusion at this time of a complete inability to synthesize thyroid hormone in absence of Duox2. No mutation was detected in Duox1.

With the increasing number of reported Duox2 mutations in CH, it becomes more and more difficult to make the correlation between genotype and phenotype as initially described.

Indeed, subsequent studies have shown a link between biallelic Duox2 defects and PIOD. Patients with compound heterozygous missense (R376W) and a nonsense mutation (R842X), leading to a presumed non functional protein showed PIOD with mild and persistent hyperthyrotropinemia. This suggests that Duox1 can compensate at least partially for the defect in Duox2 (171). Varela et al.. described also two cases of permanent CH with compound heterozygous missense and nonsense or splicing mutations (Q36H and S965fsX994; G418fsX482 and g.IVS19-2A>C conducting to inactive proteins) responsible for congenital goiter with a PIOD (172).

 

The phenotype-genotype correlation suggested by the work of Moreno et al. is no longer clear. Maruo et al. described a series of transient CH characterized by biallelic defects in Duox2: in one family, four siblings were compound heterozygous for early frameshift mutations (L479SfsX2 and K628RfsX10) resulting in a presumed complete loss of Duox2 activity (not tested at this time). Three of them had low free T4 at birth, mild thyroid enlargement. The thyroid hormone replacement therapy ceased to be necessary by 9yr of age (173). A French-Canadian patient with a transient CH initially detected by neonatal screening presented a compound heterozygozity for a hemizygous missense mutation (G1518S) inherited from the father and a deletion removing the part of the gene coding for the catalytic core of Duox2 inherited from the mother. In vitro test proved that the missense mutant protein was totally inactive (174). This case and others reported later provide further evidence that permanent or transient nature of CH is not directly related to the number of inactivated Duox2 alleles (175-177).

 

The first homozygous nonsense mutation in DuoxA2 (Y246X) that resulted in a non-functional protein tested in vitro has been found to be responsible of a permanent mild CH in a Chinese patient with a dyshormonogenic goiter (164;178). The mild phenotype can be explained by a partial maintenance of H2O2 production by Duox2/DuoxA1 as demonstrated in vitro. A high level of functional redundancy in Duox/DuoxA system could also explained the mild transient hypothyroidism in a patient with a novel biallelic DuoxA2 mutation and one allele of Duox2 and DuoxA1(179).

The variety of observerd phenotypes associated with Duox2 and now DuoxA2 mutations suggest that the manifestation of Duox2 defects could likely be influenced by the environmental factors like iodine intake or by the activation of Duox1 or DuoxA1 in peculiar circumstances.

Fig 2-6: A, Localization of Duox and DuoxA genes on chromosomes 15q15.3. B, Schematic representation of the predicted structure of DuoxA (from (156). C, Immmunolocalization of human Duox and TPO at the apical membrane of the thyrocyte (upper: Duox immunostaining, middle:preimmune serum, lower:TPO immunostaining) (130).

THYROGLOBULIN (Tg)

Thyroglobulin is the most abundant protein in the thyroid gland; its concentration within the follicular lumen can reach 200-300 g/L. Its main function is to provide the polypeptide backbone for synthesis and storage of thyroid hormones (180). It also offers a convenient depot for iodine storage and retrieval when external iodine availability is scarce or uneven. Neosynthesised Tg polypeptide chains entering the lumen of the rough endoplasmic reticulum (RER) are subjected to core glycosylation, dimerise and are transferred to the Golgi where they undergo terminal glycosylation (Fig. 2-7). Iodination and hormone formation of Tg occur at the apical plasma membrane-lumen boundary and the mature hormone-containing molecules are stored in the follicular lumen, where they make up the bulk of the thyroid follicle colloid content.

Fig. 2-7: A polarized thyroid epithelial cell synthesizing soluble proteins, Tg (▲) and lysosomal enzymes (X) and membrane proteins, NIS (┴) and TPO (°). The polypeptide chain(s) generated by RER membrane-bound polysomes, enter the lumen of RER for the former and remain inserted into the RER membrane for the latter. Inside the lumen of RER, newly-synthesized proteins undergo core glycosylation and by interacting with chaperones acquire their conformation. Proteins are then transported to the Golgi apparatus (G), where terminal glycosylation and other post-translational reactions take place. In the Trans-Golgi network (TGN), mature proteins undergo sorting processes and are packed into transport vesicles. The vesicles carrying soluble proteins (inside the vesicle) and membrane proteins (as integral vesicle membrane protein) deliver them at the appropriate plasma membrane domain: the apical domain (1) and (2) or the basolateral domain (4). Vesicles carrying lysosomal enzymes (3) conveyed their content to prelysosomes or late endosomes (LE) and lysosomes (L). Apical plasma membrane proteins may reach their final destination by an alternative route involving a transient transfer to and then a retrieval and transport (*) from the basolateral membrane domain to the apical domain.

The Tg peptide chain derives from a gene of more than 200 kbp located on chromosome 8 in humans. The human Tg gene consisting of 48 exons (181) gives rise to a 8.5kb transcript that translates a 2,749 residue peptide (in addition to a 19-residue signal peptide) (182;183). The primary structure deduced from cDNA is also known for bovine, rat, and mouse (184-186). The biochemical traits of human Tg have been reviewed in (187). The N-terminal part of Tg has regions of highly conserved internal homology (10 motives of about 60 amino acids) which appears in several other proteins and are referred to as ‘thyroglobulin type-1 domains’. Such domains have been found to be potent inhibitors of cysteine proteases (188). This finding might be of importance, because these proteases are active in Tg proteolysis (see below). It has been suggested that this region of the Tg molecule may modulate its own degradation and hormone release (189). In the Tg-type 1 repeats, cysteine and proline residues are found in constant position; they may have an important role in the tridimensional structure of the protein. The proximal region of the C-terminal half portion of Tg contains five repeats of another type of cysteine-rich motives. The presence of a high number of cysteine residues in Tg, involved for most of them in disulfide bonds, probably gives rise to peculiar structural constraints. The C-terminal portion of Tg is homologous with acetylcholinesterases (190). Because binding to cell membranes is one feature of acetylcholinesterases, perhaps Tg C-terminus has a similar role. It was reported that the acetylcholinesterase-homology region of Tg could function as a dimerization domain (178;191-193).Furthermore, three highly conserved thioredoxin boxes have been identified in mammalian Tg between residues 1,440 and 1,474; these boxes might be involved in disulfide bond formation leading to intermolecular cross-linking of Tg molecules inside the follicle lumen (194) . Tg gene expression is controlled by the same main thyroid-specific transcription factors that regulate synthesis of TPO (108):TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 that bind at the same sites in Tg as they do in TPO. Hydrogen peroxide might be a regulatory factor of Tg expression, based on experimental work showing increased Tg promoter activity with reduced Pax-8 and TTF-1 (195-198). If substantiated, this proposal offers another point of integration between H2O2 generation and transcription of NIS, Tg and TPO genes, all of which being regulated by TSH.

Maturation of the Tg polypeptide chain begins while still on the RER. It undergoes core glycosylation and then monomers fold into stable dimers. Arvan and co-workers (199-204) have mapped this process and emphasize the role of molecular chaperones. The latter are essential for folding the new Tg molecules, and those that are folded improperly are not allowed to proceed further. The principal molecular chaperones are BiP, GRP 94, ERP 72, and calnexin. Only Tg molecules that pass this quality control system unscathed can proceed towards the secretory pathway. Glycosylation is a key event in Tg maturation. Carbohydrates comprise about 10% of Tg weight (205). Human Tg may contain four different types of carbohydrate units. The "polymannose" units consist only of mannose and N-acetylglucosamine. The "complex unit" has a core of three mannose residues with several chains of N-acetylglucosamine, galactose, and fucose or sialic acid extending from them. Both these types of unit are common in glycoproteins and are linked to peptide through an asparagine-N-acetylglucosamine bond. About three quarters of the potential N-glycosylation sites in human Tg are occupied, mostly with the complex unit (206). Two additional units have been found in human Tg; one contains galactosamine and is linked to the hydroxyl group of serine, the other is a chondroitin sulfate unit containing galactosamine and glucuronic acid (207) .

Failure in Tg folding can lead to disease as in the cog/cog mouse; these animals have a large thyroid with a distended ER and sparse Tg storage in follicles (208). Their Tg shows abnormal folding and decreased export from the ER in association with increased levels of several molecular chaperones. In the Tg cDNA of cog/cog mouse, Kim et al.(185) identified a single base substitution that changes leucine to proline at position 2,263. Correction of this defect by site-directed mutagenesis returned Tg export to normal in transfected cells. The cog/cog mouse is an example of endoplasmic reticulum storage disease (209). Other examples are cystic fibrosis, osteogenesis imperfecta, familial neurohypophyseal diabetes insipidus, insulin receptor defect, growth hormone receptor defect, and a variety of lipid disorders (210). In each situation, the underlying defect appears to be a mutation in the coding sequence of exportable proteins. The ER retains the abnormal proteins, which cannot then proceed for further maturation. Several reports describe a similar pathogenesis for cases of congenital goiter and hypothyroidism in humans, although these are not as well characterized. Ohyama et al. (211) investigated a five-year-old euthyroid goitrous boy with high thyroidal radioiodine uptake, a positive perchlorate discharge test, apparently normal H2O2 generation and peroxidase activity in gland tissue, and low amounts of Tg in thyroid tissue overall, but large amounts in the RER. In another report, two hypothyroid goitrous sibs had a 138 bp segment missing between positions 5,590-5,727 in hTg mRNA, translating into a Tg polypeptide chain that lacked 46 residues (212). A third example described four subjects with congenital hypothyroid goiter from two unrelated families (213). Their thyroid tissue showed accumulation of Tg intracellularly with distension of the ER and large increases in activity of specific molecular chaperones, but with failure of Tg to reach the Golgi or the follicular lumen; this case was put forward as an ER storage disease similar to the cog/cog mouse (213).

Tg also contains sulphur and phosphorus. The former is present in the chondroitin sulfate and the complex carbohydrate units, although its form and role are not known (214). Several studies have reported phosphate in Tg, up to 12 mol. per mol Tg. Of this, about half is in the complex carbohydrate units, the remainder is present as phosphoserine and phosphotyrosine (215-217). This may relate to protein kinase A activity (218).

THYROGLOBULIN IODINATION AND HORMONE SYNTHESIS

The step preliminary to thyroid hormone formation is the attachment of iodine to tyrosyl residues in Tg to produce MIT and DIT. This process occurs at the apical plasma membrane-follicle lumen boundary and involves H2O2, iodide, TPO, and glycosylated Tg. All rendezvous at the apical membrane to achieve Tg iodination (Fig. 2-8).

Fig. 2-8: Iodination of Tg at the apical plasma membrane-follicle lumen boundary.The scheme does not account for the relative size of the intervening molecules

First, iodide must be oxidized to an iodinating form. An extensive literature has sought to identify the iodinating species, but the issue is still not resolved (see (219) for a detailed review). One scheme proposes that oxidation produces free radicals of iodine and tyrosine, while both are bound to TPO to form MIT which then separates from the enzyme (Fig.2-5C). Further reaction between free radicals of iodine and MIT gives DIT. Experimental studies by Taurog (219) and others suggest that the TPO reduction occurs directly in a two electron reaction. A second proposal, based on studies of rapid spectral absorption changes (88;220;221), is that TPO-I+ is the iodination intermediate and that the preferred route is oxidation of TPO by H2O2 followed by two electron oxidation of I- to I+, which then reacts within a tyrosine. As a third possibility, Taurog (219) proposed a reaction between oxidized TPO and I- to produce hypoiodite (OI-), which also involves a two electron reaction. Whatever the precise nature of the iodinating species, it is clear that iodide is oxidized by H2O2 and TPO, and transferred to the tyrosyl groups of Tg. All tyrosine residues of Tg are not equally accessible to iodination. The molecule has about 132 tyrosyl residues among its two identical chains; at most, only about 1/3 of the tyrosyls are iodinated. As isolated from the thyroid, Tg rarely contains more than 1% iodine or about 52 iodine atoms.

The final step in hormone synthesis is the coupling of two neighbouring iodotyrosyl residues to form iodothyronine (Fig. 2-9). Two DIT form T4; one DIT and one MIT form T3. Coupling takes place while both acceptor and donor iodotyrosyl are in peptide linkage within the Tg molecule.The reaction is catalyzed by TPO, requires H2O2 (222-225) and is stringently dependent on Tg structure (226).The generation of the iodothyronine residue involves the formation of an ether bond between the iodophenol part of a donor tyrosyl and the hydroxyl group of the acceptor tyrosyl (Fig 2-10). After the cleavage reaction that gives the iodophenol, the alanine side chain of the donor tyrosyl remains in the Tg polypeptide chain as dehydroalanine (227-229). Observations both in vivo and in vitro show an appreciable delay in coupling after initial formation of iodotyrosines. A typical distribution for a Tg containing 0.5% iodine (a normal amount for iodine-sufficient individuals) is 5 residues MIT, 5 of DIT, 2.5 of T4 and 0.7 of T3 (180). More iodine increases the ratios of DIT/MIT and T4/T3, while iodine deficiency decreases them.

Fig. 2-9: Synthesis of hormone residues (coupling of iodotyrosines) in Tg at the apical plasma membrane-follicle lumen boundary. The scheme does not account for the relative size of the intervening molecules

Fig. 2-10: Possible coupling reaction sequence. Oxidation of iodotyrosines may produce iodotyrosyl radicals. The free radicals could combine to generate the iodothyronine residue (at the tyrosine acceptor site) and a dehydroalanine residue (at the tyrosine donor site), which in the presence of H2O converts into a serine

The distribution of hormone among several sites in the Tg molecule has been studied in a number of species (180;230-233). The most important is at tyrosyl 5, quite close to Tg N-terminus. It usually contains about 40% of Tg total T4. The second most important site is at tyrosyl 2554, which may contain for 20-25% of total T4. A third important site is at tyrosyl 2747, which appears favored for T3 synthesis in some species. Tyrosyl 1291 is prominent in T4 formation in guinea pigs and rabbits and very responsive to TSH stimulation. Incremental iodination of low iodine hTg in vitro, with lactoperoxidase as surrogate for TPO, led to the identification of the favored sites for iodination (234). Small increments of iodine go first to tyrosyl residues 2554, 130, 685, 847, 1448, and 5, in that order. Further addition increases the degree of iodination at these sites, iodinates some new tyrosyls, and results in thyroid hormone formation at residues 5, 2554, 2747, and 685, with a trace found at 1291, in that quantitative order. These data identified the most important hormonogenic sites in hTg, and also the favored sites for early iodination. The same work recognized three consensus sequences associated with iodination and hormone formation: i) Asp/Glu-Tyr at three of the four most important sites for hormone synthesis, ii) Ser/Thr-Tyr-Ser associated with hormone formation, including the C-terminal hormonogenic site that favors T3 in some species and iii)Glu-X-Tyr favoring early iodination, although usually not with hormone formation (Fig. 2-11).

Fig. 2-11: Diagram of the human Tg polypeptide chain; residue numbers refer to the human cDNA sequence; (a) sites forming T4 (sites A,B,D) (solid circles) and/or T3 (site C) (solid square); (b) early iodinated sites (solid triangles); (c) other iodinated sites (open triangles).

Identifying the donor tyrosyls has attracted considerable investigational interest over the past several decades. The fact that some tyrosyls are iodinated early but do not go on to provide the acceptor ring of T4 makes them potential donor candidates (234). On the basis of in vitro iodination of an N-terminal cyanogen bromide Tg peptide, Marriq et al. (235) concluded that residue 130 was a donor tyrosine for the major hormonogenic site at Tyr5. This conclusion was challenged by Xiao et al. (236) in a similar in vitro system. A baculovirus system expressing the 1-198 fragment of Tg, either normal or mutated on tyrosyl residues, showed that iodination of a fragment containing tyrosyls only at residue 5, 107 and 130 formed T4 as did the intact normal peptide, but this fragment could also form T4 with substitutions at residue 5 or 130 (237). Dunn et al.(238) who incorporated 14C-Tyr into beef thyroid slices followed by in vitro iodination and trypsin digestion of the N-terminal portion of Tg localized pyruvate (as a derivative of dehydroalanine) to residue 130 by mass spectrometry. They proposed that Tyr130 was the donor tyrosine for the most important hormonogenic site at Tyr5. Gentile et al. (239) used mass spectrometry to identify a peptide containing dehydroalanine at tyrosine 1375 of bTg and proposed this tyrosine as the donor for the hormonogenic site at residue 1291. Donors for the other major hormonogenic sites have not yet been identified.

In addition to its role as component of the iodoamino acids, iodine is associated with cleavage of peptide bonds of Tg, at least in vitro (180). This has been attributed to generation of free radicals during oxidation (240). Exposure of Tg to reducing agents yields an N-terminal peptide of about 20-26kDa, depending on the animal species, that contains the major hormonogenic site of Tg (241). This peptide appears in parallel with iodination or may slightly precede it (242). Further addition of iodine cleaves the 26kDa further, to produce an 18kDa (on human Tg), an event that also occurs with TSH stimulation (242). Thus, iodination-associated cleavage appears to be part of the maturation of the Tg molecule. These discrete N-terminal peptides have been found in all vertebrate Tg examined so far (231).

The amount of iodine has important effects on thyroid hormone production (243). The initial reaction between TPO and H2O2 produces the so-called "compound I," which oxidizes iodide and iodinates Tg. Next, the two reactants form compound II, which is necessary for the coupling reaction to make thyroid hormones. However, if excessive iodine is present, conversion to compound II does not take place, and hormone synthesis is impaired. (Fig. 2-12) Other iodinated compounds occasionally inhibit the thyroid. Thyroalbumin excited considerable interest several decades ago. This is an iodinated albumin, shown to be serum albumin that is iodinated in the thyroid (244). Occasionally, large amounts are found in certain thyroid diseases, including Hashimoto's thyroiditis (245), congenital metabolic defects (246), thyrotoxicosis (247) and thyroid carcinoma (248). In all these cases, there are abnormalities in thyroid structure which might explain the access of serum albumin to intrathyroidal iodination sites. However, in physiological conditions, serum albumin can reach thyroid follicle lumina by transcytosis i.e. basolateral endocytosis and vesicular transport to the apical plasma membrane (249). The thyroid also iodinates lipids and many different iodolipids have been described after high doses of iodide in vitro (250;251). Of particular interest is 2-iodohexadecanal (252;253). It occurs in the thyroid of several species following administration of KI, and its amount increases linearly with additional iodine, in contrast to iodination of Tg which eventually is inhibited by excess iodide. This compound inhibits the action of NADPH oxidase, which is responsible for H2O2 production (254;255). These findings suggested that iodination of lipids impairs H2O2 production and, therefore, decreases further Tg iodination. This is the most probable mechanism for the Wolff-Chaikoff effect (128).

Fig. 2-12: Demonstration of the Wolff-Chaikoff block induced by iodide in the rat. Animals were given increasing doses of stable iodide. There was at first an increase in total organification, but then, as the dose was increased further, a depression of organification of iodide and an increase in the free iodide present in the thyroid gland occurred.

HORMONE STORAGE

Tg molecules vectorially delivered to the follicule lumen by exocytosis accumulates to reach uncommun concentrations i.e. 0.3-0.5 mM.The mechanism operating such a “packaging” is unknown. Water and ion extraction from the follicle lumen might represent an active process leading toTg concentration. As the follicle lumen is a site of Ca++ accumulation (256;257), the high degree of compaction of lumenal Tg might depend on electrostatic interactions between Ca++ and anionic residues of Tg, which is an acidic protein. Stored Tg molecules undergo iodination and hormone formation reactions at the apical plasma membrane-lumen boundary (257-259), where TPO and H2O2 generating system reside. The mature Tg molecules, now containing MIT, DIT, T4 and T3, remains extracellular in the lumen of thyroid follicles. Turnover of intrafollicular material or so-called colloid varies greatly with gland activity. For normal humans, the organic iodine pool (largely in intrafollicular material), turns over at a rate of about 1% per day (14). When the turnover increases, less Tg is stored, and with extreme hyperplasia, none is evident and the entire organic iodine content may be renewed daily (14). In this situation, secretion of Tg and resorption of Tg (see below) probably occur at similar rates and only tiny amounts of intrafollicular material are present at any time.

Thyroglobulin as usually isolated from the thyroid is chiefly the 19S 660kDa dimer that has been glycosylated and iodinated. Iodination and hormone formation of Tg is more complex than generally thought because of the slow diffusion of molecules that are in a colloidal state in the follicle lumen. It has been reported that TSH alters the hydrodynamic properties of intrafollicular Tg molecules (260;261). The diffusion coefficient of Tg which is about 26mm2 / sec in water would only be in the order of 10-100mm2 / hour in the thyroid follicle lumen. There is evidence for the presence of insoluble Tg in the form of globules of 20-120 microns, at a protein concentration of almost 600 mg/mL, in the lumen of thyroid follicles of different animal species (262). In human, about 34% of the gland Tg would be in this form (263). In pig, insoluble Tg contains more iodine than did the 660kDa Tg, and had virtually no thyroid hormone (264). Insoluble Tg has many internal crosslinks through disulfide bonds, dityrosine, and glutamyl-lysine bonds, the latter generated by transglutaminase (265). The formation of Tg multimers that probably results from oxidative processes might be limited by the presence of molecular chaperones such as the protein disulfide isomerase (PDI) and BiP in the follicle lumen (266).

THYROGLOBULIN ENDOCYTOSIS

To be useful, thyroid hormones must be released from Tg and delivered to the circulation for action at their distant target tissues. Depending on numerous factors including - the supply of iodide as substrate, the activity of enzymes catalyzing hormone formation, the concentration and physico-chemical state of Tg - the hormone content of lumenal Tg molecules varies to a rather large extent. Tg molecules newly arrived in the follicle lumen with no or a low hormone content would co-exist with “older” Tg exhibiting up to 6-8 hormone residues. The downstream processes responsible for the production of free thyroid hormones from these prohormonal molecules must therefore adequately manage the use of these lumenal heterogeneous Tg stores to provide appropriate amounts of hormones for peripheral utilization. One would expect to find i) control systems preventing excess hormone production that would result from the processing of excessive amounts of prohormonal Tg molecules and ii) checking systems avoiding the use of Tg molecules with no or a low hormone content.

Fig. 2- 13: Visualization of Tg endocytosis by in vitro reconstituted thyroid follicles obtained from porcine thyrocytes in primary culture. Purified porcine Tg molecules labeled by covalent coupling of fluorescein were microinjected into the lumen of a follicle. A and B, phase contrast and fluorescence images taken at the time of microinjection. C and D, fluorescence images of the top (C) and the bottom (D) of the follicle after 2hr of incubation. Fluorescently-labeled Tg is present inside thyrocytes.

The way the thyroid follicle proceeds to generate free hormones from stored hormone containing Tg molecules has been known for a long time. Tg molecules are first taken up by polarized thyrocytes (Fig. 2-13) and then conveyed to lysosomal compartments for proteolytic cleavage that release T4 and T3 from their peptide linkages. The first step represents the limiting point in the thyroid hormone secretory pathway. Over the last decade, there has been substantial improvement in the knowledge of the cellular and molecular mechanisms governing the internalization or endocytosis and intracellular transport of the prohormone, Tg. The evolution has first been to consider that it could proceed via a mechanism different from phagocytosis, also named macropinocytosis, evidenced in rats under acute TSH stimulation (reviewed in (267)). Results obtained in rats and dogs have been for a long time extrapolated to the different animal species including human. There is now a number of experimental data indicating that in the thyroid of different species under physiological circumstances, basal internalization of Tg, mainly if not exclusively, occurs via vesicle-mediated endocytosis or micropinocytosis (reviewed in (268)), while macropinocytosis results from acute stimulation (Fig. 2-14) (269;270).

Fig. 2-14: Schematic representation of the two modes of internalization of Tg; Micropinocytosis (on the right) and Macropinocytosis or phagocytosis (on the left). Intralumenal Tg stores potentially subjected to endocytosis are composed of (recently secreted) non-iodinated Tg, iodinated Tg (Tg-I) and iodinated Tg containing iodothyronine residues (Tg-Ith).Abbreviations are: CV, Coated Vesicle; EE, Early Endosome; LE, Late Endosome; L, Lysosome; Pp, Pseudopod; CD, Colloid Droplet; PL, Phagolysosome. The scheme on the right indicates the three possible routes of transport of internalized Tg molecules reaching the EE: transport to LE, recycling towards the follicle lumen and transcytosis i.e.transport towards the basolateral plasma membrane.

The internalization process starts with the organization of microdomains at the apical plasma membrane of thyrocytes; these microdomains or pits, resulting from the recruitment and assembly of proteins (clathrin, adaptins…) on the cytoplasmic side of the membrane, invaginate to finally generate coated vesicles after membrane fission. Lumenal Tg molecules, either free or associated to membrane proteins acting as Tg receptors, enter the pits and are then sequestrated into the newly-formed vesicles (267-269). Tg internalization via vesicle-mediated endocytosis is regulated by TSH (268). The vesicles lose their coat and, through a complex fusion process, deliver their content into a first type of endocytic compartments, the early apical endosomes (270) (Fig 2-15). In these compartments, Tg molecules probably undergo sorting on the basis of recognition of different physico-chemical parameters either linked or independent such as the hormone content, exposed carbohydrates, conformation of peptide domains… A step of sorting appears as a prerequisite for subsequent differential cellular handling of Tg molecules. It has been shown that internalized Tg molecules can follow different intracellular pathways. Part of Tg molecules are conveyed via a vesicle transport system to the second type of endocytic compartments, late endosomes or prelysosomes. This route ending to lysosomes corresponds to the Tg degradation pathway for the generation of free thyroid hormones. It is reasonable to think that Tg molecules following this route are the more mature molecules (with a high hormone content) but, this has not been firmly demonstrated. The other Tg molecules with no or a low hormone content, present in early apical endosomes, enter either of the two following routes; they are recycled back into the follicle lumen through a direct vesicular transport towards the apical plasma membrane (271) or via a two-step vesicular transport to the Golgi apparatus and then to the apical plasma membrane (272). Alternately, Tg molecules are transported and released at the basolateral membrane domain of thyrocytes via transcytotic vesicles (262;273); a process accounting for the presence of Tg in plasma. The orientation of Tg molecules towards one or the other of these three routes requires the presence of receptors. However, one route could simply convey Tg molecules that are not selected for entering the other pathways.

Receptors involved in Tg endocytosis may operate at the apical plasma membrane for Tg internalization and downstream in apical early endosomes for Tg sorting. The requirement and/or the involvement of apical cell surface receptors has long been debated. Most investigators now recognize that receptors are not needed for internalization since Tg is present at a high concentration at the site of vesicle formation. So, Tg molecules are most likely internalized by fluid-phase endocytosis and not by receptor-mediated endocytosis. On the contrary, if apical membrane Tg receptors exist, their function would be to prevent the internalization of sub-classes of Tg molecules (274;275). As it is not conceivable that internalized Tg molecules could enter the different intracellular routes, described above, at random, Tg receptors must exist in early apical endosomes. A detailed review on potential Tg receptors has been made by Marino and Mc Cluskey (276).

The first candidate receptor, initially described by Consiglio et al.(277;278) was later identified as the asialoglycoprotein receptor composed of three subunits (RLH1,2 and 3). This receptor binds Tg at acidic pH and recognizes both sugar moities and peptide determinants on Tg (279). As low-iodinated Tg molecules are known to have a low sialic acid content, this receptor could be involved in sorting immature Tg molecules for recycling to the follicle lumen. A second receptor, still not identified, named N-acetylglucosamine receptor (280;281), presumably located in sub-apical compartments, interacts with Tg at acidic pH; it could also act as a receptor for recycling immature Tg molecules back to the follicle lumen. A third receptor; megalin, has more recently been discovered in the thyroid and has been the subject of extensive studies yielding convincing data (276;282-285). Megalin is an ubiquitous membrane protein belonging to the LDL receptor family. It is located in the apical region of thyrocytes and its expression is regulated by TSH. Megalin, that binds multiple unrelated ligands, interacts with Tg with a high affinity.In vitro and in vivo data indicate that Megalin is involved in the transcellular transport or transcytosis of Tg molecules, possibly with a low hormone content (286).

From the properties and subcellular location of these receptors, one can propose an integrated view of the sorting processes that would operate in early apical endosomes. The asialoglycoprotein receptor and/or the less defined N-acetylglucosamine receptor would recognize immature Tg for recycling and megalin would interact with Tg subjected to apical to basolateral transcytosis. The remaining Tg molecules would enter, without sorting, the functionally important pathway i.e. the prelysosome-lysosome route.

Under TSH stimulation, macropinocytosis would be triggered and would become operative in Tg internalization. Pseudopods representing extensions of the apical plasma membrane project into the follicle lumen and pinch off to form a resorption vacuole known as colloid droplet (287) .The colloid droplets then deliver their content to lysosomes. Pseudopod formation is one of the earliest effects of TSH on the gland, evident within several minutes after administration (288;289). In most species but perhaps not in rat, TSH stimulates macropinocytosis through the activation of the cyclic AMP cascade (290;291).

 

Fig. 2-15: Transmission electron microscope observations of apical endocytic structures in thyrocytes. Top: coated pits at the apical plasma membrane. Bottom: an early endosome located in the apical region. Bars, 200 nm.

PROTEOLYTIC CLEAVAGE OF THYROGLOBULIN

Internalized Tg molecules that are conveyed to lysosome compartments are subjected to diverse hydrolytic reactions leading to the generation of free thyroid hormones and to complete degradation of the protein. Given its composition, Tg is likely the substrate for the different lysosomal enzymes: proteases, glycohydrolases, phosphatases, sulfatases.... Efforts have been made to identify proteases involved in the release of hormonal residues from their peptide linkage in Tg. Endopeptidases such as cathepsin D, H and L (292-299) are capable of cleaving Tg.

Initial cleavage would bring into play endopeptidases and resulting products would be further processed by exopeptidases. Dunn et al. (295) showed that cathepsin B has exopeptidase activity as well as an endopeptidase action (295;297). These investigators tested the activities of human enzyme preparations against the 20kDa N-terminal peptide from rabbit Tg, which contains the dominant T4 site at residue 5. Extended cathepsin B incubation produced the dipeptide T4-Gln, corresponding to residues 5 and 6 of Tg. The combination of cathepsin B with the exopeptidase dipeptidase I released T4 from this dipeptide, although lysosomal dipeptidase I alone was not effective. Thus, the combination of cathepsin B and lysosomal dipeptidase I was sufficient to release free thyroid hormone from its major site at residue 5. The exopeptidase lysosomal dipeptidase II may also be involved in release of free T4, but from a site in Tg other than residue 5 (297). Thus, Tg probably undergoes selective cleavage reactions at its N- and C- terminal ends to release iodothyronines that are located nearby (297;300). Starting from highly purified preparations of thyroid lysosomes, Rousset et al. (301-303) have identified intralysosomal Tg molecules with very limited structural alterations but devoid of hormone residue. One may think that proteolysis of Tg occurs in two sequential steps; i) early and selective cleavages to release T3 and T4 residues and ii) delayed and complete proteolysis. The reduction of the very high number of disulfide bonds might be the limiting reaction between the two steps. The nature and the origin of the reducing compounds acting on Tg are not known. Noteworthy, the possibility of proteolytic cleavage of Tg inside the follicle lumen, before internalization, has been proposed (304-307) but not yet confirmed by other groups.

After Tg digestion, T4 and T3 must go from the lysosomal compartments to the cytoplasm and from the cytoplasm out of the cell to enter the circulation. It has been postulated for decades that thyroid hormones are released from thyrocytes by simple diffusion. There are many objections to this view (308). One of these comes from the chemical nature of iodothyronines; T4 and T3, which are generally considered as lipophylic compounds possess charges on both their proximal (amino acid side chain) and distal (phenolate) parts. As now known for the entry of thyroid hormones in peripheral target cells, the exit of thyroid hormones from thyrocytes probably involves membrane transporter(s). Details of hormone transport across the lysosomal membrane and then across the basolateral plasma membrane are unknown, including whether it is an active or passive process. At present, only a lysosomal membrane transporter for iodotyrosines has been reported (309;310). Nevertheless the role of newly cloned peripheral tissue thyroid transporters (311;312) in this process remains to be defined.

The type I and type II iodothyronine 5'-deiodinase is present in the thyroid (63;313;314) and deiodinate about 10% of T4 to T3. The extent of this intrathyroidal deiodination is increased when the thyroid is stimulated by TSH (315;316). Estimates of average normal secretion for euthyroid humans are 94-110 µg T4 and 10-22 µg T3 daily (317). The thyroid may also convert some T4 to 3,3'5'-T3 (reverse T3) within the thyroid. About 70% of the Tg iodine content is in the form of DIT and MIT, so this represents an important part of the intrathyroid iodine pool. Rather than lose it to the circulation, the thyroid deiodinates MIT and DIT and returns most of iodide to the intrathyroidal iodide pool. The responsible enzyme i.e. the iodotyrosine deiodinase is an NADPH-dependent flavoprotein with a estimated molecular weight of about 42kDa (318) and recently identified as DEHAL1(319-322). About 3-5 times more iodide is formed inside the gland each day by this deiodinase than enters the cell from the serum (14). The importance of the internal recycling of iodide is demontrated by congenitally goitrous subjects who harbour mutations in DEHAL gene (322) and cannot deiodinate iodotyrosines. These patients are successfully treated with large amounts of iodide (323). Some iodine is lost from the gland through inefficiency of its recycling by the iodotyrosine deiodinase (14;87;317;324). This leak may increase as the thyroid adapts to a high daily iodine intake (325), possibly as an autoregulatory process to prevent excessive Tg iodination. Much more iodide can be lost from diseased glands. Ohtaki et al. (87) found that some iodide leaks from all glands, including normal ones, but that the amount increases markedly with gland iodine content, presumably reflecting a dependence on dietary iodine intake. Fisher et al. (317) reported that about 38 µg iodide was released when the mean T4 secretion was 53 µg/day.

Among other products which are released or leak out from the thyroid, there is Tg (326;327). The secretion of Tg is clinically important. Its presence in serum can be detected by a routine assay and provides a sensitive (although not always specific) marker for increased thyroid activity. Attempts have been made to determine the biochemical characteristics of circulating Tg molecules in terms of iodine content (328), structural integrity (329) and hormone content (330). Serum levels are elevated in patients with hyperplastic thyroid or thyroid nodules including differentiated thyroid cancer. Tg measurement can identify congenital hyperplastic goiter, endemic goiter, and many benign multinodular goiters, but its greatest application is in the follow-up of differentiated thyroid cancer (331). Most papillary and follicular cancers retain some of the metabolic functions of the normal thyrocyte, including the ability to synthesize and secrete Tg. Subjects who have differentiated thyroid cancer treated by surgery and radioiodine should not have normal thyroid tissue left, and therefore, should not secrete Tg. If Tg is found in their serum, it reflects the continuing presence either of normal tissue, unlikely after its previous ablation, or of thyroid cancer. The depolarized cancer cells presumably secrete Tg directly in intercellular space. Tracking serum Tg levels is probably the most sensitive and practical means for the follow-up of such patients. It is more sensitive when the subject is stimulated by TSH. Until recently, this could only be done by withdrawal of thyroid hormone and consequent symptomatic hypothyroidism, but now recombinant human TSH can be administered to enhance the sensitivity of the serum Tg and thyroid scan (332-337).

CONTROL OF HORMONE SYNTHESIS

The most important controlling factors are iodine availability and TSH. Inadequate amounts of iodine lead to inadequate thyroid hormone production, increased TSH secretion and thyroid stimulation, and goiter in an attempt to compensate. Excess iodide acutely inhibits thyroid hormone synthesis, the Wolff-Chaikoff effect (243), apparently by inhibiting H2O2 generation, and therefore, blocking Tg iodination (127). A proposed mechanism is that the excess iodide leads to the formation of 2-iodohexadecanal (255), which is endowed with an inhibitory action on H2O2 generation.

TSH influences virtually every step in thyroid hormone synthesis and release. In humans the effects on secretion appear to be mediated through the cAMP cascade (see chapter 1) while the effects on synthesis are mediated by the Gq/phospholipase C cascade (338). Elsewhere in this chapter, we have mentioned instances of TSH regulation. To summarize, TSH stimulates the expression of NIS, TPO, Tg and the generation of H2O2 , increases formation of T3 relative to T4, alters the priority of iodination and hormonogenesis among tyrosyls and promotes the rapid internalization of Tg by thyrocytes. These several steps are interrelated and have the net effects of increasing the amount of iodine available to the cells and of making and releasing a larger amount and a more effective type of thyroid hormone (T3).

Anti-thyroid drugs are external compounds influencing thyroid hormone synthesis. The major inhibitory drugs are the thionamides: propylthiouracil and methimazole. In the thyroid, they appear to act by competing with tyrosyl residues of Tg for oxidized iodine, at least in the rat (219). Iodotyrosyl coupling is also inhibited by these drugs and appears more sensitive to their effects than does tyrosyl iodination.

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Primary Testicular Failure

Abstract

Primary testicular failure may result in endocrine failure, leading to testosterone deficiency or exocrine failure causing impaired spermatogenesis and subsequently male infertility. While some aspects of primary testicular failure are described in detail in separate chapters of Endotext.com, this chapter focuses on congenital or acquired anorchia, Leydig cell hypoplasia, and spermatogenic failure including germ cell aplasia (Sertoli cell only syndrome), spermatogenic arrest, hypospermatogenesis, and mixed atrophy. In addition, genetic causes for primary testicular failure are described such as numerical chromosome aberrations including Klinefelter syndrome, XX-Male syndrome, and XYY syndrome, structural chromosome aberrations of the autosomes or sex chromosomes, and Y chromosome microdeletions. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org.

Key messages

  • Bilateral anorchia is defined as a complete absence of testicular tissue in genetically and phenotypically male patients.
  • Leydig cells hypoplasia is caused by inactivating mutations of the LH receptor.
  • Primary spermatogenic failure has to be considered as a description of certain histopathologic phenotypes, and not as a manifestation of single disease entities.
  • Several genetic causes for primary spermatogenic failure have been elucidated recently.
  • Modern management of patients with primary testicular failure caused by numerical chromosome aberrations such as Klinefelter syndrome can ameliorate symptoms of testosterone deficiency and – at least in some patients – can overcome infertility.
  • Up to date clinical guidelines are available for molecular diagnosis of Y chromosome microdeletions.
  • Novel technologies such as whole-genome sequencing will help to greatly increase the fraction of men suffering from primary testicular failure with a clear genetic diagnosis.

INTRODUCTION

The testis has an endocrine as well as an exocrine function. Endocrine testicular failure results in testosterone deficiency. In primary endocrine testicular failure, a decline in testosterone secretion (resulting in a condition termed hypoandrogenism) is caused by a deficiency or absence of Leydig cell function. Clinically relevant diseases described in this chapter are anorchia, Leydig cell hypoplasia and numerical chromosome abnormalities. Testicular dysgenesis is another cause for primary testicular failure that is described in depth in Endotext.com, Pediatric Endocrinology, Chapter 7: Sexual Differentiation. In contrast to primary endocrine testicular failure, secondary endocrine testicular failure is caused by absent or insufficient bioactivity of GnRH or LH (see Endotext.com, Endocrinology of Male Reproduction, Chapter 5: Hypogonadotropic hypogonadism and gonadotropin therapy).

The phenotype of primary exocrine testicular failure is male infertility. A comprehensive review on causes and treatment of male infertility is given in Endotext.com, Endocrinology of Male Reproduction, Chapter 7: Clinical management of male infertility. Cryptorchidism as a clinically relevant cause for primary exocrine testicular failure is discussed in Endotext.com, Endocrinology of Male Reproduction, Chapter 19: Cryptorchidism and hypospadias and testicular tumors as a cause and/or sequelae of testicular failure is discussed in Endotext.com, Endocrinology of Male Reproduction, Chapter 13: Testicular cancer pathogenesis, diagnosis and endocrine aspects.

This chapter focuses on anorchia, germ cell aplasia, spermatogenetic arrest, hypospermatogenesis, numerical chromosome abnormalities, structural chromosomal abnormalities, as well as Y chromosome microdeletions causing primary exocrine testicular failure.

ANORCHIA

Bilateral anorchia is defined as complete absence of testicular tissue in genetically and phenotypically male patients. In unilateral anorchia testicular tissue is still present on the contralateral side.

Pure anorchia has to be differentiated from conditions with ambiguous and intersex genitalia (see Endotext.com, Pediatric Endocrinology, Chapter 7: Sexual Differentiation). A clinically important differential diagnosis is cryptorchidism and testicular atrophy where testicular tissue is still detectable (see Endotext.com, Endocrinology of Male Reproduction, Chapter 19: Cryptorchidism and hypospadias).

Congenital Anorchia

Bilateral congenital anorchia is rare; the incidence appears to be 1:20,000 males. Unilateral congenital anorchia is about 4 times as frequent.

As male differentiation of the genital tract and development of the penis and scrotum is dependent on the production of anti-Mullerian hormone (AMH) and androgens, the testis must have disappeared after initial activity in cases of bilateral anorchia. For the development of Wolffian duct structures, an ipsilateral testis must be present at least up to the 16th week of gestation ("the vanishing testis syndrome") (1). Intrauterine infarction of a maldescended testis or testicular torsion appears to be the major contributor to anorchia (2).

In patients with congenital bilateral anorchia serum gonadotropins are already elevated in childhood and rise to very high levels from the age of puberty onwards. Testosterone levels remain within the castrate range. In patients with suspected bilateral anorchia it is mandatory to rule out cryptorchidism, as cryptorchidism is associated with an increased risk for testicular cancer and should definitively not be overlooked (see Endotext.com, Endocrinology of Male Reproduction, Chapter 13: Testicular cancer pathogenesis, diagnosis and endocrine aspects). Both the hCG stimulation test, that examines testosterone secretory capacity, and serum AMH measurement can be used for differential diagnosis. During hCG administration testosterone levels remain unchanged in patients with bilateral anorchia even after a 7-day period of stimulation, while a rise can be detected in patients with cryptorchidism (3). In comparison to the hCG test, measurement of AMH, which is undetectable in anorchia, has a higher sensitivity, but equal specificity for differentiation of bilateral anorchia from bilateral cryptorchidism (4; 5). Endocrine tests are not useful for differential diagnosis of unilateral anorchia. In these cases imaging techniques such as computer tomography or MRT and finally exploratory surgery or laparoscopy have to be applied.

Unilateral anorchia does not require therapy. In phenotypically male patients with bilateral congenital anorchia, testosterone substitution has to be implemented at the time of expected puberty. For psychological or cosmetic reasons, implantation of testicular protheses could be offered to the patient although these are often expensive. To date, there is no treatment of infertility in bilateral anorchia.

Acquired Anorchia

Surgical removal of both testes in patients with androgen-dependent prostate carcinoma is the most prevalent cause for bilateral acquired anorchia. Other reasons include unintended removal or devascularisation during herniotomy, orchidopexy or other testicular surgery, testicular infarction, severe trauma and self-mutilation. If only one testis is lost then fertility and testosterone production will normally be maintained by the remaining testis and no specific therapy is required. However, patients with a single testis require careful management when surgery is planned on the remaining testis.

The clinical appearance in patients with bilateral acquired anorchia depends on the time when testicular loss occurred. Acquired anorchia before puberty leads to the characteristic phenotype of male eunuchoidism and after puberty to the phenotype of post-pubertal testosterone deficiency (see Endotext.com, Endocrinology of Male Reproduction, Chapter 2: Androgen physiology, pharmacology and abuse).

Untreated acquired bilateral anorchia seems to have no effect on life expectancy, but clearly has an adverse effect on the quality of life (6). If both testes have been removed for therapeutic purposes, e.g. in a patient with prostate carcinoma, androgen supplementation is contraindicated. All other patients have to receive permanent testosterone substitution from the time of the expected onset of puberty in order to induce pubertal development, and in an adult immediately after testicular loss to maintain the various androgen-dependent functions.

LEYDIG CELL HYPOPLASIA

Leydig cell hypoplasia is a rare disease with an autosomal recessive pattern of inheritance and estimated incidence of 1:1,000,000. The Leydig cells are unable to develop because of inactivating mutations of the LH receptor that fails to provide the necessary stimulation of intracellular pathways. The underlying gene defect in Leydig cell hypoplasia was first described by Kremer et al (7) and various other defects have since been described (8–21). Men with Leydig cell hypoplasia present with very low serum testosterone and high LH levels. Leydig cell hypoplasia belongs to the group of the disorders of sex differentiation (DSD) and is currently classified as 46,XY DSD.

The phenotype is dependent on the extent of intrauterine testosterone secretion. Two types of Leydig cell hypoplasia have been described. Type I is the most severe form, resulting in a female phenotype of the external genitalia with blind ending vagina, primary amenorrhea, and absence of secondary sex differentiation at puberty. It is caused by inactivating mutations in the LH receptor that completely prevent LH and hCG signal transduction and thus testosterone production. Leydig cell hypoplasia type II is characterized by milder signs of androgen deficiency with a predominantly male habitus but signs of hypogonadism with micropenis and/or hypospadia. This milder form is derived from mutations of the LH receptor, which only partially inactivate signal transduction and retain some responsiveness to LH (16). Testicular histology reveals seminiferous tubules, whereas Leydig cells are not present or appear only as immature forms. Epididymides and deferent ducts are usually present, whereas the uterus, tubes or upper vagina are not found. In a patient with Leydig cell hypoplasia type II lacking exon 10 of the LH receptor, maternal hCG synthesized during pregnancy probably led to the development of a normal male phenotype, whereas LH was unable to stimulate the mutant receptor at the time of puberty (22; 23). HCG treatment of this patient was capable of inducing testosterone biosynthesis and complete spermatogenesis (22). This case, however, represents an exception. Therapy of 46,XY DSD with complete feminization requires both orchidectomy because cryptorchid gonads are prone to malignant degeneration, and estrogen substitution therapy.

Spermatogenic failure

Whereas endocrine testicular failure causes hypogonadism, spermatogenic failure - defined as exocrine testicular failure - leads to male infertility. Spermatogenic failure might be caused by hypothalamic, pituitary, or testicular disorders. A comprehensive review on causes and treatments of male infertility is given in Endotext.com, Endocrinology of Male Reproduction, Chapter 7: Clinical management of male infertility. Various testicular etiologies of spermatogenic failure may lead to the same histopathological pattern. In this sense, spermatogenic failure such as germ cell aplasia (Sertoli cell only syndrome), maturation arrest (MA) at different levels of early round spermatids, primary spermatocytes, or spermatogonia, and hypospermatogenesis have to be clearly differentiated from normal spermatogenesis. A key point is that primary spermatogenic failure has to be considered as a description of certain histopathologic phenotypes, and not as a manifestation of single disease entities.

GERM CELL APLASIA (SERTOLI CELL ONLY SYNDROME)

Germ cell aplasia or Sertoli cell only syndrome (SCO) is a histopathologic phenotype that was first described by Del Castillo et al. in 1947 (24). In complete germ cell aplasia the tubules are reduced in diameter, and contain only Sertoli cells but no other cells involved in spermatogenesis [Figure 1]. Germ cell aplasia can also be focal with a variable percentage of tubules containing germ cells, but in these tubules spermatogenesis is often limited in both quantitative and qualitative terms (25), and such cases should be referred to as hypospermatogenesis (see below). Germ cell aplasia or SCO is one common cause of non-obstructive azoospermia (NOA).

Figure 1. Germinal cell aplasia or Sertoli cell only Syndrome: Seminiferous tubules exhibit only Sertoli cells (SCO). Note the thickening of the lamina propria, focal hyperplasia of Leydig cells (hypLc) and interstitial infiltration of lymphocytes (ly). Primary magnification, x 20.

The lamina propria of SCO tubules is often found to be thickened due to increased collagen type IV and increased thickness of the basal lamina. The latter is associated with an overabundance of the beta2 chain of laminin and thought to be related to spermatogenic dysfunction (26).

In congenital germ cell aplasia, the primordial germ cells do not migrate from the yolk sac into the future gonads or do not survive in the epithelium of the seminiferous tubule. Anti-neoplastic therapy with radiation or chemotherapy may cause complete loss of germ cells. Other reasons include viral infections of the testes such as mumps orchitis. Germ cell aplasia can occur in maldescended testes.

Chromosomal abnormalities, especially microdeletions of the Y chromosome, are important genetic causes for complete germ cell aplasia (27). These deletions have been characterized and deletions in the AZFb or AZFb+c regions were identified to be important genetic causes of SCO and/or MA resulting in azoospermia (28). Sertoli cells, although showing normal histology, have an increased apoptotic index (29–31).

Several studies tried to identify other genetic risk factors which are associated with SCO. SEPTINS belong to a family of polymerizing GTP-binding proteins being required, for example, for membrane compartmentalization, vesicle trafficking, mitosis and cytoskeletal remodeling. SEPTIN12 participates in male infertility, especially SCO. Although no mutations were found in patients with SCO, 8 coding single-nucleotide polymorphisms (SNP1-SNP8) could be detected in these patients and the genotype and allele frequencies in SNP3, SNP4, and SNP6 were notably higher than in the control group (32). Most recently, Miyamoto et al. (33) analyzed the human LRWD1 gene whose translated protein mediates the origin recognition complex in chromatin which is critical for chromatin organization in post-G1 cells. Again, no mutations in SCO patients were found, but allele frequencies of two of three SNPs (SNP1 and SNP2) were notably higher compared to controls.

A hint to genetic risk factors leading to SCO was given by Tüttelmann et al. (34). They evaluated copy number variants (CNVs) in patients with severe oligozoospermia and Sertoli-cell-only syndrome and found that sex-chromosomal CNVs were significantly overrepresented in patients with SCO.

Diagnosis of germ cell aplasia can only be made by testicular biopsy. However, the testicular biopsy may not be representative in certain patients, as testicular sperm have been retrieved by testicular sperm extraction (TESE) in patients with apparently "complete germ cell aplasia" following a diligent review of the testicular histology (35). In addition, it has been demonstrated in a large consecutive series of bilateral biopsies from 534 infertile men that a marked discordance of spermatogenic phenotype pattern between both testes can be detected in about 28% of patients (36). Therefore, multiple testicular biopsies of both testes must be scrupulously screened before a diagnosis of complete germ cell aplasia can be made (37).

Patients with the complete form of germ cell aplasia are always azoospermic. Currently, there is no therapy for exocrine testicular failure of patients with complete germ cell aplasia. In general, testosterone production in the Leydig cells is not affected and patients are normally androgenized, and only few patients have hypoandrogenism requiring treatment.

Some patients have the appearance of complete germinal cell aplasia in some tubules but with complete spermatogenesis in adjacent tubules (sometimes called ‘focal’ germinal cell aplasia) while others have the appearance of an excess number of precursor germ cells in relation to the number of mature spermatids in the epithelium. Such cases have been described as incomplete or focal germinal cell aplasia which implies, perhaps falsely, a commonality between these disorders and those with complete germinal cell aplasia in all tubules.

SPERMATOGENIC ARREST

Spermatogenic arrest is also not a specific diagnosis for primary exocrine testicular failure, but a histopathological description of the interruption of normal germ cell maturation [Figure 2] at the level of a specific cell type including that of spermatogonial arrest [Figure 3], spermatocyte arrest [Figure 4], and spermatid arrest [Figure 5]). Sometimes, seminiferous cords/nodules with immature Sertoli cells can be found. These Sertoli cells still exhibit anti-muellerian hormone expression indicating their prepubertal state of differentiation. A definite diagnosis can only be made by multiple testicular biopsies.

Figure 2. Normal spermatogenesis: Seminiferous epithelium in stage I (I) and stage III (III) of spermatogenesis showing spermatogonia (sg), pachytene spermatocytes (p), round step 1 and 3 spermatids (rsd) and elongating step 7 spermatids (elsd). Primary magnification, x 40.

Figure 3. Arrest of spermatogenesis: Seminiferous tubule showing arrest of spermatogenesis at the level of spermatogonia. Note multilayered spermatogonia (spg). Arrow: Sertoli cell nuclei. Primary magnification, x 40.

Figure 4. Arrest of spermatogenesis: Seminiferous tubule showing arrest of spermatogenesis at the level of primary spermatocytes in pachytene stage (p). Primary magnification, x 40.

Figure 5. Arrest of spermatogenesis: Seminiferous tubule showing arrest of spermatogenesis at the level of early round spermatids (rsd). Note prominent multinucleated spermatid (mrsd). Primary magnification, x 40.

Meiotic arrest is regularly found in patients showing non obstructive azoospermia being considered as idiopathic, because no genetic or other origin can be detected. There are numerous studies showing lack of expression of several genes in meiotic maturation arrest compared to normal spermatogenesis. A major subgroup of patients lacks BOULE protein expression in primary spermatocytes, which is key factor of meiosis (38). The defect seems to be due to factor(s) upstream of BOULE being involved in the transcription and/or translation of BOULE. Heat shock protein levels are low or absent, such as heat shock transcription factor, Y chromosome (HSFY) (39) or HSPA2 that is involved in DNA mismatch repair (MMR) (40). SYPC3, a gene responsible for the synaptonemal complex is also involved in MMR and was found to be reduced. There is increasing evidence that alterations of the SYPC3 gene are involved in spermatocyte maturation arrest. Although expression of SYCP3 mRNA is found in patients showing normal spermatogenesis and spermatocyte maturation arrest, the lack of expression in men with spermatogonial arrest, Sertoli Cell Only syndrome, and testicular atrophy suggests negative effect on spermatogenesis and male fertility (41). However, data concerning the involvement of SYCP3 mutations related to spermatocyte arrest are inconsistent. A mutation analysis of the SYCP3 gene for 58 patients revealed only polymorphisms (42). Miyamoto et al. found a 1 bp deletion (643delA) resulting in a truncation of the C-terminal region of the SYCP3 protein in two of 19 azoospermic men with maturation arrest versus 75 patients showing normal spermatogenesis (43). Recently Stouffs et al. detected one change present in an evolutionary important functional domain of the SYCP3 gene in only one male patient that was absent in more than 200 controls (44).

MicroRNA-383 was shown to be down-regulated in maturation arrest (45). It was associated with a hyperactive proliferation of germ cells in patients with mixed patterns of maturation arrest, indicating that miR-383 functions as a negative regulator of proliferation. The authors concluded that abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor (46). There is a possible feedback loop between the fragile X mental retardation protein (FMRP) and miRNA-383, and FMRP acts as negative regulator for miRNA-383 functions, a loop that seems to be disturbed in maturation arrest (47).

Increased apoptotic index associated with spermatocyte maturation arrest was reported (29–31), data that correspond to the lack of expression of survivin, an inhibitor of apoptosis (48). These data correspond to the reduction of cyclin A, required for both the mitotic and meiotic divisions, in meiotic arrest (49).

In tubules showing meiotic arrest, there is also disturbance of the expression pattern of genes that are required for spermiogenesis. For example, BET (bromodomain and extra terminal) genes encode for transcriptional regulators and for histone-interacting chromatin remodelers. BRDT (bromodine testis specific), a key molecule participating in chromatin remodeling, is required for creation and/or maintenance of the chromocenter in round spermatids, a structure that forms just after completion of meiosis (for review see (50). The BRDT protein is localized in the nuclei of spermatocytes, spermatids, and ejaculated spermatozoa, and transcription is almost zero in primary spermatocytes of testes showing meiotic arrest (51). These data indicate that genes being important for postmeiotic spermiogenesis are already disturbed in the premeiotic stage.

In some patients with predominant round spermatid maturation arrest, the expression of cAMP Responsive Element Modulator (CREM) is significantly reduced or undetectable (52). Most recently, different expression of chromatin remodeling factors between normal spermatogenesis and round spermatid maturation arrest were found and suggest that impaired epigenetic information and aberrant transcription represents one reason for spermatid maturation arrest (53). Studies of the numerous mouse knock out models that display a spermatogenic phenotype, including sperm cell arrest, has contributed little of clinical relevance to the large number of men with idiopathic infertility. The possible role of several gene mutations and polymorphisms has been extensively investigated but no clear-cut genetic factor could be identified so far (54; 55). Spermiogenesis is a complex process with numerous different factors being involved. Thus it should be noticed that many factors are described and will be found to be related or responsible for spermatid maturation arrest, such as Krüppel-like factor 4 (KLF4), a transcription factor which is involved in many cellular and developmental processes including terminal differentiation of cells and carcinogenesis. A significant altered subcellular localization in arrested spermatids gives a first hint at a role for KLF4 during spermiogenesis (56).

Data concerning the topic of possible epigenetic alterations related to spermatogenic defects are rare. Khazamipour et al. analyzed the methylation status in the specific CpG island of the promoter region of MTHFR (Methylenetetrahydrofolate reductase) and found a significant hyper-methylation in 53% of the patients showing NOA compared to 0% of patients with obstructive azoospermia and normal spermatogenesis, indicating that hyper-methylation is specific and not due to a general methylation defect (57). Authors suggest that epigenetic silencing of MTHFR may be involved in azoospermic infertility. A similar study analyzing the CpG island containing tissue specific differentially methylated regions (TDMRs) in the VASA gene revealed significantly higher methylation in maturation arrest compared to normal spermatogenesis (58). Hyper-methylation associated silencing of PIWIL2 and TDRD1 was reported by Heyn et al. in human infertile patients showing maturation arrest (59).

Adiga et al. evaluated the expression pattern of a DNA methyltransferase (DNMT3B) which is important for germ cell methylation (60). Although they found a reduced number of DNMT3B positive primary spermatocytes in the case of bilateral maturation arrest, the few mature spermatids did not reveal any alterations of global methylation status.

Additionally, there may also be extratesticular factors such as long standing ischemia due to malformation of valves in spermatic veins responsible for maturation arrest (61). Secondary factors for spermatogenetic arrest are toxic substances (radiotherapy, chemotherapy, antibiotics), heat or general diseases (liver or kidney insufficiency, sickle cell anaemia) (62).

Testicular volume, FSH and inhibin B may be in their respective normal range, but may also be elevated or decreased. When these clinical parameters are normal, the differential diagnosis includes obstructive and non-obstructive azoospermia and this distinction made by diagnostic biopsy.

The arrest may be caused by genetic or by secondary influences. Genetic etiologies include trisomy, balanced-autosomal anomalies (translocations, inversions) or deletions in the Y chromosome (Yq11). It is likely that many genetic factors exist but have not yet been identified.

Complete arrest of spermatogenesis results in azoospermia. To date, there is no known therapy for uniform spermatogenic arrest (63).

HYPOSPERMATOGENESIS

The histological phenotype “hypospermatogenesis” shows complete spermatogenesis, but the number of elongating or elongated spermatids is moderately or severely reduced and the composition of the seminiferous epithelium is often incomplete because of missing generations of germ cells.

There are numerous reports showing functional impairment or alterations in seminiferous tubules showing hypospermatogenesis. Hypospermatogenesis is often associated with multinucleated spermatids indicating failure in spermiogenesis, or with so-called “megalospermatocytes” that are the morphological representation of missing synaptonemal complexes during meiotic prophase (64; 65). Whereas mitotic activity of spermatogonia is reduced (66), the apoptotic index indicating increased germ cell degeneration is elevated as shown by caspase immunohistochemistry (31) or TUNEL analysis (30). Both are also true in the case of maturation arrest at different levels of germ cell development.

Concentric spherical concrements deriving from the basal lamina are often found, when ultrasonographic examination of the testis reveals “microlithiasis”. These concrements may be associated with carcinoma in situ (syn: testicular intraepithelial neoplasia: TIN).

During spermiogenesis, protamine mRNA, being associated with the prognosis of successful ICSI therapy, is reduced in early round spermatids (67; 68). The histone to protamine transition during spermiogenesis is due the transcription factor CREM (cAMP responsive element modulator) and CREM activators. There are different isoforms functioning as activators and repressors and the expression pattern is related to impaired spermatogenesis (69–71).

Sertoli cell function is impaired, which has been described by Bruning et al. (72) by three dimensional reconstruction indicating functional dedifferentiation. This phenomenon, found to be associated with numerous aspects of Sertoli cell function, was later reviewed by Sharpe et al. (73). Most recently, Fietz et al. (74) could show a reduced mRNA expression of the androgen binding protein by quantitative RT-PCR. Huthaniemi et al. (75) found increased testosterone levels associated with androgen receptor CAG repeat length and because of a constant testosterone to estrogen ratio, authors suggested increased estrogen levels to be responsible for impaired spermatogenesis. Contrary data were reported by Nenonen et al. (76) who found a non-linear association between androgen receptor CAG repeat length and risk of male subfertility. This meta-analysis including almost 4000 patients revealed that androgen receptors with both either short or long repeats displayed lower activity than the receptors with repeats of median length. On a cellular level, Fietz et al. (74) analyzed androgen receptor mRNA of Sertoli cell populations associated with defined spermatogenic impairment using laser assisted cell picking and did not find any correlation of CAG repeat length to testicular histology or AR expression, suggesting factors other than CAG repeat to be responsible for severe spermatogenic impairment including mixed atrophy. This was also found by Hadjkacem-Loukil et al. (77) in a cohort of Tunesian azoospermic men showing Sertoli Cell Only syndrome or maturation arrest.

The lamina propria looks mostly unaffected in routine histological sections. However, functional defects resulting in a loss of contractility i.e. such as myosin heavy chain (MHY11) (78) or smooth muscle actin (79) were associated with hypospermatogenesis or mixed atrophy.

Functional dedifferentiation was found in Leydig cell hyperplasia and adenoma indicated by downregulation of the Leydig cell specific relaxin-like factor using in situ hybridysation and immunohistochemistry (80).

In most patients with hypospermatogenesis, testicular volume is reduced. FSH is elevated in most, but not all patients, with serum levels correlating positively with the proportion of tubules with germ cell aplasia (81). Several studies have demonstrated that inhibin B is a more sensitive and specific endocrine marker of hypospermatogenesis (82; 83). However, even the combined measurement of inhibin B and FSH provides no certainty concerning the presence or absence of sperm in multiple testicular biopsies (84; 85).

Mixed Atrophy

In most oligozoo- or azoospermic patients, testicular biopsy reveals a pattern of different spermatogenic defects in adjacent tubules: “mixed atrophy” being first described by Sigg (86): the simultaneous occurrence of seminiferous tubules includes SCO tubules or even only lamina propria (tubular shadows). This requires a detailed score-count analysis (35; 37). Additionally, functional mRNA or protein analysis of gene expression pattern described above can help to optimize the diagnosis of the underlying defects.

From a practical clinical perspective, the differentiation is important as patients with hypospermatogenesis or mixed atrophy may have azoospermia or varying degrees of oligoasthenoteratozoospermia, and sperm may be retrieved from testicular biopsies (TESE) (35). Pregnancies can be achieved with sperm retrieved by TESE that are injected into mature oocytes by intracytoplasmic sperm injection (ICSI). It has been suggested that residual sperm production could be improved by FSH therapy in incomplete germ cell aplasia. Clinical studies performed so far have demonstrated some increase in sperm concentration in the ejaculate and improvement of pregnancy rate (87; 88).

NUMERICAL CHROMOSOME ABERRATIONS

Klinefelter Syndrome

Harry Klinefelter first described this syndrome in 1942 as a clinical condition with small testes, azoospermia, gynecomastia and an elevated serum FSH (89). Only in 1959 was the chromosomal basis of the disorder elucidated as the chromosomal constitution with a supernumerary X-chromosome. Subsequently, the diagnosis of Klinefelter syndrome is made by chromosome analysis demonstrating the 47,XXY karyotype or one of its rarer variants.

The prevalence of Klinefelter syndrome is approximately 1 in 1,000 to 1 in 500 males (90). It is the most frequent form of primary testicular dysfunction affecting spermatogenesis as well as hormone production and is found in about 3% of unselected infertile men and >10% of men presenting with azoospermia (91; 92). It appears that at least half of the cases remain undiagnosed and untreated throughout life (90).

A non-mosaic 47,XXY karyotype is found in 80 - 90 percent of Klinefelter patients and mosaicism is seen in another 5 - 10 percent. The 47,XXY/46,XY mosaicism is most common. The 48,XXXY, 48,XXYY and 49,XXXXY karyotypes constitute 4 - 5 percent of all Klinefelter syndrome karyotypes, structurally abnormal extra X chromosomes are found in less than one percent of patients. Apart from karyotype analysis, molecular genetics methods can be used to quantify the number of X chromosomes, for example by quantitative PCR analysis of the androgen receptor gene located on the X chromosome (93).

The numerical aberration in non-mosaic 47,XXY is derived with equal likelihood from maternal or paternal meiotic error (94; 95). Most cases are caused by meiosis without X/Y or X/X recombination. Advanced maternal age seems to be a risk factor (90). It is not known whether the 47,XXY karyotype is slightly over-represented among spontaneous abortions and stillbirths. However, in contrast to many other aneuploidies, Klinefelter syndrome seems to be only a minor risk factor and most pregnancies result in a live-birth.

Patients with Klinefelter syndrome are usually inconspicuous until puberty. Interestingly the velocity of height gain can be increased in the pre-pubertal years. Men with Klinefelter syndrome tend to be tall (mean adult height is about the 80th percentile for the population) and to have relatively long legs compared to their overall height. Previously, the tall stature in KS was mainly thought to be a consequence of the hypogonadism, i.e. lower testosterone/estradiol levels not stopping long-bone growth by inducing epiphyseal growth plate fusion. However, more recent data comparing gonosomal aneuploidies support that increased body height is caused by excessive expression of growth-related genes. In this respect, the SHOX-gene is the leading candidate as it is located in the pseudoautosomal region and therefore present in three copies in Klinefelter men (96).

In most patients, early stages of puberty proceed normally. Post-pubertally the syndrome is characterized by small testes with firm consistency remaining in the range of 1 - 4 ml. Most patients with Klinefelter syndrome are infertile because of azoospermia. Testicular histopathology in adult men with Klinefelter syndrome displays various patterns. Classically, germ cell aplasia, total tubular atrophy or hyalinizing fibrosis and relative hyperplasia of Leydig cells are found. However, in some adult Klinefelter patients, foci of spermatogenesis up to the stage of mature testicular sperm can be detected ((97), and see below).

The degree of virilization varies widely. In early puberty, LH and FSH increase while serum levels of testosterone plateaus at or just below the lower limit of the normal range. After the age of 25, about 80% of patients have reduced serum testosterone levels and complain of decreasing libido and potency. On average, serum estradiol levels are high normal or may exceed the normal range. LH and especially FSH levels are exceedingly high, serum levels of inhibin B are very low or undetectable (98; 99).

During puberty, bilateral painless gynecomastia of varying degrees develops in about half of the patients. In a large Danish study covering 696 men with Klinefelter syndrome, no evidence for a substantial increase in the overall cancer rate was found (100). The risk of developing mammary carcinoma may be increased relative to normal men but remains a rare occurrence and routine surveillance is not recommended (100; 101). A significantly increased risk was found for the rare mediastinal malignant germ cell tumors, which occur preferentially at the age of 14 to 29 years (100).

The intelligence of Klinefelter patients is very variable. The group difference between boys with Klinefelter syndrome and controls amounts to 11 points in full scale IQ (92 versus 103), and deficits are observed primarily in verbal and cognitive abilities (102). Some of the young patients attract attention because of learning difficulties and school problems. They may fail to reach the level of achievement or professional expectations of their families (103; 104). Compared with their classmates, certain abnormal physical and psychological characteristics of the patients become obvious and they may become socially alienated. Higher-grade aneuploidy of the sex-chromosomes (48,XXXY, 48,XXYY and 49,XXXXY) is associated with mild to severe mental retardation while Klinefelter patients with chromosomal mosaicism (47,XXY/46,XY) may show very few clinical symptoms.

In general, the variability of the clinical features in patients with Klinefelter syndrome is related to the degree of androgenisation, which, in turn, partly depends on the pattern of inactivation of one copy of the androgen receptor gene. In particular, a significant genotype-phenotype association exists in Klinefelter patients and androgen effects on appearance and social characteristics are modulated by the androgen receptor CAGn polymorphism (105; 106).

Regarding infertility treatment, it should be noted that in rare cases sperm could be found in the ejaculate and, exceptionally, spontaneous paternity has been described (107). The rate of diploidy of sperm as well as disomy for gonosomes and autosomes has been reported to be increased in patients with Klinefelter syndrome, however, the majority of sperm appear to be normal (108–111). Almost two decades of experience with TESE/ICSI in patients with Klinefelter syndrome demonstrates that testicular sperm can be recovered in about 50% of the patients (112–115). Increasing age may be a negative predictive factor for successful TESE and some advocate to offer TESE and cryopreservation of tissue/spermatozoa already to teenaged patients. To what extent other factors such as previous testosterone treatment influence the chances of successful TESE remains under debate, as does the suggested treatment with drugs increasing FSH prior to TESE (116). So far, over 170 babies were born using testicular sperm for ISCI, all showing normal karyotype, although aneuploidies can be occasionally found by preimplantation or prenatal diagnosis (117). However, since the birth of normal children conceived by assisted reproductive techniques seems to be the rule (115), preimplantation diagnosis is not per se indicated. Based on indirect clues, it was postulated that 47,XXY spermatogonia are able to complete meiosis (118). However, Sciurano et al. nicely showed by fluorescence in situ hybridization (FISH) in testicular tissue of Klinefelter patients that all meiotic spermatocytes were euploid 46,XY(119). Fittingly, the common birth of children with normal karyotype suggests that the few sperm which can be found in patients with Klinefelter syndrome derive from the clonal expansion of spermatogonia with normal karyotype.

When testosterone serum levels are reduced, substitution with testosterone is necessary. To avoid symptoms of androgen deficiency, hormone replacement therapy should be initiated as early as needed. In particular, Nielsen et al. (120) showed that early testosterone replacement not only relieves biological symptoms such as anemia, osteoporosis, muscular weakness and impotence, but also leads to better social adjustment and integration. However, concurrent testosterone treatment severely reduces the chances of successful TESE and, therefore, the option of TESE should be considered before starting the first testosterone substitution and otherwise treatment should be stopped before the biopsy. Testosterone replacement must be considered a lifelong therapy in Klinefelter patients to assure quality of life. Usually gynecomastia is not influenced by hormone therapy. If it disturbs the patient, a plastic surgeon experienced in cosmetic breast surgery could perform a mastectomy.

XX-Male Syndrome

The XX-Male Syndrome is characterized by the combination of male external genitalia, testicular differentiation of the gonads and a 46,XX karyotype by conventional cytogenetic analysis. This disorder shows a prevalence of 1:9,000 to 1:20,000.

Applying fluorescence in situ hybridization or molecular methods it has been demonstrated that about 80% of XX-males have Y chromosomal material translocated onto the tip of one X chromosome (121). Translocation of a DNA-segment which contains the testis-determining gene (SRY = Sex Determining Region Y) from the Y to the X chromosome takes place during paternal meiosis (122). The presence of the gene is sufficient to cause the initially indifferent gonad to develop into a testis. The breakpoints and consecutively the size and content of the translocation seem to influence the severity of the phenotype (123).

Most SRY-positive patients are very similar to patients with Klinefelter syndrome. In general, however, 46,XX males are significantly shorter than Klinefelter patients or healthy men, resembling female controls in height and weight, which is in line with the recent view that the number of sex-chromosomes (most likely copies of the SHOX-gene) largely determines final height (96). The incidence of maldescended testes is significantly higher than that in Klinefelter patients and controls (124). The testes are small (1 - 4 ml) and firm, and endocrine changes of primary testicular failure with decreased serum testosterone and elevated estrogen and gonadotropin levels are observed. About every second patient develops gynecomastia. XX-males seem to have normal intelligence, however, exact data are lacking. Ejaculate analysis reveals azoospermia. The testicular histology of postpubertal SRY-positive XX males shows atrophy and hyalinization of the seminiferous tubules devoid of germ cells.

In SRY-negative XX-males (about 20% of XX-males), mutations in SOX9, RSPO1 or other candidate genes may be responsible for the sex reversal, but these are very rare and the mechanism underlying the majority of cases currently remains unclear (125). SRY-negative XX-males are generally less virilized than SRY-positive men and may show additional malformations of the genital organs such as maldescended testes, bifid scrotum or hypospadias (126).

Today, there is no therapy for infertility of men with XX-male syndrome. Patients with reduced testosterone production have to receive appropriate testosterone replacement therapy.

XYY-Syndrome

Most 47,XYY males have no health problems distinct from those of 46,XY males. The diagnosis relies entirely on the cytogenetic demonstration of two Y chromosomes with an otherwise normal karyotype. The non-mosaic chromosomal aneuploidy is caused by non-disjunction in paternal meiosis. Usually the finding is incidental, occurring when karyotyping has been undertaken for unrelated issues. The prevalence among unselected newborns appears to be 1:1,000.

Men with 47,XYY-syndrome have serum levels of testosterone and gonadotropins, as well as testicular volumes, comparable to those of normal healthy men. Most men with 47,XYY-syndrome have normal fertility. Onset of puberty seems to be delayed by 6 months, adult height is 7 cm in excess of the male population mean. The intelligence quotient lies within the normal range, but men score an average of ten points less than age-matched peers. Behavioral problems are more common in 47,XYY males, however, a history of violent behavior is exceptional (127; 128).

Most 47,XYY-men do not need any specific therapy. Men who achieve fatherhood can expect chromosomally normal offspring probably with the same likelihood as normal men. Nevertheless, to be safe, prenatal diagnosis can be offered.

STRUCTURAL CHROMOSOME ABERRATIONS

Structural chromosome abnormalities encompass alterations of chromosome structure that are detectable through light-microscopic examination of banded metaphase preparations as well as smaller, sub-microscopic deletions and duplications that are only detectable with molecular genetics (e.g. array Comparative Genomic Hybridization, aCGH). Structural rearrangements such as Robertsonian translocations, that also imply a change in chromosome number, are also regarded as structural abnormalities.

Structural anomalies of the autosomes are distinguished from anomalies of the sex chromosomes (gonosomes). Especially reciprocal and Robertsonian translocations, inversions, marker chromosomes, X and Y isochromosomes, and Y chromosomal deletions are of practical importance for andrology. When evaluating a structural chromosomal anomaly for clinical purposes, the distinction between balanced and unbalanced structural aberrations is pivotal. The former are characterized by a deviation from normal chromosome structure but without a net loss or gain of genetic material. If no important gene is disrupted at the breakpoints, balanced structural aberrations exert no negative effect on general health but may cause spermatogenic failure (oligo- or azoospermia) and independent of that, an increase in the risk for unbalanced karyotypes in the offspring (91; 129; 130).

In unbalanced structural chromosomal abnormalities, genetic material is either missing or there is an overall net excess of material in the cell. Unbalanced chromosomal aberrations may be incompatible with life and lead to abortion or cause a broad spectrum of disease. Exceptions are deletions of the Y chromosome that may limit reproductive functions selectively, and are therefore of importance in reproductive medicine (see below).

The majority of male individuals carrying structural aberrations is probably fertile and need no specific therapy. Conversely, men with impaired spermatogenesis show an increased prevalence of structural chromosomal abnormalities (129; 91; 92). Infertile patients with structural chromosomal aberrations may conceive naturally while more severe cases may require 'symptomatic' treatment modalities such as intracytoplasmic sperm injection, however, success rates may be lower than in couples with normal karyotypes (131). It should also be considered that unbalanced karyotypes of the embryo may result from balanced parental chromosomal anomalies (132). For any carrier of a structural chromosome abnormality who considers fatherhood by any means, genetic counselling is strongly recommended, and it should be obligatory prior to any infertility treatment (133; 134). It should be mentioned that in many countries karyotyping of men with idiopathic infertility and decreased sperm concentration is recommended prior to ICSI therapy although an evidence based screening threshold does not exist (135). The risk of spontaneous pregnancy loss, congenital malformations regularly associated with developmental delay as a result of an unbalanced karyotype in the offspring, options of prenatal and preimplantation genetic diagnosis, and - for certain aberrations - the possibility that other family members are also affected should be discussed with the patient.

Structural Aberrations of the Autosomes

Balanced autosomal anomalies may interfere with the meiotic pairing of the chromosomes and thus adversely affect spermatogenesis. These abnormalities often do not display a typical clinical phenotype. The presence and extent of disturbed fertility cannot be foreseen in individual cases. The same balanced autosomal aberration can have a severe effect on spermatogenesis in one patient and none at all in another patient. Even brothers with the same pathological karyotype can have widely differing sperm densities. So far no clinical or laboratory parameter in an infertile male is known which reliably indicates the presence of an autosomal structural anomaly. Therefore, in cases of unclear azoospermia or (severe) oligozoospermia, karyotyping is generally advised (135).

Translocations and other structural chromosomal aberrations can be either a de novo occurrence in the subject or inherited. Therefore, testing in family members should be encouraged, as the presence of a chromosomal aberration is regularly associated with a higher rate of abortion and the risk for the birth of a severely handicapped child.

Structural Aberrations of Sex Chromosomes

An intact Y chromosome is essential for the male reproductive system. The male-specific region of the Y chromosome (MSY) differentiates the sexes and comprises 95% of the chromosome length (136). The SRY gene is localized on the short arm of the Y chromosome and it influences differentiation of the embryonic gonad into the testicular pathway. The long arm of the Y chromosome contains areas responsible for establishing regular spermatogenesis.

When speaking of deletions of the Y chromosome, those of the short and the long arm must be distinguished (137). Short arm deletions of the Y chromosome that encompass the sex determining SRY gene result in sex reversal. Clinically, affected subjects appear as phenotypically female individuals with somatic signs of Turner's syndrome. If the deletion affects the long arm, the phenotype will be male. Loss of the heterochromatic part of the Y chromosome's long arm (Yq12) leaves general and reproductive health unaffected. Deletions of the euchromatic part of the Y chromosome's long arm (Yq11) may affect spermatogenesis, because Yq11 harbors loci essential for spermatogenesis (136).

In addition to deletions, a series of further structural anomalies of the Y chromosome are known. Pericentric inversions generally remain without consequence. An isodicentric Y chromosome is a more complex aberration nearly always occurring as a mosaic with a 45,X-cell line. The phenotype may be male, female or ambiguous. Patients with a male phenotype are usually infertile. These patients have an increased risk of developing testicular tumors (see Endotext.com, Endocrinology of Male Reproduction, Chapter 13: Testicular cancer pathogenesis, diagnosis and endocrine aspects). Reciprocal translocations between the Y chromosome and one of the autosomes are rare. In most cases, spermatogenesis is severely disturbed. However, several men with these aberrations have been reported as fertile. Translocations between the X- and Y-chromosomes occur in several variations; often the karyotype is unbalanced. The correlation between karyotype and clinical presentation is complex. The phenotype may be male or female; fertility may be normal or disturbed.

The X chromosome contains numerous genes essential for survival. Every major deletion of this chromosome has a lethal or severe effect in the male sex. Translocations between the X chromosome and an autosome usually result in disturbed spermatogenesis, whereas inversions of the X chromosome do not substantially affect male fertility.

Y CHROMOSOME MICRODELETIONS

The human Y chromosome is not only the dominant sex determinator, but plays an essential role in the genetic regulation of spermatogenesis (138). The long arm of the Y chromosome contains three partially overlapping but discrete regions that are essential for normal spermatogenesis (136; 139). The loss of one of these regions, designated as AZF (azoospermia factor)a, AZFb (P5/proximal P1), AZFc (b2/b4), and AZFbc (with two variants differing in the proximal breakpoint: P5/distal P1 and P4/distal P1) can lead to infertility (136). The deleted regions are usually of submicroscopic dimensions and are known as Y chromosomal microdeletions. Their prevalence in azoospermic men lies between 5 - 10% and between 2 - 5% in cases of severe oligozoospermia (140). Clearly, the frequency of Y microdeletions is related to the criteria by which men have been selected (141; 142), whereas ethnic differences might exist as well (143). Deletions of the AZFc region represent about 80% of all AZF deletions (143). The type and mechanism of deletions have been recently clarified and result from homologous recombination between retroviral or palindromic sequences (144). The AZFc region includes 12 genes and transcription units, each present in a variable number of copies making a total of 32 copies (145). The classical complete deletion of AZFc (b2/b4 deletion), removes 3.5 Mb, corresponding to 21 copies of genes and transcription units (146). Even more gene copies are removed by more extensive deletions (7.7 Mb and 42 copies removed in P5/distal P1 deletions; 7.0 Mb and 38 copies removed P4/distal P1 deletions) (147). It remains unclear if any of the genes of the respective regions are indeed pathologically relevant.

Clinically the patients present with severely disturbed spermatogenesis; endocrine testicular function may or may not be affected by the microdeletion as in other cases of spermatogenetic failure. Testicular histopathology varies from complete or focal Sertoli-cell-only syndrome (SCO) to spermatogenic arrest or hypospermatogenesis with qualitatively intact but quantitatively severely reduced spermatogenesis (148; 143). In azoospermic men, the presence of a complete deletion of AZFa seems to be associated with uniform germ cell aplasia (complete SCO), while a histological picture of SCO or spermatogenic arrest seems common in men carrying complete AZFb or AZFbc deletions. However, in exceptional cases, complete AZFb-deletions seem compatible with finding, albeit very few, spermatozoa (149; 150). Overall, the chances for successful sperm retrieval in carriers of complete AZFa as well as AZFb and AZFbc deletions has still to be considered virtually zero. On the other hand, men carrying complete AZFc deletions have severe oligozoospermia in about 50% of cases and in azoospermic carriers, successful TESE seems possible in about half of them (148; 151). No clinical parameter can help distinguishing patients with microdeletions of the Y chromosome from infertile men without microdeletion and, therefore, screening of all men with severe oligo- or azoospermia and without other causes is indicated (143; 135). It should be noted that Y chromosome microdeletions have also been described in proven fertile men (152).

A positive result of the analysis, which should be carried out according to the standard recommended by the current guidelines (153), provides a causal explanation for the patient's disturbed spermatogenesis. Beyond this, the test also has prognostic value, as TESE is possible in about 50% of men with AZFc deletion and every son of such a patient will carry the paternal Y chromosomal microdeletion and thereby inherit disturbed fertility (154). Hence, genetic counseling is indicated for all carriers of Y chromosomal microdeletions (133; 148).

Smaller deletions removing only part of the AZFc region have been identified as a polymorphism significantly associated with infertility, especially oligozoospermia (145). These so-called gr/gr deletions arise by the same mechanism (homologous recombination) and have been extensively studied in large groups of men in different countries. Overall, they are found in about 6.8% of infertile men but also in 3.9% of the controls and four meta-analyses have reported significant Odds Ratios, reporting on average 2-2.5 fold increased risks of reduced sperm output/infertility (55; 155–157). Although they represent a significant risk factor for male infertility, they should be regarded as a polymorphism and for the time being this type of diagnostics offers no advantage in male infertility workup. Concerns have been raised that a gr/gr (partial AZFc) deletion may expand to a complete AZFc deletion in the next generation and gr/gr deletions have also been reported as risk factor for testicular cancer (158; 159). Currently, however, no general agreement to advise routine testing has been reached (55; 157; 160; 153).

OUTLOOK: NEW TECHNOLOGIES, NOVEL GENETIC CAUSES

For many years, single candidate genes have been evaluated - usually by genotyping single nucleotide polymorphisms or sequencing - with the goal of identifying causal mutations for spermatogenic failure. Most of these approaches were, however, not successful most probably because 1) “male infertility” as well as “spermatogenic failure” are highly genetically heterogeneous and 2) selection of patient groups is often not stringent. Conversely, with the advances in genetic technologies, namely array-Comparative Genomic Hybridization (array-CGH) and whole-exome or even -genome sequencing, it is now possible to perform unbiased genome-wide analyses. These novel methodologies easily outperform the previous candidate gene approach which is illustrated by an increasing number of recent publications of so-called Copy Number Variations (CNVs), larger DNA regions that may be duplicated or deleted, as well as single genes causing spermatogenic failure. Examples are studies presenting CNVs on the autosomes as well as sex-chromosomes that are associated with azoo- or severe oligozoospermia (34; 161–163) as well as genes that are frequently mutated in specific phenotypes like meiotic arrest (163). In the near future, these novel technologies will help to greatly increase the fraction of men with a clear genetic diagnosis.

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Abnormalities of Female Pubertal Development

ABSTRACT

Puberty is the period of growth that bridges childhood to adulthood and results in physical and sexual maturity as well as the capacity for reproduction. Over half of pubertal timing is considered heritable. Significant pathology can result in both advanced and delayed puberty and can result in altered attainment of adult height, secondary sexual characteristics and reproductive capacity. The age for evaluation of precocious puberty has changed in the recent past due to greater understanding of the timing of pubertal development and important racial differences. The early detection of significant intracranial pathology underscores the importance of the workup in young girls with true precocious puberty, and the close follow up of girls in whom a brain MRI is not initially indicated. GNRH agonists have become a mainstay of therapy in girls with precocious puberty. The optimal method of delivery and age of cessation is not known, but increases in adult height and no obvious reproductive sequelae have been demonstrated. Unlike precocious puberty, the definition of delayed puberty has not changed in recent years, and large studies suggest that the most common diagnosis after evaluation is constitutional delay, however, this is more common in boys presenting with delayed puberty than girls. The most common diagnosis in girls with delayed puberty is gonadal failure. Advances in reproductive technologies have allowed women with Turner’s syndrome and MRKH to build their families. Among phenotypic women with all or part of a Y chromosome, gonadal extirpation is recommended, the timing of which varies with their genetic analysis which is the greatest predictor of risk for germ cell tumors. Molecular research and newer techniques of genetic analysis such as genome wide association studies and next generation sequencing have allowed the identification of genetic mutations that may be responsible for some of the complex diseases that cause both delayed and precocious puberty. For complete coverage of this and related areas in Endocrinology, visit the free online web-textbook, www.endotext.org.

Introduction

The pubertal process is the period of transitional growth bridging the childhood years and adulthood. The genetic blueprint housed within the genome of the individual has long before set in motion a number of critical processes. The end result is the maturation of a multitude of endocrine axes necessary for (1) secondary sexual development and, (2) the attainment of the immediate capacity for reproduction. Intrinsic to this reproductive maturation is yet another important process of puberty: (3) a secondary wave of skeletal growth and the attainment of adult stature. Abnormal puberty, whether premature or delayed, may adversely influence each of these events resulting in an untimely or altered ability for spontaneous secondary sexual development and spontaneous reproduction or abnormal growth.

In recent years numerous advances have been made in molecular medicine and the assisted reproductive technologies. The impact of these advances has had a tremendous effect on the care of patients with abnormal puberty by: changing the initial counseling provided to our patients; allowing for new treatments during the time of altered pubertal growth; and, providing reproductive options to individuals previously known to be infertile and some considered sterile. In addition, new insight about the physiology of puberty and the genetics of these disorders has accumulated. The focus of this chapter will be on our expanded knowledge of both the genotypes and phenotypes of the disorders presenting as abnormal puberty.

NORMAL PUBERTY FOR GIRLS (IT’S OCCURRING EARLIER!): A basis for the Definition of Abnormal Puberty

Onset of Normal Pubertal Landmarks

The first somatic change associated with the initiation of puberty in girls is an increase in growth velocity. It is during the initial increment in growth velocity that the first sexual sign of puberty occurs. The initial standards of puberty were published in approximately 1970 by Marshall and Tanner. These standards reported that in British girls thelarche (breast budding) developed at an average age of 11 years, followed by adrenarche, the appearance of pubic hair. After thelarche and adrenarche, growth velocity continues to increase and peak, a landmark termed the adolescent growth spurt. A peak height velocity of 9 cm/year is attained at that time. Subsequently, with near closure of the epiphyses there is a deceleration phase for growth. It is in this deceleration phase of growth that menarche occurs. It is often at least 5 years after menarche until most of menstrual cycles are ovulatory; clinicians cannot consider that puberty is normal until this reproductive mechanism is established as it represents the final step in maturation of the HPO axis.

The sequence and timing of pubertal development may vary by ethnicity. The classic description of the normal sequence of pubertal signs as published by Marshall and Tanner was taken from studies of British Caucasian girls not long after WW II.(1,2) They noted that breast development was the first sign of puberty occurring on average at 11 years of age in the British girls. In contrast, a study of African girls in the 1970s noted that for the majority of them adrenarche preceded thelarche.

Several larger studies conducted in the United States have given further insight regarding the timing of pubertal events and suggest that the age of puberty may be decreasing. (3,4) The Pediatric Research in Office Settings (PROS) data were taken from a cross-sectional study of 17,077 American girls of whom 9.5% were African-American and 90.4% were Caucasian. It should be noted that Hispanic girls were included in both African-American and Caucasian groups. Surprisingly, nearly 30% of the African-American girls had evidence of breast and/or pubic hair development at age 7 years and nearly 50% by age 8 years. For Caucasian girls, 15% had started puberty by age 8 years and nearly 40% by age 9 years. The mean ages for breast and pubic hair growth were 10.0 and 10.5 years for Caucasian girls, respectively, and 8.9 and 8.8 years for African-Americans, respectively. The average age of menarche for Caucasian girls remained unchanged at approximately 12.8 years with the African-American girls starting menstruation earlier and at a mean age of 12.16 years. (3) The PROS results may have been skewed slightly given the fact that inspection rather than palpation was utilized to determine thelarche. The NHANES studies did not collect onset of pubic hair or breast data in girls prior to the age of 12 years, centering their analyses on the timing of menarche and the attainment of completed puberty. (4)

In most studies taken from the US, an earlier time of menarche was reported when compared to the older data with ranges from 2 to nearly 5 months earlier depending on the ethnic group studied. While it is reasonable to consider that the original British normatives published by Marshall and Tanner are likely different from the heterogeneous American population at the end of the 20th century, tremendous debate about the shortcomings and interpretations of these American data has continued over the last 10 years in a number of different forums. An expert panel overall agreed that the weight of the evidence supports a secular trend toward earlier breast development and menarche but not for other female pubertal markers. (5) Some evidence exists that malnutrition in certain socioeconomic groups of US children may currently be reversing this trend. (5)

Determinants of Normal Pubertal Growth

From conception to the fusion of epiphyses during the later stages of puberty, a number of maturational processes occur for formation and modeling of the skeleton. Intrinsic to somatic growth is the initial mesenchymal cell condensation and differentiation into cartilage that serves as a template for subsequent bone formation. Osteoblast differentiation occurs on the surface of this cartilaginous template and endochondral bone formation results when such differentiation occurs on calcified cartilage at the growth plate.

Genetic, environmental (i.e., nutrition), and hormonal determinants exist which are critical for the attainment of adult stature. The long held tenets that adult height is polygenic have been supported by genome-wide association studies for height (6). It has been estimated that 50 or more loci are associated with final adult stature (6-8). If all of these genes are functional, these parental-inherited growth genes determine the final adult height attained by an individual. Minimal changes by any number of these genes may result in height variation within the predicted height distribution. One can estimate this height by a calculation of mid-parental height. For females this is determined by subtracting 13 cm from the father’s height, adding this to the mother’s height in cm and then dividing by 2.

Under pathophysiologic situations, an individual may be taller or shorter than would be dictated by parental height determinants. Sometimes these differences are genetically determined and in other situations abnormal hormonal influences alter an otherwise intact genetic predisposition, and in other cases environmental factors play a role.

Genetic Influences of Growth

Some statural genes are present on both X and Y-chromosomes with Y individuals being taller than X individuals. From tallest to shortest one can generalize the following: XYY > (taller than) XY > (taller than) XXX > (taller than) XX > (taller than) X individuals. A few genes have been implicated in these differences. One set of genes, the SHOX genes, exist on the distal X chromosome. (9-12) Mutations have resulted in short stature and deletion of this locus is associated with short stature in Turner syndrome (45,X). (9)

Hormonal Determinants of Growth (Some gene mediated)

No doubt, a normal endocrine environment critically influences bone growth. For example it is essential that intact and normal growth hormone and thyroid hormone production, among others, be present. This is demonstrated by the fact that growth hormone and thyroid hormone deficiency separately result in short stature until corrected. (13) Growth hormone excess results in such conditions as a gigantism and acromegaly.

In addition to these known growth-promoting hormones, sex steroids are essential for mediating the pubertal growth spurt and attainment of final adult stature. Premature sex hormone production in children with congenital adrenal hyperplasia causes premature epiphyseal growth and fusion: thus, tall as children and short as adults. Early onset precocious puberty similarly causes premature pubertal growth with the risk of short adult stature unless corrected. The lack of pubertal development (delayed puberty) allows for continued long bone growth since the epiphyseal centers remain open longer than normal. Usually, in these situations, growth is normal until the expected age onset of puberty and the growth spurt is not noticed; however, linear growth continues in the absence of epiphyseal closure. This results in eunuchoid body proportions: an arm span which exceeds the height by more than 6 cm and disproportionately long legs.

While it had always been accepted that estrogen mediates pubertal bone growth in females, it was not until this era of molecular medicine that it was determined that estrogen and not testosterone mediates the same function for males. Inactivating mutations in either the estrogen receptor gene or the aromatase gene (preventing conversion from androgens to estrogens) in males have resulted in lack of normal bone growth at puberty and lack of epiphyseal closure with resultant tall stature (i.e., taller than predicted). (14-17) These findings establish that estrogen is essential for initiation of pubertal growth, closure of the growth plate, and augmentation accrual of bone during puberty. The presence of both alpha and beta estrogen receptors have been identified in the growth plate and studies are underway to understand the exact mechanism of estrogen action. (18)

DEFINITION OF ABNORMAL PUBERTY

The classic definitions of abnormal puberty, whether premature or delayed, are based on timing that is considered to be 2.5 standard deviations removed from the mean. Previously, the definition of precocious development for girls was the appearance of secondary sexual development before the age of 8 years, an age felt to represent 2.5 standard deviations earlier than the mean.

Revised recommendations have been made based on the findings of the PROS Network. (19) These guidelines propose that precocious puberty be defined by the presence of breast or pubic hair development before age 6 years in African-American girls and age 7 years in Caucasian girls.

However some experts disagree with the PROS recommendations. A few girls with puberty starting between 6 and 8 years of age for African Americans and between 7 and 8 for Caucasians were initially reported with endocrine or CNS pathologic etiologies of early puberty. As a result, concerns emerged that the PROS definitions may miss significant pathology and that strict enforcement of the new guidelines will lead to missed diagnoses. (20-22) Data suggest that between 2-9% of girls in this age group will have underlying pathology and 1% will have a tumor. (23,24) Missed diagnoses have included CNS tumors, neurofibromatosis, hypothyroidism, congenital adrenal hyperplasia, and hyperinsulinism. Thus, for such children beyond the recommended age of evaluation with presenting symptoms of precocious puberty, a complete history and physical exam are warranted to ensure that a serious underlying condition is not missed. Some experts recommend a bone age evaluation and careful longitudinal follow-up for girls younger than age 8 years that do not fall into the PROS guidelines for evaluation for precocious puberty. (22)

Recommendations based on the findings of the PROS Network have not been made for revising the definition of delayed puberty in girls as they have for precocious. As such, the absence of thelarche by age 13 years for girls signifies an abnormality, and remains the definition of pubertal delay. The classic definition for delayed menarche, i.e., primary amenorrhea, has been the absence of menarche by age 15 or 16 years, which is approximately 2.5 to 3 standard deviations from the mean, respectively.

While some patients present strictly with the absence of the onset of pubertal development, others have abnormalities in the tempo and sequence of puberty that has seemingly begun on time. Menarche usually occurs within 3 years of thelarche, when most girls have tanner stage 4 breast development. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists have jointly published guidelines that recommend evaluation of delayed puberty if menarche does not occur within 3 years of thelarche. (25)

These guidelines also recommend evaluation of girls with the following characteristics:

  • No breast development by age 13 years (delayed puberty)
  • Absence of menarche by age 14 years in the presence of hirsutism or history or exam suggestive of eating disorder or excessive exercise or an outflow abnormality
  • Absence of menarche by age 15 years.

Age definitions should be seen only as general guidelines. Rather than require a young woman meet the strict definitions of menarche by age 15 or 16 years to initiate an evaluation for delayed puberty, it has been suggested that all adolescents be followed annually throughout the pubertal process. (26)

For example, if a young woman presents concerned because of no menses at age 14 years, some of the major etiologies of primary amenorrhea could be recognized at an office visit without adding any significant costs. Screening at an age prior to 15 years should, as discussed in the previous paragraph, include screening for eating disorders and consideration of an excessive androgen disorder such as polycystic ovary disease. Exclusion of outflow tract disorders such as vaginal agenesis or imperforate hymen / transverse vaginal septum would require gentle pelvic examination. The physical exam should also be directed to identify findings that are typical of some associated endocrinopathies or syndromes such as gonadal dysgenesis. It would be better to begin a partial evaluation (i.e., FSH level and use of growth velocity curve) during earlier adolescent years at the time that abnormalities are first suspected than it would to wait until these young women are significantly different from their peers. No doubt, adolescence is one of the most difficult time periods in growth and development. It is potentially very harmful for an individual’s psychosexual development to allow significant delays in secondary sexual development or onset of menses to continue without evaluation, treatment and appropriate counseling. Young women are particularly likely to be worried about delayed breast development. 

PRECOCIOUS PUBERTY

Overview

The overall incidence of sexual precocity among American children has been estimated to be between 1:5,000 to 1:10,000. (27) The female to male ratio is approximately 10:1. Early activation of pulsatile gonadotropin-releasing hormone (GnRH) secretion is the most common mechanism of precocious puberty; usually it is idiopathic but it can be from serious conditions such as hypothalamic tumors. While the classic definition of sexual precocity is the appearance of secondary sexual characteristics before the age of 8 years in girls, newer guidelines as discussed above suggest that puberty is not considered precocious unless it occurs prior to age 6 years for African-American girls or age 7 years for Caucasian girls. (19) However, many pediatric endocrinologists in the United States routinely evaluate all girls with precocious development prior to the cutoff at age 8 years (28). As discussed above, even when puberty occurs between ages 6-7 and 8 years, it is important to consider evaluation of all children. (20-22) The child may be suffering from a serious CNS disorder associated with precocious puberty. (21)

Long term implications of early puberty include an increased risk of breast cancer, metabolic diseases (e.g., type 2 diabetes, obesity), endometrial cancer and cardiovascular disease. (29,30) In addition, psychosexual maturation remains concordant with chronological age, and unfortunately early physical sexual maturation at any age places these young girls at a high risk for sexual abuse. Clinicians should routinely screen children with early development for sexual abuse. Direct questioning in age appropriate language should be used and the history should include questions about behavioral markers including new onset bedwetting, nightmares, or other behavioral issues. It is thus important not only to make a reasoned judgment as to when to initiate an evaluation, but also to institute the appropriate therapy and support to prevent these potential long-term sequelae, even in selected girls who fall outside the new recommendations. It is also prudent to remember that early maturing girls, who may not “fit” the criteria of having premature puberty, may elect to engage sooner in coitus and other risk taking behaviors such as drugs than later maturing girls.(31,32)

Precocious puberty represents the appearance of the secondary sexual characteristics from increased sex steroid production. This increase may be secondary to aberrant gonadotropin stimulation or intrinsic disease of the ovary or adrenals. Many terms have been used to describe the types of precocious puberty, and some are less used in contemporary literature.

True precocious puberty, also known as complete precocious puberty, refers to puberty that appears early and either progresses through each of the pubertal landmarks including menarche or, in the absence of treatment, would likely progress through each of these stages. In the majority of children presenting for precocious development this early evidence of puberty is not the result of true precocious puberty and will halt or even regress; treatment is unnecessary (33). Classically a GnRH challenge test that demonstrated a pubertal response of gonadotropins (i.e., LH response > FSH response) was the hallmark of this diagnosis. The usual ability to suppress pubertal development with GnRH agonists remains the hallmark of treatment.

Incomplete precocious puberty refers to the appearance of one phase of the pubertal process: thelarche, adrenarche, or menarche. Isolated precocious thelarche, isolated precocious adrenarche, and isolated menarche are the three forms of incomplete precocious puberty.

Sexual precocity has been further categorized according to whether the pubertal signs are concordant or discordant with the sex of the individual: isosexual precocity referring to early sexual development consistent with the sex of the individual (i.e., feminization of a female); heterosexual or contrasexual precocity indicating precocious pubertal development that is limited to those physical signs not characteristic for the sex of the individual when presenting as isolated findings (i.e., virilization of a female). GnRH dependent and GnRH independent precocious puberty (GIPP) refer to those causes of precocity that are or are not secondary to GnRH production. Central precocious puberty (CPP) refers to precocity of CNS origin.

A summary of the causes of sexual precocity is presented in Table I below, followed by a numeric breakdown of the frequency of occurrence of these disorders in Table II.

Table I. Classification of Female Precocious Puberty
  • I.     Complete isosexual precocity (true precocious puberty: gonadotropin dependent)

    A.    Idiopathic

    B.    CNS lesions: Hamartomas, Craniopharyngioma, etc

    C.    Primary hypothyroidism

    D.    Post treatment for CAH

    E.     Genetic

     

    II. Incomplete isosexual precocity (GnRH independent)

    A.    Isolated precocious thelarche

    B.    Isolated precocious menarche

    C.    Estrogen-secreting tumors of the ovary or adrenals in girls

    D.    Ovarian cysts

    E.     McCune-Albright syndrome

    F.     Peutz-Jeghers syndrome

    G.    Iatrogenic

     

    III. Contrasexual precocity (Isolated virilization)

    A.    Isolated precocious adrenarche

    B.    Congenital adrenal hyperplasia

    C.    Androgen-secreting ovarian or adrenal neoplasm

    D.    Iatrogenic

     
Table II. Numeric breakdown of etiologies for precocious puberty in a large series of girls (N=438) evaluated from 1988-1999 by the classic definition (pubertal onset < 8 years) (24)
I. Central Precocious Puberty                      428 (97.7%)Incompletely Evaluated                     124Completely Evaluated                        304                          Idiopathic                                                226 (74.4%)

CNS Pathology                                        56 (18.4%)

Hydrocephalus                                                                11 (19.6%)

Encephalocele                                                                 2 (3.6%)

Neurofibromatosis                                                           3 (5.4%)

Encephalitis                                                                     1 (1.7%)

Intracranial hemorrhage                                                  1 (1.7%)

Hypothalamic hamartoma                                                7 (12.5%)

Pituitary microadenoma                                                   5 (8.95%)

Optic chiasma astrocytomas                                           3 (5.4%)

Optic chiasm glioma                                                         1 (1.7%)

CNS Vascular Malformation                                            1 (1.7%)

Other miscellaneous CNS disorders/lesions                   21 (37.5%)

(100%)

Coincidental/Associated Disorders                                         22 (7.2%)

                                                                                                 (100%)

 

II. GnRH Independent (GIPP)                         10 (2.3%)

McCune Albright syndrome                3 (30%)

            Ovarian “hyperfunction”/

               follicular cyst                                    4 (40%)

            Ovarian tumors                                   3 (30%)

Juvenile granulose cell tumor            (2)

Theca-granulosa cell tumor               (1)

In this review of 438 girls examined between 1988-1998, prior to the newer PROS definitions, the incidence of central precocious puberty (CPP) was noted to be 97.7% and GnRH independent precocious puberty (GIPP) was 2.3%. (24) Neurogenic abnormalities were noted in 18.4%, and idiopathic CPP in 74% of the girls in this study. The frequency of neurogenic CPP tended to be higher in the youngest girls (i.e., those under age 4 years) and the frequency of idiopathic CPP tended to be higher in girls presenting at older ages (i.e., between ages 7-7.9). Those girls identified with idiopathic precocious puberty after age 7 may, in fact, represent the recent observations of earlier onset of normal puberty by Herman-Giddens. (3)

Central Precocious Puberty

Central precocious puberty results from early maturation of the hypothalamic- pituitary-gonadal axis. Serum gonadotropins, gonadal pulsatility and sex steroid concentrations are in the normal postpubertal range. As mentioned previously, idiopathic precocious puberty seems to be the most common cause of CPP. Neurogenic CPP seems to be found more frequently in extremely young girls with the earliest onset of puberty. CNS lesions identified include neoplasms, trauma, hydrocephalus, post infectious encephalitis, congenital brain defects, and such genetic disorders as neurofibromatosis type 1 and tuberous sclerosis, and granulomas of tuberculous origin. The most commonly identified neurogenic neoplasms found in CPP include hamartomas, astrocytomas, and pituitary microadenomas. (24) Hamartomas are congenital hypothalamic malformations that histologically contain fiber bundles, glial cells, and GnRH- secreting neurons and often act as a mini-hypothalamus. Less frequently identified tumors include epipendymomas, gliomas, and pinealomas. While the craniopharyngioma has usually been associated with delayed puberty, it can rarely cause precocity as well.

Known genetic causes of CPP are rare and are currently limited to the KISS1 and the MKRN3 genes. The former gene produces a peptide that many currently believe to be the primary stimulatory signal of puberty and the later gene seems to be related to an inhibitor of GnRH secretion that exists prior to puberty, Activating mutations have been found in the genes encoding kisspeptin 1 (KISS1) and its receptor (KISS1Rr1.(34-37) In addition, as in normal puberty, higher levels of kisspeptin 1 have been identified in children with CPP compared to controls.(38) Recent research on 15 families with central precocious puberty utilizing whole exome sequencing identified loss of function mutations in MKRN3 genes which encode the makorin RING-finger protein 3 in 5 of the 15 families. The gene is maternally imprinted and likely plays a vital role in developing cells, particularly in the central nervous system. Interestingly, a larger deletion of 15q11-q13 which contributes to Prader-Willi syndrome encompasses the MKRN3 gene. The protein, makorin RING finger protein 3, is involved with RNA binding and ubiquination and degradation. Further research in 215 unrelated children with sporadic CPP identified 8 children with mutations in MKRN3, all on the paternal allele. (39) While these mutations are rarely a cause of CPP, this research does suggest an inhibitory role of MKRN3 in GnRH secretion. (40)

Other chromosomal abnormalities associated with CPP have also been described, such as 9p deletion, Williams-Beuren syndrome (1q11.23 microdeletion), and 1p36 deletion. Also, maternal uniparental disomy of chromosome 14 (Temple syndrome) and 7 (Silver-Russell syndrome) have been identified. The latter two genomic imprinting disorders, taken into consideration with MKRN3 mutations and Prader Willi, suggest the importance of epigenetic alterations in the pathogenesis of precocious puberty. (41)

Girls with severe primary hypothyroidism can develop true precocious puberty. These girls have elevated gonadotropins in addition to high TSH levels. The associated precocity may result from cross-activation of the FSH receptor by the high circulating TSH or from direct stimulation of the ovary by the gonadotropins. Large ovarian cysts are not uncommon in patients with primary hypothyroidism and precocious puberty. These girls will have the atypical finding for precocious puberty of delayed bone maturation.

Occasionally, treatment and correction of long standing virilizing congenital adrenal hyperplasia will be followed by the development of true precocious puberty. It has been hypothesized that GnRH secretion and gonadotropin stimulation of the ovary may ensue in these patients after the removal of hypothalamic androgenic suppression.

Contemporary Issues for Management of CPP

The evaluation of true precocious puberty requires confirmation of true puberty, a careful physical examination with attention to growth charts, and evaluation for a central lesion. If a CNS lesion is present, the child will typically have a pubertal gonadotropin response to GnRH that is usually associated with idiopathic true precocious puberty and occasionally with a hamartoma. The mainstay of CNS evaluation is imaging of the CNS.

In addition, bone age X-rays are helpful to identify the advanced physiologic age associated with true precocious puberty. Precocious development that continues to progress is almost always associated with a marked increase in growth velocity and sometimes this rapid growth occurs prior to the presentation of precocious development (42).

The long standing gold standard in the diagnosis of central precocious puberty has been the GnRH stimulation test. Peak levels of LH greater than 3 - 5 mIU/ml 30 – 40 minutes following stimulation are highly suggestive of central precocious puberty. (43) After GnRH was no longer available, in the United States, a GnRH-agonist was substituted as the stimulus. Either test remains today the gold standard for diagnosis. The measurement of a single LH value 30 - 60 minutes after administration of a GnRH agonist (leuprolide acetate at 20 mcg/hg) was considered adequate for diagnosing CPP; an LH value greater than 9.2 mIU/ml at 30 minutes was diagnostic in one study. (43,44). Today, however, with the use of ultrasensitive LH assays, it has become standard to use basal LH serum levels as the routine for diagnosis, saving the gold standard stimulation test for those patients with inconclusive unstimulated basal results. (39) Generally speaking, LH values are unmeasurable before pulsatile GnRH is secreted in the prepubertal period. Random LH levels greater than or equal to 0.3 mIU/ml were 100% specific in one study for distinguishing CPP. (45) An unstimulated LH value of 1.1 IU/L or greater has been considered sufficient to assume that endogenous GnRH is being secreted and diagnostic for CPP in another study. (46,47) One should remember that exclusion of central precocious puberty does not rule out gonadotropin independent puberty.

Ovarian imaging and thyroid testing may also complement the evaluation. Estradiol levels are not really helpful in the diagnosis of precocious puberty with one exception. Levels vary tremendously and estradiol levels may be in age appropriate normal ranges in girls with central precocious puberty. If, however, levels are markedly elevated (above 100 pg/ml) then it is likely that the patient either has an ovarian cyst or an ovarian steroid producing tumor such as a granulosa cell tumor.

While some CNS lesions will need treatment (often surgery), the majority of remaining causes of true precocious puberty (i.e., idiopathic) respond to GnRH analogues. It has also been demonstrated that precocity associated with hamartomas, which may intrinsically produce GnRH, may be effectively treated with GnRH agonists. (48) Analogues work by desensitizing the pituitary and decreasing the release of luteinizing hormone and follicle stimulating hormone. (49)

GnRH agonist therapy initially increases circulating gonadotropin and estradiol concentrations for short periods of time. Chronic therapy is associated with suppression of pulsatile gonadotropin secretion and a blockade to the LH response of endogenous GnRH. Suppression is best monitored with GnRH challenge tests although basal LH values may be substituted when there is no doubt about suppression. Some children who are initially suppressed will escape suppression and require increased dosages. Additionally, measurement of serum estradiol (if elevated on prior analysis), height, bone age, and assessment of secondary sexual characteristics may be helpful. Evaluation of ovarian morphology and uterine size by pelvic ultrasonography may, in some cases, provide additional evidence of such suppression.

Cessation of menses, regression in physical pubertal signs (i.e., breast size and pubic hair), and a diminution of uterine and ovarian size usually occur within the first 6 months of therapy. (50) Optimal time for discontinuation of treatment has not been established, however, discontinuation at age 11 appears to result in optimal height outcomes.(51) Pubertal changes reappear within months after cessation of therapy with a mean time to menarche of 16 months.(52)

Analogues can be given in Depot formulations (IM or SC injections q4-12 weeks), as an implant (q4week to 12 month) or as a nasal spray (1-3 times daily). Leuprolide intramuscular injection is the only available depot preparation in the United States, and no studies have documented greater adherence to the multi-monthly dose compared to monthly dose. Injection site reactions occur in 10-15% of patients.(53) Histrelin, the once yearly subcutaneous implant, can suppress gonadotropin secretion for up to 2 years. (54) A minor surgical procedure is necessary for implantation and some site reactions have been reported, even a risk of infection. A recently published open label phase 3 multicenter histrelin study documented the efficacy in sustained gonadotropin suppression with yearly histrelin implants for up to 6 years of use. 52.8% of participants experienced site reactions, all of which were mild to moderate in sequelae. Additional difficulties with implant breakage (22%) at removal were noted. Gonadotropin levels returned to puberty levels within 6 months of implant removal.(50)

The literature does not include randomized controlled trials of long term outcomes for children with central precocious puberty treated by GnRH analogues. Predicted height has been shown to often improve after long-term GnRH agonist therapy; the absence of treatment has been associated with reductions of these height predictions (51,55). In one large study mean gains ranged from 3-10 cm in girls treated up to age 11 years after treatment with GnRH therapy (56). In comparison, one small study of children followed for 12 years with slowly progressive precocious puberty did not demonstrate a loss of adult height without treatment. However, these studies often have flaws such as the calculations of gained height based on unreliable predicted heights.

A consensus document of 30 experts from Europe, the US, and Canada concluded that: “The efficacy of GnRH analogs in increasing adult height is undisputed in early-onset (i.e., girls under age 6 years) precocious puberty” (57). Those children who do not benefit may have the following characteristics: slowly progressive puberty, the precocity of which does not adversely affect the child; a normal predicted height prognosis; and a lack of evidence for gonadal activation (58). While consideration should be given to withholding treatment for these children, studies consistently demonstrate that girls presenting under age 6 years are able to subsequently achieve normal adult height because of the GnRH agonist therapy (59,60). Two of the most difficult decisions in the treatment of central precocious puberty are whether to initiate treatment in girls between ages 6-8 years and to decide what age to stop treatment (61).

Since GnRH agonists decrease the aberrantly increased GH secretion seen in precocious puberty, some have suggested that these analogues may significantly suppress growth velocity enough to compromise the predicted improvement in height which could explain the ambiguity in studies regarding analogue impact on adult height. Some studies have evaluated the benefit of GnRH agonists with growth hormone (GH) and a recent meta-analysis suggested greater final height and predicted adult height with combination therapy, but no difference in final height standard deviation scores. (62) A prospective cohort study evaluated GnRH agonist alone (n= 17) vs GnRH agonist and GH (n=23) and followed subjects until final adult height was achieved. Final adult height was significantly greater than target adult height in the combination treatment group (4.86 +/-0.9cm vs 1.51 +/- 1.0cm, p<0.05) suggesting benefit to the addition of growth hormone to GnRH analogues in CPP. (63)

The psychological effects of central precocious puberty have not been adequately studied (57). Therefore, decisions regarding whether and when to initiate treatment or stop treatment based on psychosexual concerns rely on clinical expertise and expert opinion.

Incomplete, Isosexual, or Gonadotropic Independent Precocious Puberty (GIPP)

GIPP can originate from the gonads, the adrenals, from extragonadal or intragonadal sources of human chorionic gonadotropin, or from exogenous sources. In girls, functionally autonomous ovarian cysts are the most common cause of GIPP. An ovarian follicle up to 8 mm in diameter are common in normal prepubertal girls and may appear or regress spontaneously, but rarely secretes significant amounts of estrogen (64,65). An intriguing finding of the somatic cell mutation associated with McCune-Albright syndrome in the cells of one such cyst sheds light on this occurrence (66). GnRH agonists are not effective in treating autonomous cysts.

Juvenile granulosa cell tumors or theca cell tumors of the ovary are a rare cause of GIPP. Tumor markers for granulosa cell tumors include Inhibin B and müllerian inhibiting substance. Other ovarian neoplasms even more rarely seen in this age group that may also secrete either estrogens and/or androgens include gonadoblastomas, lipoid tumors, cystadenomas, and ovarian carcinomas (67). Peutz-Jeghers syndrome has been associated with GIPP; the mucocutaneous pigmentation and gastrointestinal polyposis seen in this disorder has been rarely associated with gonadal sex-cord tumors (68).

McCune-Albright syndrome (MAS) classically includes the triad of hyperpigmented café-au-lait spots, progressive polyostc fibrous dysplasia of the bones and GnRH-independent sexual precocity (69). Some girls will present with vaginal bleeding preceding thelarche. Bone lesions and café-au-lait spots may increase over time. The actual clinical phenotypes vary markedly.

This disorder is caused by postzygotic somatic cell mutations of the gene encoding the alpha-subunit of the stimulatory guanine nucleotide binding protein Gs. These activating mutations stimulate constitutive G protein activation in affected cells with aberrant cyclic AMP production (70). The mutations may occur at various times in fetal development with a patchy tissue distribution of affected cells. Each of the associated findings is affected by these mutations: granulosa cells in the ovary, melanocytes of the skin (71), and the dysplastic bone cells (72,73). In addition to the classic triad, other endocrine cells may also be similarly affected and associated with their autonomous hyperfunction: pituitary adenomas, usually growth hormone secreting, hyperthyroid goiters (74), and rarely adrenal hyperplasia (75). Another recent finding is the presence of these same somatic cell mutations in cells from isolated hyperfunctioning ovarian cysts of GIPP patients who do not exhibit other findings of McCune-Albright Syndrome (66). This may account for the findings of “ovarian hyperfunction” in patients with GIPP as reported in the series of Table II above (24).

The sexual precocity of McCune-Albright syndrome is due to autonomously functioning follicular cysts. These patients can progress from GnRH independent to GnRH dependent puberty; when their bone age reaches the physiologic age of the normal time-onset for puberty, awakening of the arcuate nucleus for pulsatile GnRH secretion may occur and progress to the establishment of ovulatory cycles.

Approaches to treatment have included aromatase inhibitors such as Testolactone and selective estrogen-receptor modulators. Studies evaluating the efficacy have been uncontrolled. One study with Testolactone showed only early effectiveness, with loss of efficacy over time (76). Another study showed success with Tamoxifen in reducing vaginal bleeding (77). However the effect of Tamoxifen on height has not been adequately evaluated. One international multicenter trial evaluated the efficacy of monthly fulvesterant in 30 girls with MAS and followed them for a year. Days of vaginal bleeding, and bone advancement were less in the treatment patients. However, there were no changes in predicted adult height or frequency of ovarian cysts.(78) An open label study evaluating the effectiveness of letrazole on 9 girls with MAS for 12-36 months demonstrated reduction in rates of growth, vaginal bleeding and bone age. Ovarian volume, estradiol, and bone metabolism indices which showed initial improvement, began to rise after 24-36months of treatment. (79) When the shift from gonadotropin independent to gonadotropin dependent puberty takes place, GnRH analog therapy then becomes the first line therapy.

Iatrogenic sexual precocity

In prepubertal children, exogenous intake of estrogen has been shown to cause precocious pubertal development. Estrogen containing products may include variety of health or nutritional supplements and personal products such as hair products, lotions, and creams. Ingestion of estrogen containing meat has also been implicated although controversial. In actuality, these causes of precocious development appear to be extremely rare.

Premature Thelarche

Isolated precocious thelarche is a common entity and is associated with unilateral or bilateral breast enlargement without other signs of sexual maturation. It generally occurs at early ages up to 4 years, with approximately 80% presenting prior to age 2 years. The thelarche regresses spontaneously after diagnosis in over half of girls (80).

In all girls gonadotropin levels rise in the newborns after delivery and remain elevated for up to 4 years of age. While most newborns rarely exhibit a dramatic ovarian response to these elevated levels, it is likely that isolated precocious thelarche is a result of this physiologic process. The uterus remains prepubertal in size during this time, however, the ovaries may develop temporary follicular activity, and estradiol levels will be slightly higher than is seen in control girls. This is usually a benign self-limiting disorder not associated with bone age progression. However, clinical consideration should be given that the breast development could be the first sign of precocious puberty. A careful history and physical assessing for neurological symptoms and signs and assessment for growth by growth charts and a bone age should usually be performed.

Premature Menarche

Premature menarche has been reported as periodic vaginal bleeding without other signs of secondary sexual development (81). While this entity has been repeatedly yet rarely reported, pediatric vaginal bleeding can occur as the first manifestation of sexual precocity in most causes of GIPP listed above. These etiologies should be excluded before one considers premature menarche as the diagnosis. The differential diagnosis of vaginal bleeding in a child without other signs of sexual maturation is quite different than precocious development and includes foreign objects in the vagina (common) and vaginal tumors (rare).

Contrasexual precocity

Virilizing precocious puberty in girls and isolated precocious adrenarche

Most girls with contrasexual precocious puberty present with early appearance of pubic hair or hirsutism. The most common cause is a mild form of 21-hydroxylase deficiency, which is present in 0.1-1.0% of the population. Other more rare forms of congenital adrenal hyperplasia have also been identified in these patients. Virilizing adrenal (occasionally malignant) and ovarian tumors (e.g., Leydig or Sertoli cell tumors) in young girls can similarly present with virilizing precocious puberty. In actuality, most girls with appearance of pubic hair likely have isolated precocious adrenarche. While many of them have only early yet normal pubertal development (3), evidence exists that the prevalence of ovarian hyperandrogenism, hyperinsulinism and dyslipidemia is increased in this population (82). These findings suggest that premature pubarche in some girls may be a childhood marker for insulin resistance and polycystic ovary syndrome.

DELAYED PUBERTY

An Overview of Delays within the H-P-O Circuit (Delays of Secondary Sexual Development and Menarche)

Several large descriptive studies have been published which have categorized the causes of pubertal/ menarchal delay. In 1981, a series of 252 female adolescents evaluated over 20 years at the Medical College of Georgia from a large referral area in Georgia was published (83). It included all patients seen with either delay of the onset of puberty or menarchal delay. The series was subsequently expanded to include 326 patients. In this series the most common causes of abnormal puberty were: (1) ovarian failure (now called ovarian insufficiency) (42%); (2) congenital absence of the uterus and vagina (14%), and (3) constitutional delay of puberty (10%). While these 3 disorders comprised two-thirds of all patients seen, a host of less frequent disorders was also diagnosed (see Table III below); the most common of these included PCOD and idiopathic hypogonadotropic hypogonadism (IHH), both at 7% each.

Table III. Etiologic breakdown of 326 patients with abnormal puberty (pubertal and menarchal delay) (Medical College of Georgia Series) (84)
Group total No. %
Hypogonadism (Pubertal Delay) Hypergonadotropic hypogonadism:
Turner Syndrome 84 26
Chromosomally Normal 57 16
46,XX 48 15
46,XY 9 2
Total 141 57 43
Hypo (eu) gonadotropic hypogonadism:
Reversible 62 18
Constitutional delay 32 10
Systemic illness 7 2
Eating disorders 9 3
Primary hypothyroidism 4 1
CAH 3 1
Cushing syndrome 1 0.5
Pseudopseudohypoparathyroidism 1 0.5
Hyperprolactinemia 5 1.5
Irreversible 37  13
Congenital Deficiency Syndromes
Isolated GnRH deficiency 23 7
Forms of hypopituitarism 6 2
Congenital CNS defects 2 0.5
Acquired anatomic lesions
Unclassified pituitary adenoma 2 0.5
Craniopharyngioma 3 1
Unclassified malignant tumor 1 0.5
Total 99 31
Eugonadism: (Menarchal Delay)
Anatomic 59 18
Mullerian aplasia 45 14
Outlet obstruction
Transverse vaginal septum 10 3
Imperforate hymen 2 0.5
Cervical atresia 1 0.5
Inappropriate feedback 22 7
Intersex disorders 5 1.5
Androgen insensitivity 4 1
17-ketoreductase deficiency 1 0.5
Total 86 26

In April of 2002, a more recent series of both male and female patients evaluated for delayed puberty at Children’s Hospital in Boston between January 1996 and July 1999 was published (85). This study, like the MCG study, included patients with delayed onset of puberty; it, however, did not include patients with menarchal delay. For the females reported (N=74), the 3 most common causes were: (1) constitutional delay of puberty (30%); (2) ovarian failure now called ovarian insufficiency (26%); and permanent hypogonadotropic hypogonadism (20%). Over 20 other numerically less frequently reported disorders were identified and listed below (see Table IV).

Table IV. Etiologic breakdown of 74 females with delayed puberty (Children’s Hospital Series, 2002) Revised from Sedlmeyer, et al. (85).
Group total No. %
Hypogonadism (Pubertal Delay) Hypergonadotropic hypogonadism:
Turner Syndrome 5 7
Chromosomally Normal 14 19
46,XX 13 17
46,XY 1 2
Total 19 14 26
Hypo (eu) gonadotropic hypogonadism:
Reversible (Functional)
Constitutional delay 22 10
Systemic illnes 1
Giardiasis 1
Rheumatoid Arthritis 1
Systemic lupus erythematosis 1
Sickle cell disease 1
Congenital heart disease 1
Isolated seizure disorder 1
Eating disorders
Endocrine disorders 2
Growth hormone deficiency 1
Hyperprolactinemia 1
Irreversible (Permanent) 15 20
Congenital/ Genetic Syndromes
Kallmann syndrome 1
Idiopathic Hypo Hypo 2
CHARGE syndrome 2
Forms of hypopituitarism
Rathke's pouch 2
Hypophysitis 1
Hypopituitarism 1
Panhypopituitarism with hearing loss 1
Acquired anatomic lesions
Craniopharyngioma 3
Germinoma 1
Ologodenrdroglioma 1
Total 51 67
Other 4 5

Numerical and physical clues to the disorders presenting with delays in pubertal development: organizing the approach to the patient.

The numerical findings in these series point out several useful facts. First, most practitioners confronted with females presenting with pubertal delay can identify a few disorders that present in the majority of patients: ovarian insufficiency, constitutional delay, and permanent hypogonadotropic hypogonadism (as frequent causes of delayed onset of puberty) and vaginal agenesis (as the most frequent cause of menarchal delay). Rather than wait until the ages defining female pubertal or menarchal delay (ages 13 and 15 or 16 years, respectively), a physical examination with inspection of the introitus, plotting the patients on growth charts (longitudinal and velocity), and obtaining gonadotropins values will identify many of these disorders even before these age definitions are met. Idiopathic hypogonadotropic hypogonadism (IHH), however, is the exception being more difficult to diagnose in the younger patients. It is often a diagnosis of exclusion in the late teenage years. Secondly, constitutional delay occurs in less than one-third of patients in any series. While constitutional delay is a frequent cause of delayed puberty it occurs with higher frequency in males, and less frequently in females. Two thirds of all females presenting with delayed puberty historically have had underlying pathology. Lastly, pubertal delay can be an ascertainment for the identification of a rare disorder (See Table II). Similarly, should any diagnosis be made during childhood years and in advance of the time for normal puberty, plans can be made prior to the pubertal years to begin treatment and to allow for the most normal pubertal progression as is possible. At least in the Children’s Hospital setting, this appears to be the case for Turner syndrome for which the frequency of presentation with delayed puberty was decreased from the earlier MCG series.

The physical findings of the patients in these series also provide clues for helping us to form a differential diagnosis and organize our diagnostic approach. First, classification according to estrogen as in the MCG series allows for a separation of major etiologies.

Table V. Classification of Pubertal Abnormalities
I. Hypoestrogenism/ Hypogonadism (Delayed Onset of Puberty)

A. Ovarian failure (Hypergonadotropic)

B. Hypothalamic-Pituitary Immaturity or Suppression (Hypogonadotropic)
II. Normal estrogen milieu/ Eugonadism (Delayed Menarche)

A. Congenital absence of uterus and vagina (CAUV)

B. Chronic Anovulation (e.g., PCOD)

C. Intersex Disorders (e.g., Androgen Insensitivity)

The absence of breast development suggests a cause of hypogonadism: ovarian failure / ovarian insufficiency or a hypothalamic-pituitary problem. The practitioner can further narrow these possible etiologies by obtaining an FSH level; high levels suggest ovarian insufficiency and low normal values direct one to etiologies that have their effect at the level of the hypothalamus or pituitary. The presence of breast development usually directs one towards causes of menarchal delay suggesting the ongoing production of estrogen. One should remember, however, that some patients may have initiated puberty only then to have this process (and estrogen production) suppressed. Historically, biological evidence for estrogen or its lack has been more helpful than a single estradiol assay. A vaginal smear which demonstrates greater than 15% superficial cells, a positive progestin challenge test, the presence of endometrium on ultrasound measuring 1.5 mm or greater, or the presence of copious cervical mucus will usually confirm the suspicion of ongoing estrogen production.(86)  There are currently no available studies for which evidence supports or refutes any one best method of determining the presence of sufficient ongoing estrogen production. Patients demonstrating breast development in the absence of evidence of ongoing estrogen production by any of these methods should be treated like any other hypogonadal patient.

Second, absence of pubic hair after age 13 years is a very significant clue of several specific abnormalities. Pubic hair growth results from both adrenal and gonadal androgen production. One should remember that even when the H-P-O circuit appears delayed, the H-P-A (adrenal) circuit should still be functioning and providing adrenal androgens. For most disorders of delayed onset of puberty, at least some pubic hair should be present because this H-P-A circuit is unaffected by the defect (ovarian insufficiency and IHH). When pubic hair is absent after 13 years, it suggests a defect of: (1) pituitary function (i.e., the inability to stimulate both ovarian and adrenal androgen production as in pituitary insufficiency); (2) steroidogenesis (i.e., the inability to convert cholesterol to androgens as in 17-hydroxylase deficiency); or (3) androgen receptors (i.e., the inability to translate the hormone signal into end organ androgenization as in Androgen Insensitivity Syndrome (AIS)). The first two of these disorders occur in the 46,XX hypogonadal patients (Tables III and IV) and demonstrate defects within both H-P-O and H-P-A circuits, the common denominator being pituitary insufficiency or a steroid enzyme block. When examined they are found to have a normal müllerian system. 46,XY patients with 17-hydroxylase deficiency will present with absence of: (1) pubic hair; (2) breast development; and (3) a müllerian system. Androgen receptor defects are found in patients with normal breast development and absence of the vagina (i.e., AIS). Thus, for the patient with absent pubic hair after age 13 years, the most critical portions of the examination include the breasts and introitus.

Third, the apparent absence of a müllerian system (i.e., vaginal agenesis) can occur for either 46,XX or 46,XY patients. However, an examination, not a karyotype, is the most cost effective initial screen. Patients may present with absence of the vagina yet also demonstrate normal pubertal breast and pubic hair development. If a rectal examination is unrevealing for them, the likely diagnosis is congenital absence of the uterus and vagina (CAUV) also known as müllerian aplasia or Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. If, instead, a bulging midline mass is identified just above the “absent vagina,” the patient likely has either a transverse vaginal septum (TVS) or imperforate hymen. None of these findings warrant chromosomal studies as they clinically suggest the presence of a 46,XX karyotype. The patient found to have breast development and absence of both pubic hair and a müllerian system likely has AIS. These latter findings alone warrant a karyotype to confirm the 46,XY compliment and the need for gondadal extirpation. As stated above, the patient with absence of the müllerian system as well as thelarche and adrenarche likely has 46,XY 17-hydroxylase deficiency.

Fourth, identification of stature significantly shorter than one would expect for an individual whose growth was interrupted only by the delayed onset of puberty often reveals a genetic cause for both short stature and delayed puberty (e.g., Turner syndrome). Alternatively these findings could be the result of an endocrine cause which stopped growth several years earlier than the usual time onset for puberty in addition to preventing or slowing the onset of secondary sexual development (i.e., growth hormone deficiency, thyroid deficiency, or pituitary insufficiency).

DISORDERS IDENTIFIED IN PATIENTS WITH EITHER DELAYED PUBERTY OR MENARCHE

The remainder of this chapter will address specific concerns of the most common causes of the pubertal abnormalities identified in the two series described above. It will primarily refer to the data of the MCG updated series of 326 patients presenting with either delayed pubertal onset or delayed menarche tabulated in Table III and classified according to Table V above (84). In addition to discussing the common findings associated with these etiologies it will point out recent findings from molecular medicine and summarize contemporary treatment strategies.

Hypogonadism

Hypergonadotropic Hypogonadism

The single most common cause of delayed puberty in all prior delayed puberty series has been primary ovarian insufficiency (83,84). Forty-three percent of all patients seen in the MCG series had hypergonadotropic hypogonadism. The fact that ovarian insufficiency presenting at puberty was numerically less frequent (i.e., 26%) in the recent Children’s Hospital series suggests that more children are being diagnosed with Turner syndrome and other forms of ovarian insufficiency before the adolescent years and that treatment may be presently initiated at an earlier age (85). In future series of delayed puberty, primary ovarian insufficiency may all but disappear as an etiology; ideally these patients being diagnosed before the usual time onset of puberty with earlier initiation of treatment.

Turner Syndrome

Numerically, more patients with ovarian insufficiency and delayed puberty have had a form of Turner syndrome than were diagnosed with either 46,XX or 46,XY gonadal dysgenesis. Approximately 30% of the Turner patients have the classic 45,X karyotype with the remainder of patients having mosaic forms of Turner syndrome (Table VI below). Mosaicism refers to the presence of two or more cell lines, both of which originated from a single cell line. Patients with mosaic forms of Turner syndrome usually have a 45,X cell line associated with another cell line such as 46,XX or 46,XY. Other cell lines exist which represent structural abnormalities of the X chromosome such as isochromosome for the long arm of X, i.e., [i(Xq)] ; they may occur either as single cell lines or as mosaicism in association with 45,X.

Table VI. Karyotypes of patients with CIOF.
Reproduced with permission (83)
Classical Turner Syndrome (45,X) 28*
Y Cell Lines 16
46,XY 1*
45,X/46,XY 12
45,X/47,XY 1
45,X/46,X?del(Y) 1
45,X/46,X,i dic(Y)/47,XY,i dic(Y)/ 46,XY/47,XYY 1
Structural abnormalities of X  31
Isochromosome
46,X,i(Xq) 7*
45.X/46,X,i(Xq) 10
45,X/46,X,i dic(Xq) 2
45,X/46,X,i (Xq)/46,i (Xq),i (Xq) 1
45,X/46,X,i (Xq)/47,X,i (Xq),i (Xq) 2
Other
46,X,t (X;X)qter-p22 1*
45,X/46,X,del X (q13) 2
46,X,Xq+ 1*
45,X/46,X,Xq+ 1
45,X/46,X,r(X) 1
45,X/46,XX/46,X,r (X)/ 47,X,r (X),R (X) 1
45,X/46,X,r 1
46,X,del X (q25) 1*
Other X mosaic cell lines 9
45, X/46, XX 8
45,X/47,XXX 1
Total 84
* Single cell lines.+ Turner phenotype with intra-abdominal streak gonad and contra-lateral intra-abdominal testis.

All of the chromosomal findings in mosaic and non-mosaic patients with Turner syndrome have a common denominator: privation of either the entire X chromosome or a portion of the X chromosome. Fetuses with Turner syndrome have as many germ cells at mid gestation as do 46,XX fetuses. It is commonly believed that the loss of critical X chromosome-linked ovarian determinant gene(s) (87-89) is the cause of accelerated loss of germ cells (90) due to a defect of follicular development as noted by Jirasek et al. Many of these individuals lose all of their follicles with associated germ cells before birth. Some of them lose the remaining germ cells during childhood years and before puberty. Less than 15% of patients with Turner syndrome will lose their follicles (with germ cells) either during or after the pubertal process (83). Five percent of patients with Turner syndrome will have enough follicles (i.e., germ cells and surrounding granulosa cells) remaining at puberty to not only initiate the pubertal process but also to allow them to have regular, cyclic menses during at least a portion of their adolescent or adult years; 2-5% may spontaneously become pregnant.(91,92)

Once the germ cells are prematurely depleted from the ovaries, the only remaining tissue present is the connective stroma of the gonads. It usually appears as a ribbon of white connective tissue located beneath the fallopian tubes and along the pelvic sidewalls (90). These residual gonads have the appearance of “streaks” and are referred to as streak gonads. The presence of a Y cell line in a patient with Turner syndrome brings with it a 15-25% risk of developing malignant germ cell tumors within those streak gonads. In those particular patients the streaks need to be surgically removed as soon as a diagnosis is made. For all patients with Turner syndrome, privation of X chromosomal material is associated with the variable Turner stigmata, cardiovascular and renal abnormalities, and the development of a number of specific medical problems. Turner stigmata include short stature, high arched palate, low hair line and webbed neck, multiple pigmented nevi, short fourth metacarpals, shield chest, increased carrying angle of the arms (cubitis valgus), and lymphadema of ankles, to name a few.

These stigmata related to loss of X-chromosomal material are variably present in Turner patients. Furthermore, reports of phenotypic-karyotypic correlations have been inconsistent (83,93). Several observations and hypotheses have been made that help understand these relationships or lack thereof. First, it has often been felt that the presence of physical findings associated with Turner syndrome is dose dependent, i.e., the higher the percentage of 45,X cells the greater the likelihood of such abnormalities. While this makes the greatest sense intuitively, not all studies have been able to demonstrate a relationship between karyotype and phenotype (83). Recently, when ascertainment was considered, better correlations were made dependent on the degree of mosaicism. Patients found incidentally by prenatal karyotyping had fewer phenotypic features of Turner syndrome than those diagnosed after birth because of a clinical suspicion (94). Another explanation suggests that X chromosome gene imprinting exists and that some of the findings of Turner syndrome are related to the parental origin of the missing X chromosome in Turner patients (95).

Short stature is the one consistent phenotypic finding of Turner syndrome (83). The MCG series was reported prior to the treatment of Turner patients with growth hormone. The fact that none of the patients in that series was taller than 63 inches (160 cm) in height supported the tenet that statural genes are located on both arms of the X chromosome. The knowledge of consistent short adult stature, often under 5 feet (152 cm), and the potential psychological effect it has in combination with other features of Turner syndrome, provided impetus for identifying therapies independent from estrogen treatment for these patients. Many hundreds of Turner patients have now been treated with growth hormone pushing the final adult stature beyond this 63-inch (160 cm) mark for some and certainly past the predicted final height for many other Turner women.

The most serious somatic abnormalities found in patients with Turner syndrome are those involving the heart and great vessels. Cardiovascular disease is the primary cause of early mortality in women with TS with standard mortality ratios of 3.5 (CVD) to 24 (congenital anomalies).(96) Most of the mortality results from cardiac malformations, which have been reported in up to 50% of patients and include coarctation, pseudocoarctation, bicuspid aortic valves (separately between 30 and 45% incidence), and a host of other anatomic variants of the vascular tree, especially in the area of the ascending aorta. The high prevalence of these abnormalities has been reported in the years following the NIH consensus panel as has the recommendations for routine MRI screening (97-99). 1.4% of Turner patients have been estimated to develop dilation of the ascending aorta with subsequent dissection and rupture; most have died after being misdiagnosed with another cause of the chest or epigastric pain (97-101). Most patients with dissection and rupture of the ascending aorta have had a cardiac congenital malformation, hypertension, or pre-existing dilation. At least 10%, however, have had neither an identifiable risk factor including aortic dilation nor an aorta diameter above the previously held risk size (i.e., > 5 cm) (101). Several explanations have been given for dissection and rupture in patients not felt to be at risk. First, this occurrence has been associated with the pathohistologic entity of cystic medical necrosis of the vessel wall, the culprit of similar clinical outcomes in patients with Marfan syndrome. This suggests that there is an inherent defect of the vessel wall that predisposes all Turner women, with or without risk factors, for this occurrence (100). Second, prior measurements have not taken into account the fact that women with Turner syndrome are smaller and thus should have smaller size aortas. When the aorta size was normalized to body surface area in a study of 166 adult Turner patients and compared to a control population (n=26), over 30% of the Turner women had an ascending aorta measurement that was larger than that of 95% of the control population (99,101). As a result, new guidelines have been suggested for those aorta measurements above which significant risk for rupture exists (99,101,102).

Pregnancy may be the largest single risk factor for dissection and rupture of the aorta in Turner patients. There are nearly a dozen reports in the literature of death occurring during, immediately after or even more remotely removed from pregnancy in Turner patients who became pregnant from oocyte donation and embryo transfer. This gathering body of literature supports the fact that the cardiovascular (i.e., increased blood volume and stroke volume) and potential hormonal changes of pregnancy (perhaps remodeling of vessel wall by estrogen or progesterone) place these patients at a high risk of dissection, rupture of the ascending aorta, and death (101,103-105). A conservative estimate of a 2% maternal mortality rate has been reported from a US national survey and is 100 fold greater than the death rate for all causes during pregnancy (103). Similarly, a French study demonstrated a maternal mortality of 2.2%.(106) While death usually occurs during pregnancy, some evidence suggests that changes of the aorta during pregnancy may increase the risk of rupture in future years as well. The report of a more recent Nordic cohort study of pregnant Turner women did not find maternal deaths but did report: 35% hypertensive disorders; 20% of patients with pre-eclampsia; and a 3.3% potentially life threatening problem, (107) Further prospective longitudinal data are needed to understand the absolute risk to these women during pregnancy. It would be ideal if IVF registries included this information.

A number of other medical conditions may also be found in Turner patients. Horseshoe kidney is the most common renal abnormality observed and a number of autoimmune disorders, commonly Hashimoto thyroiditis, are diagnosed. Given the higher incidence of specific medical conditions for women with Turner syndrome than the general population, the NIH study group guidelines recommend continued monitoring of hearing and thyroid function, screening for hypertension, diabetes, and dyslipidemia as well as aortic enlargement (98).

Normal Chromosomes

The second largest group of young women with primary ovarian insufficiency has a 46,XX karyotype (46,XX gonadal dysgenesis). For them, some have a genetic etiology. An autosomal recessive form of this disorder was previously suggested by the presence of sibships reported in which several non-twin sisters are affected with ovarian insufficiency (83). The reports of mutations in candidate autosomal genes of affected patients provides support for the belief that autosomal etiologies exist for patients with 46,XX gonadal dysgenesis and premature ovarian insufficiency (POI). However, the more consistent finding that approximately 2% of sporadic and 14% of familial cases of 46,XX ovarian insufficiency have premutations for the fragile X syndrome makes this the current most likely explanation for the presence of 2 or more sisters with ovarian insufficiency (108). In addition, a number of other known genetic disorders have also been associated with POI including myotonia dystrophica, ataxia telangectesia, galactosemia, blepharophimosis-ptosis-epicanthus inversus syndrome, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome, and proximal symphalangism. In addition, infiltrative diseases such as mucopolysaccharidoses and environmental etiologies such as childhood viral illnesses may also cause premature depletion of oocytes from the ovaries. This is suspected in identical twins reported to be discordant for ovarian insufficiency (83). While mumps can cause orchitis in males, it is suspected that viruses such as mumps may cause oophoritis and loss of oocytes as well. Patients previously treated for childhood malignancies such as Wilms tumor, may develop germ cell depletion as a result of radiation therapy or chemotherapy (e.g., alkylating agents).

Probably the most common cause of premature primary ovarian insufficiency in women with a 46,XX karyotype is autoimmune. For the group of patients for whom an abnormality is not identified, autoimmune is considered the most likely cause. These patients have an increased risk for developing other autoimmune endocrine abnormalities such as thyroiditis with thyroid dysfunction, hypoparathyroidism, and adrenal insufficiency. In addition, pernicious anemia has been reported in some of these patients. They should be screened on a routine basis for thyroid dysfunction and the other endocrinopathies, if symptomatic. Previous recommendations for patients with 46,XX POI included annual screening with a.m. cortisol levels followed by an ACTH stimulation test in those whose a.m. cortisol levels measured less than 17 – 20 mcg%. Subsequently, given the low prevalence of adrenal insufficiency in these patients, it was suggested that such screening be contemplated only when Addisonian symptoms presented. The NIH has a high referral ascertainment of POI patients with adrenal insufficiency. Studies of these patients have now shown that routine screening for the presence of adrenal steroid or 21-

hydroxylase antibodies is effective to identify patients at-risk for adrenal insufficiency and, once identified, ACTH stimulation testing can follow (109).

As one would suspect, in the absence of an identifiable genetic etiology for depletion of the oocytes, more of the 46,XX gonadal dysgenesis patients present at puberty with residual germ cells after the initial insult than do those with Turner syndrome. In the MCG series of patients, nearly 40% of them had enough follicles at puberty to mount a pubertal response before presenting with amenorrhea and ovarian insufficiency (83). A number of patients with 46,XX gonadal dysgenesis will actually go through the pubertal process and have cyclic menses before developing ovarian insufficiency and amenorrhea in their late teens or 20’s. Some of these patients who spontaneously go through puberty will also have reversal of ovarian insufficiency for indeterminate periods of time and rarely become pregnant during these times of spontaneous menstrual function. It is because of this natural history of POI that includes the reversal of the disease process in some patients that the term previously used for this condition by Fuller Albright, ovarian insufficiency, has been revived by some current authorities (109,110).

It is difficult to understand accurately the numeric breakdown of the different etiologies of ovarian insufficiency in pubertal delay patients. Reports of large series of such patients exhaustively studied to determine cause do not currently exist. There is, however, information regarding the breakdown of different etiologies in a large French cohort (N=357) of ovarian insufficiency patients spanning the ages of 11 to 39 years from which inferences may be made for the younger population (111). In that series, 7.8% of patients with POI had an identifiable genetic cause including chromosomal abnormalities (not Turner syndrome) (2%), FMR1 pre-mutations (2%), molecular alterations of genes thought to be etiologic (i.e., FSHR, GDF9, BMP15) (2%), congenital disorders of glycosylation (0.2%), and autoimmune polyglandular syndrome (APS) type 2 and multiple autoimmune disease (0.8%). In addition, 10% of the patients presented with an autoimmune disorder not identified as genetic. Ovarian insufficiency in the remainder of women was considered idiopathic.

Rare patients present with 46,XY gonadal dysgenesis. These are patients who likely have mutations in a gene controlling testicular morphogenesis such as the SRY gene, often referred to as the master switch for testicular development. While only approximately 15% have SRY mutations, there are now a number of genes both upstream and downstream in expression of SRY for which mutations may alter testicular development. As a result, the germ cells that arrive at the genital ridge will organize in the cortical, rather than medullary region of the undifferentiated gonad. For these classic patients with 46,XY gonadal dysgenesis, however, germ cell loss is complete before birth. Since they never develop testes, they will not produce müllerian inhibiting substance to ablate the developing müllerian system. They will also not produce androgens to allow for masculinization of the external genitalia.

Historically these 46,XY individuals were labeled with Swyer syndrome; at birth they have a normal female phenotype with a normal vagina, uterus and fallopian tubes., i.e., complete 46,XY gonadal dysgenesis or sex reversal (112). At puberty, they do not initiate pubertal development and are found to have elevated gonadotropin levels. They do not have other phenotypic abnormalities like the patients with Turner syndrome. They are often tall because of the presence of a Y chromosome. 46,XY individuals with gonadal dysgenesis have the highest risk for developing germ cell tumors of their streak gonads of any individuals with gonadal dysgenesis and a Y chromosome cell line. The streaks must be removed as soon after diagnosis as is reasonable. Less frequently, partial forms of this syndrome have been found to exist often in association with other systemic anatomic or medical conditions such as polyneuropathy, adrenal insufficiency, and even sudden infant death syndrome (113,114).

Molecular Findings

Turner syndrome. While Turner syndrome is considered to result from haploinsufficiency of critical loci or regions of the X chromosome and a number of putative genes have been identified, a molecular understanding of mechanisms involved is far from understood. A number of the stigmata and malformations of Turner syndrome have been thought to be caused by edema present during development because of an abnormal lymphatic vascular system and thus abnormal lymphatic drainage. As such, the abnormalities are actually deformations. For example, edema of the nail beds causes nail hypoplasia, edema of the neck causes cystic hygromas and webbed neck, and edema of the kidneys prevents them from migrating around the aortic bifurcation and results in horseshoe kidney. The presence of cystic hygromas during fetal life is also associated with coarctation of the aorta; lymphatic drainage back to the heart is sufficiently abnormal during development to cause this cardiac malformation and likely some of the other anatomic variations of the vascular tree that have been found in these patients.

One region of the X chromosome, Xp11.2-p22.1, has been thought to include “Turner syndrome loci”, as a number of associated features including ovarian insufficiency, short stature, high-arched palate, and autoimmune disease have been mapped here (115). Deletions of the X-chromosome linked SHOX gene has explained many of the dysmorphic skeletal features of Turner syndrome including the short stature (11). While not consistently reported, it has generally been thought that the number of phenotypic findings of Turner syndrome are related to the percentage of cells that are 45,X; the implication being that mosaic patients have fewer findings than do those with a single 45,X cell line. As stated above, a recent correlation between some of the findings associated with Turner syndrome suggested an imprinting effect with the variation in phenotype at least partially explained by the parent of origin of the remaining X chromosome. Renal abnormalities, for example, were exclusively found in patients retaining their maternal X chromosome (95).

Prior karyotypic/phenotype correlations have suggested that the proximal regions of both the p and q arms of the X chromosomes are most critical for maintenance of the germ cell compliment (93). However, terminal deletions at the telomeric regions of these arms are also associated with oocyte depletion, although to a lesser degree. Deletion of these regions are more likely to result in POI after some period of ovarian function rather than a complete loss of germ cells evident at the start of the teenage years as is more commonly seen with the proximal deletions.

Early molecular studies of patients with POI and translocations between the X- chromosome and autosomes identified 2 regions of the long arm of the X chromosome within the translocation breakpoints which were felt to harbor important ovarian determinant genes. POF1 (Xq26-q28) (116) contains several candidate genes (HS6ST2, TDPF3, GPC3) (116) and one known to be associated with POI, the Fragile site Mental Retardation 1 (FMR1) gene. POF2 (116,117) (Xq13.3-q22), the human homologue of the Drosophila melanogaster diaphanous gene, contains several candidate genes for which one, (DIAPH2), has been disrupted in POI (118,119). Other loci on the X chromosome have also been identified as important in maintenance of a normal oocyte compliment. Members of the Transforming Growth Factor-β (TGF-β) superfamily proteins are known to have key functions within the oocytes and granulosa cells. Of them, Bone Morphogenetic Protein 15 (BMP15 or GDF9) is produced by a gene (BMP15) mapped to Xp11.2 (120). Mutations within this gene have been associated with POI (121-123). While the list of X-chromosome candidate genes for ovarian determinants is ever growing, 2 genes known to be important in drosophila ovarian development or oogenesis are the DEAD-box 3 (DBX) and the Ubiquitin-Specific Protease 9 (USP9X) genes. Both of these genes, are located within the human Xp11.4, an area known to escape X inactivation. It would appear that a double dosage of all of these genes, especially DBX and USP9X, is required for normal ovarian function. Mutations, interruption, or loss of one of these genes results in premature loss of germ cells from the ovaries. It is possible that mutations within these loci are responsible for ovarian insufficiency in women with intact X chromosomes as they likely are in patients with Turner syndrome. All in all, there appear to be numerous gene loci on both arms of the X-chromosome responsible for ovarian development and function. It is no wonder that all of the Turner variant chromosomes, each with different portions of the X chromosome missing, result in POI.

The most studied of the X-chromosome genes associated with POI is the FMR1 gene. When mutated by a CGG triple nucleotide repeat expansion the result is fragile X syndrome. As in many triple nucleotide repeat disorders, areas of normal repeat sequence may be predisposed to expansion during or before meiosis. Function of the gene is maintained within a given number of these triple repeats but when a certain threshold is reached gene function may be adversely altered. For the fragile X gene (FMR1), a CGG repeat sequence occurs with up to 60 such repeats being normal. Expansion to over 200 such repeats leads to fragile X syndrome; the high level of repeats causing hypermethylation of the promoter and silencing of the gene. Interesting observations were made that female carriers of the premutation of this locus with an unstable intermediary level of repeats (i.e., 60-199), often had POI. Best evidence suggests that this premutation is associated with a 21 fold greater chance of developing POI and that 2% of sporadic and 14% of familial ovarian insufficiency patients harbor this unstable intermediate trinucleotide repeat. Similarly, microdeletions of the FMR2 gene are associated with the same predisposition to POI (124).

46,XX Gonadal Dysgenesis.

The list of genes involved in ovarian development and maintenance of the germ cell compliment has continued to expand as molecular analysis of patients with 46,XX gonadal dysgenesis and POI has revealed etiologic mutations. Some patients have mutations within one of the X-chromosome loci. For others, mutations have been found within autosomal genes, some that are associated with syndromic POI and others with nonsyndromic forms. Additionally, many, but not all POI or gonadal dysgenesis etiologies are associated specifically with the premature loss of germ cells. Examples of known genes for which mutations have been shown to cause syndromic forms of premature loss of germ cells include the Autoimmune Regulator (AIRE) gene causing autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or APECED (125), the Forkhead-Transcription-Factor-Like 2 (FOXL2) gene causing blepharophimosis-ptosis-epicanthus inversus syndrome (126), and the Galactose-1-Phosphate Uridylytransferase (GALT) gene causing galactosemia (127) located on chromosomes 21, 3, and 9, respectively. Myotonic dystrophy is an autosomal triple repeat disorder, like the fragile X premutation carrier state, that is similarly associated with premature loss of germ cells from the ovary. Autosomal genes for which mutations have been associated with nonsyndromic premature loss of germ cells include Inhibin A (INHA) (another member of the TGF-β family), NR5A1 (SF1), and NOBOX (128-131). Of these, mutations in SF1 have been most commonly found, first in 46,XY gonadal dysgenesis patients, and more recently in patients with primary and secondary amenorrhea with 46,XX ovarian insufficiency (130). Other autosomal candidate genes are currently under study (e.g., DAZL). It would appear that all of these mutations cause loss of germ cells.

Most previous studies have focused on single gene mutations and POI. However, it is likely that some etiologies of POI are multifactorial in nature with synergism between different genes and other epigenetic factors, particularly in complex diseases such as POI. After a recent GWAS study found associations between ADAMTS19 gene mutations and POI, further interest in the role of ADAMTS genes in ovarian development and function spurred additional genetic studies. (132) ADAMTS is expressed in the embryonic phase of gonadal development being important for angiogenesis and organ morphogenesis. SNP analysis found significant epistasis between SNPS in IGF2R and specific diplotypes for ADAMTS19 in women with POI. The authors hypothesized that since IGF2R is important in steroidogenesis and ADAMTS genes are regulated by progesterone, women with SNPs and diplotypes for these two genes are at higher risk of POI.

Our molecular understanding of hypergonadotropic hypogonadal patients has revealed a number of patients with seemingly normal ovarian development for whom germ cell depletion is not the cause of the elevation of gonadotropins. Rather, in these patients, the inability for steroid production is usually the cause of the hypergonadotropic state; and, hence the classification of ovarian insufficiency. The first such classic syndrome, Savage syndrome, was originally described as gonadotropin resistance. Initially, a number of families were identified in Finland in which 46,XX individuals with gonadotropin resistance were found to be homozygous for a single mutation of the FSH receptor gene (133-135). Reports of additional mutations have since accumulated throughout other parts of the world (136,137). Subsequently, other 46,XX hypergonadotropic patients have been identified with mutations in the LH receptor (138-142), the FSH b (143,144), and the LH b genes. Overall the result of these disorders is a lack of estrogen production and variable hypergonadotropic states. 46,XY individuals with homozygous or compound heterozygous mutations of the LH receptor gene do not masculinize in-utero and present during adolescence with a female phenotype, delayed puberty, and hypergonadotropic hypogonadism. Their gonads, however, are testes not ovaries.

The second classic hypergonadotropic state that has long been described in association with otherwise normal gonads is 17 a-hydroxylase deficiency. Both 46,XX and 46,XY individuals present with delayed puberty and a female phenotype, and ovaries and testes, respectively. Mutations have been also found in this gene (145,146). Similarly, mutations of the aromatase gene in 46,XX individuals have been identified and associated with delayed puberty and hypergonadotropism in these individuals (14,147-149). In contradistinction to the other hypoestrogenic syndromes, aromatase deficiency, however, is associated with elevations of androgens in-utero and at puberty and the predictable but variable degrees of masculinization in these otherwise phenotypic females. Finally, the fascinating report of a 46,XY patient identified with a mutation in the CBX2 gene suggests a new syndrome for which hypergonadotropism is associated with seemingly a normal ovarian architecture (150). When reported, this child was under 5 years of age. A more recent cohort series of 47 patients with disorders of sexual development did not find any pathogenic mutations in CBX2 mutations within their subjects. It is likely that mutations in CBX2 are a rare cause of gonadal disorders of sexual development.(151)

46,XY Gonadal Dysgenesis.

Our understanding of Swyer syndrome (46,XY gonadal dysgenesis/sex reversal) together with 46,XX sex reversal helped to identify the SRY gene on the Y chromosome short arm (152). Common thought has held that SRY expression is the essential signal in the process of testicular morphogenesis. Hence, SRY has been seen as the master switch for this process. However, only 15% of women with 46,XY gonadal dysgenesis have been found to harbor mutations in this gene (153,154). The fact that the remaining 46,XY gonadal dysgenesis patients have intact Y chromosomes and that most 46,XX true hermaphrodites studied have not been found to harbor SRY sequences provides evidence that other genes are present and necessary for testicular development either upstream or downstream in expression to SRY. Such conjecture has been replaced with an ever growing list of now known genes operative in this pathway of testicular morphogenesis. Mutations of the WT1 (155,156), SOX9 (155,157-160), DSS (161), SF-1 (114,162), DAX-1 (160), Desert Hedgehog (DHH) (113,163), TSPYL1 (164), and CBX2 (150) genes have all been associated with specific syndromes and 46,XY sex reversal. Of these, the most frequently reported and best characterized involves the SOX-9 gene and the accompanying syndrome of Campomelic dwarfism.

Contemporary Issues for Management

Patients identified with ovarian insufficiency will need evaluation for associated medical disorders. For Turner syndrome, the most commonly identified acquired medical condition is thyroiditis. For them, the most dangerous abnormalities involve cardiovascular malformations. While previously it has been well known that coarctation of the aorta occurs more frequently for these patients as does bicuspid aortic valves, it is now evident that these patients are also at increased risk of developing dilation of the ascending aorta (and less commonly at other vascular sites) with subsequent dissection and, if undiagnosed and untreated, rupture. Like patients with Marfan syndrome, they appear to have cystic medial necrosis as the predisposing vascular histopathology. Similar to Marfan syndrome, the increased cardiovascular demands of pregnancy also appear to increase significantly this risk. The NIH consensus panel has suggested that all Turner patients have a baseline echocardiogram and, if normal, then a cardiac MRI (98). Additional evidence suggests that the MRI measurements of the aorta should be normalized for body surface area (99). Subsequent studies should be repeated every 3-5 years and perhaps during each trimester of pregnancy if patients are willing to take a risk estimated to be at least 2% for maternal mortality and, for those who survive a potentially increased risk after exposure to pregnancy.

All Turner patients should be counseled about their increased risk of dilation, dissection and rupture of the ascending aorta that is increased with pregnancy. Since most previous deaths occurred after misdiagnosis, Turner patients should be counseled to make health care providers aware of this possible diagnosis when being evaluated for disproportionate symptoms of indigestion and upper abdominal or chest pain. During dissection, the patient may have abnormal phonation and experience unusual coldness and sensations in their legs. It is possible that most deaths could have been avoided with timely diagnosis and surgical repair. Turner syndrome patients need evaluation for horseshoe kidney and for other less frequently diagnosed autoimmune disorders such as diabetes, hypertension, dyslipidemia, and hearing impairment (98).

Treatment of patients with Turner syndrome includes not only hormone replacement for pubertal progression and health maintenance at least through age 50 years, but an even earlier consideration for growth hormone treatment. While there were some initial conflicting reports, general consensus is that the use of growth hormone for enhancing adult stature is a worthwhile endeavor (165-178). The initiation of estrogen therapy at an age concordant with normal endogenous ovarian production (i.e., at least by ages 9 to 11 years) has always been considered important for normal psychosexual development of the adolescent. However, it is also believed that such early estrogen replacement might also result in an earlier closure of epiphyses and a potential limitation of final adult stature.   The use of growth hormone therapy initiated during the childhood years may allow a more normal childhood stature (concordant with mid parental height) and the earlier initiation of estrogen therapy obviating these concerns (168,179,180). Synergistic benefits of low dose estradiol and GH treatment for these patients when begun as early as 5 years of age can add 2.1 cm to adult height, beyond the 5cm gain expected from GH therapy when combined with estradiol at 12 years of age. However, inappropriate feminization at a young age and unknown long term consequences of early estradiol supplementation limit the widespread use of adding estradiol to GH therapy in these young patients.(181) Other techniques to increase adult height include the delay of pubertal induction or the addition of oxandrolone until 15 years of age. Studies suggest that this technique can add up to 4cm of adult height but also raise concerns about effects of delayed puberty on bone health and the psychologic impact of delayed secondary sex characteristics.(174,182-184) Oxandrolone, an anabolic steroid, has significant side effects such as virilization and liver dysfunction, limiting its use.(185)

Most women with TS build their families utilizing oocyte donation due to premature oocyte depletion and ovarian insufficiency; an increasing number of them with gestational carriers due to the risk of death from aortic dissection (2%) during pregnancy. (186) A Nordic cohort of 106 women with TS who had a live birth after donor oocyte IVF reported 20% risk of preeclampsia, and potential life threatening complications in 3.3%, however no deaths occurred. The one woman with an aortic dissection had normal pre-pregnancy imaging. 9% of this cohort had a known cardiac defect before pregnancy. (107)

Due to the potential delayed depletion of oocytes in some TS women, there may exist a potential for fertility preservation in those women with regular menses. Case reports document the feasibility of oocyte cryopreservation in post pubertal girls (ages 13-15) with Turners syndrome who already had evidence of diminished ovarian reserve. A range of 4-13 oocytes have been cryopreserved. (187-189)

Given all of these considerations for natural reproduction in these women with TS, counseling is critical to provide them the most accurate information regarding risks and benefits.   Turner syndrome remains a relative contraindication to pregnancy, and if risk factors are present it becomes an absolute contraindication. Until better data are available or prophylactic treatment of the aorta is developed that provides protection for pregnancy, counseling should be provided that includes alternatives such as use of a gestational carrier or adoption.

Patients with 46,XX gonadal dysgenesis should be evaluated for premutations of the fragile X (FMRI) gene. This finding should prompt counseling for themselves and other family members and prohibit use of their similarly affected sisters as oocytes donors. In addition, 46,XX ovarian insufficiency patients should be screened regularly for the development of Hashimoto thyroiditis and at least at baseline for adrenal steroid cell or 21-hydroxylase antibodies. Continued surveillance should be considered for the presence of hypoparathyroidism, adrenal insufficiency, and other autoimmune disorders such as pernicious anemia. All gonadal dysgenesis patients with a Y cell line need extirpation of their gonads including Turner patients with 45,X/46,XY (or those with a Y chromosome fragment) gonadal dysgenesis and the 46,XY gonadal dysgenesis patients. One should remember that rare Turner patients with seeming a single 45,X cell line might have undetected mosaicism for a Y cell line. Screening 45,X single cell line patients and those individuals with an unidentified chromosomal fragment with Y-DNA centromeric probes may be prudent to uncover those additional individuals at-risk for gonadal malignancies.

All patients with premature gonadal failure need estrogen therapy for initiation and completion of pubertal progression and subsequently for the maintenance of a multitude of health processes. While the continued accrual and remodeling of bone is of utmost importance, it remains likely that numerous other physiologic processes are dependent on normal estrogen status as well, at least through 50 years of age. The findings and concerns for long term hormone replacement of the Women’s Health Initiative do not apply to these or any other patient prior to the age of 50 years and should not be used to prematurely stop their hormone replacement.

Counseling is of utmost importance for these individuals and should cover expectations for all aspects of these young women’s lives including alternatives for reproduction. While the use of donor oocytes and IVF has proven safe for 46,XX and 46,XY gonadal dysgenesis patients, an estimated maternal death rate of at least 2% exists for Turner syndrome patients and pregnancy may increase the risk for rupture in future years. While it is often easier to include pregnancy by donor oocyte as an alternative during counseling, until more information is available such discussions should be framed with the above concerns. One should also turn to patient guidelines of national organizations such as the American Society for Reproductive Medicine (ASRM) and the American College of Obstetricians and Gynecologists (ACOG) as they are developed about these issues. The use of “buddy programs” in which these patients are paired with others who have previously confronted the same issues during adolescence and support groups (e.g., Turner Syndrome Society) is an excellent complement to this counseling.

Hypogonadotropic Hypogonadism

A number of young women will present with delay of the onset of pubertal development who have no evidence of ongoing estrogen production, because something has interrupted either GnRH or gonadotropin secretion from the hypothalamus/pituitary. Patients with constitutional delay of puberty represent the most common of these disorders. Other disorders are clearly congenital or acquired.

Constitutional delay

Constitutional delay of puberty refers to a common condition for which patients will go through puberty but at a time that is more than 2.5 standard deviations delayed from the mean (Tables III and IV) (83-85). A number of these patients often have a family history of delayed puberty (85). Their physiologic age (i.e., bone age) lags behind that of their peers and is manifested by a delay in the adolescent growth spurt and temporary short stature. Most of these patients present between 13 and 16 years of age and at that time have very early signs of thelarche. Their gonadotropin levels are in the low to normal range and their workup is otherwise unrevealing.

Until recently, no specific mutations had been identified as causing constitutional delayed puberty, despite the observation that 50-80% of those with this disorder have a positive family history. A recent proband study evaluated families with delayed puberty for some of the common mutations found in idiopathic hypogonadotropic hypogonadism ( IHH). They found that subjects with constitutional delayed puberty more commonly shared the same mutation with affected family members compared to non-affected family members (53% vs 12%, p = 0.03). They even found that subjects with delayed puberty without a similar family history were more likely than controls to carry mutations commonly seen in IHH (14.3% vs 5.6%, p =0.01)(190).

In males, 60% of pubertal delay is constitutional. In females, however, no more than 30% have this benign reproductive condition. While constitutional delay represents a leading cause of female pubertal delay, prior emphasis on this statistic has led to the false diagnosis for many young women and the misguided reassurance that they were simply “late bloomers.” As many as two-thirds of females presenting with delayed puberty will have an irreversible etiology for reproductive failure, not constitutional delay (83). For this reason, any patient presenting with delayed puberty and given the label of constitutional delay should be scrutinized very carefully for other etiologies, especially if they are beyond age 16 years and have yet to initiate pubertal development.

It can be challenging to differentiate constitutional delay from IHH. Given the finding of similar mutations observed in some of both groups of patients, these disorders may, in fact, fall in the same spectrum, one being reversible and the other not. Numerous tests have been proposed to help distinguish the two; however, none have been particularly helpful. When previously performed, an intravenous GnRH challenge test usually confirmed early awakening of the hypothalamic-pituitary-ovarian circuit by demonstrating a pubertal gonadotropin response, i.e., a greater release of LH than FSH. Such a response is seen only after endogenous GnRH secretion occurs and puberty is at or beyond its very early stages. At the same time, this early gonadotropin release produces the multifollicular ovarian appearance of early puberty; the ultrasound appearance of which is likely as reassuring that puberty is marching onward as is the LH response of a GnRH challenge. The most helpful distinction between IHH and delayed puberty is the failure to enter puberty by the age of 18 years. However, many patients and their parents may not readily adopt the wait and see tactic and instead may prefer additional periodic assessments. One option is to follow with pelvic ultrasound studies looking for the appearance of the multifollicular ovary associated with the early stages of pubertal progression. It would be ideal that no adolescent would reach mid teenage years without spontaneous or exogenously-induced pubertal development! 

Acquired Abnormalities

A number of acquired medical conditions may interfere with either the production of GnRH and/or gonadotropin secretion producing a hypogonadotropic hypogonadal state (Tables III and IV) (83,85). The Children’s Hospital series refers to many of these as functional disorders (85). Endocrine disorders such as hypothyroidism, congenital adrenal hyperplasia, Cushing syndrome, and hyperprolactinemia that begin before or during the early pubertal process may interfere with gonadotropin secretion. While only some cases of growth hormone deficiency are acquired, this disorder is included here with the other endocrinopathies.   Patients with unusually short stature, pubertal delay, and low gonadotropin levels should be considered as having one of the endocrinopathies that also affects growth (i.e., hypothyroidism and growth hormone deficiency). Treatment of these disorders will allow the resumption of puberty.   Systemic illnesses including malabsorption states, eating disorders, active autoimmune diseases, and the rare hypoxemic states related to congenital heart malformations or severe anemias (i.e., sickle cell) are also occasionally etiologic for hypogonadotropism and pubertal delays. Most of these conditions are similarly reversible. Finally, pituitary tumors are consistently reported in rare patients of all descriptive delayed puberty series (83). The craniopharyngioma occurs usually between the ages of 6 and14 years prior to the usual time onset of puberty. It is an aggressive tumor that causes early destruction of the pituitary and suprasellar regions and usually delays any pubertal development. On the other hand, it can also be an indolent tumor not becoming apparent until the late teenage years or even the mid 20’s. The typical calcification of these tumors makes them easily diagnosed radiologically.   Unlike the craniopharyngioma, the prolactinoma usually does not develop until after puberty is initiated.   Estrogen is known to increase messenger RNA for prolactin and its increase at puberty is seemingly associated with the development of prolactinomas in at-risk individuals.   For these patients, the prolactinoma usually arrests a pubertal process that has begun on time. These tumors are extremely slow growing and rarely interfere with other pituitary functions, if at all. If a dopamine agonist is given to lower the prolactin levels, puberty or menstrual function will usually proceed normally. The prolactinoma now outnumbers the craniopharyngioma as a cause of hypogonadotropic hypogonadism (83).

Congenital Abnormalities

A number of disorders classically felt to be irreversible are found in patients with hypogonadotropic hypogonadism. Some of these patients present with fractional or complete pituitary insufficiency. The majority of patients have been historically categorized with idiopathic hypogonadotropic hypogonadism (IHH) and, despite the fact that specific causes have now been identified for as many as 30% of them, the label of IHH has persisted. Such patients have absence of spontaneous pubertal development that persists beyond age 18 years; hypogonadotropism is usually the isolated pituitary deficiency for them. Specifically they have functional GnRH deficiency. Numerous studies involving frequent blood sampling have demonstrated 4 different aberrant patterns of gonadotropin secretion. The majority of patients with IHH demonstrated apulsatile secretion and the remainder were divided between sleep entrained pulsitility, decreased pulse frequency, and decreased pulse amplitude (191).

Both syndromic and nonsyndromic etiologies exist. Kallmann syndrome (KS) refers to IHH with anosmia or hyposmia. The association of IHH with anosmia is not surprising given that the GnRH secretory neurons originate within the olfactory placode and then migrate to the hypothalamus extending their axons to the median eminence. Normosmic IHH (nIHH) refers to those IHH patients with a normal sense of smell. A number of genes have been identified that regulate development and migration of GnRH neurons, the production, processing and secretion of GnRH, and its expression at the receptor. (192) Mutations have been identified within these genes which result in both KS and IHH and will be discussed further in this chapter. X-linked KS and some of the patients with mutations in these other genes may have unilateral renal agenesis (KAL1 mutations in males), midline facial defects, or neurologic and skeletal abnormalities (193,194).

It has always been intriguing that variable phenotypes have existed within families harboring the same IHH mutation (193-195). Perhaps more intriguing have been the reports that 10% of males with IHH, some with mutations within genes regulating GnRH neuronal development or secretion, have reversal of their disorder and spontaneous continued reproductive function after discontinuation of treatment that may have been given for months or years (196). Recent studies of adult onset hypogonadotropic hypogonadism in males with prior reproductive function have also reported finding the same mutations shared by those men who never initiated puberty. A series of 32 male patients with hypogonadotropic hypogonadism were assessed after treatment withdrawal and 6% had recovery of gonadal function.(197) Similarly, reversible hypogonadotropic hypogonadism has been reported after years of treatment in women, one who also had anosmia (personal patient, reported only in abstract form, Goldstein, Fertil Steril 2011,96: S116, ), and an adult onset form has been identified in women with hypothalamic amenorrhea sharing similar mutations.(198) Taken together, with the prior information about similar mutations in some patients with constitutional delay of puberty, what was previously labeled as IHH appears often to be a part of a spectrum disorder with overlap between constitutional delay of puberty (spontaneous early resolution), irreversible forms in both males and females, late onset forms in individuals who first established reproductive function, and late reversible forms in patients with prior longstanding hypogonadotropic hypogonadism. All of these forms of hypogonadotropic hypogonadism have been shown to have some patients with mutations in the same genes.

A number of other genetic defects have been found to cause hypogonadotropic hypogonadism such as leptin deficiency and adrenal hypoplasia congenital (193,199-206). Besides IHH, forms of hypopituitarism also exist and result in delayed puberty with hypogonadotropism. Included are septo-optic dysplasia (SOD) (207,208), combined pituitary hormone deficiency (CPHD) (209-212), CHARGE syndrome (213,214), Prader-Willi Syndrome, and Laurence-Moon-Bidel-Bardet Syndromes. Finally, other forms of hypopituitarism exist, some of which are associated with anatomic abnormalities such as Rathke’s pouch cysts, anterior encephalocele, and hydrocephalus (83).

Molecular Findings

As in the patients with hypergonadotropic hypogonadism, molecular research has provided new insight into the clinical findings of a number of patients with hypogonadotropism. In particular, these studies have helped to better understand the variation of clinical presentation and gonadotropin levels, and the different responses to exogenous GnRH reported in these patients. For men with Kallmann syndrome, the first mutations found were those involving a cell surface adhesive gene, the KAL1 gene (215-217). The initial identification of these mutations began our understanding of the anosmia and hypogonadotropic state for KS patients; such mutations prevent normal development of the neurologic tract responsible for transport of GnRH to the median eminence and the olfactory bulb (193,218-221). Subsequently, a number of these men were also found to have unilateral renal agenesis. While similar mutations have not yet been identified in anosmic females, it is likely that a few will ultimately be uncovered. The second molecular finding involved nIHH patients and was the identification of mutations in the GnRH receptor gene (222-225). Since then, a host of mutations have been identified in hypogonadotropic patients; genes involved generally have their adverse effects in the hypothalamus, pituitary, or both.

Hypothalamic defects that are etiologic for KS and/ or nIHH involve mutations in genes responsible for GnRH production (GNRH1 gene) (226), GnRH processing (PCSK1 gene) (227-229), GnRH neuronal development that prevents subsequent normal transport through the neuronal pathways to the median eminence [FGFR1 (215,230-235), FGF8 (236), PROK2, PROKR2 (237) and CHD7 (238) genes in addition to the KAL1 gene], and GnRH secretion (GPR54 or KISS1 and receptor genes) (33,34,190,239,240) into the portal circulation.

Those genes for which mutations have been identified as a cause of IHH primarily at the level of the pituitary include the GNRHR, HESX1 (207,208), PROP1 (209,210,241), SOX2 (242), SOX3 (243), LHX3 (211,212), LHX4 (244,245), LHβ (246), and FSHβ (144) genes. Except for GNRHR or gonadotropin β gene mutations, the other mutations produce a host of phenotypic findings that often include other pituitary or endocrine deficiencies. Mutations within the leptin (201,202), leptin receptor (204,206), and NROB1 (DAX1) (247,248) genes appear to cause IHH within both the hypothalamus and pituitary. The former mutations are associated with extreme obesity (201,202). Finally, additional mutations yet to be fully understood have been found in IHH patients in the TAC3, TACR3, and nasal embryonic LHRH factor (NELF) genes (249). Numerically, the most commonly found mutations among IHH patients are those within KAL1 (men only), the FGFR1, CHD7 (CHARGE syndrome), and GNRHR genes. Interestingly, the least common and last to be identified are the mutations in the GNRH gene.

The identification of all of these mutations gives us tremendous insight into the requirements and signals for normal pubertal development It appears that the pubertal process is well orchestrated between a number of different genes and a mutation in any one of them may result in the absence of pubertal development. Given the findings of KISS1 and KISS1R mutations in a few patients with central precocious puberty, if there is a single signal for the pubertal process among all of the genes identified it is likely kisspeptin. The other genes identified in these patients with hypogonadotropic hypogonadism appear to provide the framework within which the reproductive system works. We now know that a number of genes are involved in laying down the normal neuronal transport pathway for GnRH. Some are sufficiently tightly involved with the optic bulb development (KAL1) that all patients with mutations have anosmia. Mutations in others (FGFR1) may result in either anosmic or normosmic IHH. It also appears that if a mutation exists in one of the genes that prevents normal neuronal development (e.g., FGFR1), rarely sufficient development may ultimately occur in the absence of this seemingly critical protein either with time or induced from hormone therapy such that reversal of this disorder may occur in a few patients (196). There seems to be overlap between these genes as well, given that patients may be compound heterozygotes with two mutations and each in a different gene (249). In addition, several patients have presented with a KS-like phenotype and found to have mutations in CHD7 gene, usually etiologic for the CHARGE syndrome (238).

Contemporary Issues for Management

As has been elaborated, numerous different disorders exist for patients presenting with hypogonadotropic hypogonadism. Many of these are rare and best managed by specialists who treat the specific disorder, each disorder having very specific individual clinical concerns. It should be determined early whether treatment of the disorder will allow subsequent pubertal progression or whether a form of hypogonadotropism exists for which puberty will not progress without sex steroid replacement. Early hormone therapy is critical for the management of such patients. Similarly important is the individual counseling about expectations for pubertal development, associated problems, reproductive options, and chance of recurrence or reversal. No doubt, this may require a multidisciplinary team approach. An interesting finding of the Children’s Hospital study was that it provided evidence that there may be an association between hypogonadotropic hypogonadal causes of delayed puberty and attention deficit disorder with or without hyperactivity (85). Finally, as more and more gene mutations are identified in IHH patients, an understanding of minor phenotypic findings associated with them may make earlier diagnosis possible. When seen, for example, in an extremely obese adolescent, leptin or leptin receptor mutations should be considered.

Eugonadism

The MCG series presented a third group of females with pubertal abnormalities and evidence of ongoing estrogen production. These patients primarily present with delayed menarche.

Anatomic Abnormalities

Congenital absence of the uterus and vagina (CAUV), also known as müllerian aplasia or Meyer-Rokitansky-Kuster-Hauser-syndrome (MRKH), is the second most common cause of pubertal aberrancy in the MCG series (84). In particular, these patients present with delayed menarche. They have fusion failure of the two müllerian anlagen during embryogenesis. The normal fusion process is usually followed by canalization of the vagina. In its absence, small uterine remnants and their attached normal fallopian tubes remain; the vaginal plate and uterine remnant(s) are uncannalized. Rare patients will have a variable degree of uterine fusion and/or variable foci of functional endometrium (250). These patients progress through puberty at the normal time. They present with normal pubertal development and delayed menarche and on examination are found to have isolated absence of the vagina. They have normal ovarian function. Nearly 30% of these patients have concomitant renal abnormalities, including unilateral renal agenesis, horseshoe kidneys and urethral duplication. From 12 to 50% of these patients will have associated skeletal abnormalities, scoliosis being the most common and limb defects such as lobster claw hand deformity and phocomelia rarely present (83). Other abnormalities may also occur.

Another group of patients who may present with an anatomic cause of delayed menarche are those with an imperforate hymen or rarely a transverse vaginal septum (TVS). Given the average age of menarche, most girls with an imperforate hymen will present several years before the age of 15 years and thus may not be “labeled” as presenting with primary amenorrhea. While a complete TVS causes a presentation similar to imperforate hymen, the majority of patients with a TVS will have perforations in their septum and will not present with absence of menses.

Patients with an imperforate hymen or complete TVS initiate puberty at the normal time and present with cyclic pain, on average, within 1 to 2 years after menarche. Being obstructed, they develop an hematocolpos with or without an hematometra. On examination they are found to have an obstructing membrane, the thin imperforate hymen often bulging on valsalva maneuver or a thicker TVS. The latter is usually located at the junction of the upper one-third of the vagina but can be at lower levels as well and because of its thickness usually does not bulge on valsalva. Once these obstructing membranes are surgically excised normal menstrual function usually follows. In contrast to patients with outlet obstruction, those with vaginal agenesis will usually have normal hymeneal tissue and either an absent vagina or a small pouch created by attempts at coitus. For them, there is never a midline mass on rectal exam.

Molecular Findings

Because patients with CAUV were never previously able to have children, the inheritance pattern for most of them has been generally unknown and clues for potential candidate genes have remained elusive. The majority of these patients are sporadic occurrences within their family. Rare sibships with several non-twin sisters affected have been reported and twins both concordant and discordant for CAUV also exist (83). A report of the outcome of pregnancy for these patients who were able to have their own biological children through IVF utilizing a gestational host suggests that this condition is not commonly autosomal dominant; none of the female babies were found to be similarly affected (251).

A number of genes have been proposed as candidate for harboring germ-line mutations etiologic for the syndrome of CAUV. The anti-Müllerian hormone (AMH), anti-Müllerian hormone receptor (AMHR), and other genes involved in the pathway of AMH directed müllerian regression (e.g., the β-catenin gene) have been considered likely candidates. Since a number of somatic systems are involved in this syndrome, studies have centered around developmental genes and in particular, the HOX family of genes. In addition, HOXA10 is expressed in the developing paramesonephric ducts. Mutations in HOXA13 have been associated with the hand-foot-genital syndrome and in HOXD13 have caused synpolydactyly in humans. Furthermore, the PBX1 gene protein is thought to be a HOX cofactor during müllerian and renal development. Other developmental gene candidates considered have included the PAX2, Wilms tumor transcription factor (WT1), and WNT4 genes as well as genes controlling the synthesis of retinoic acid receptors, the RAR-gamma and RXR-alpha genes. The latter 3 of these genes, when mutated in mice, have produced müllerian abnormalities. Finally, given that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations cause congenital absence of the vas in men and that the early wolffian anlagen seemingly direct müllerian development in females, this gene too has entered the list of suspects.

Our laboratory has performed mutation analyses for a number of these candidate genes in müllerian aplasia patients including CFTR (252), WNT7, AMH (253), AMHR (253), HOXA10 (254,255), HOXA13 (256), galactose-1-phosphate uridyl transferase (GALT), PAX2 (257), WT1 (258), and WNT4 (259). Studies by others have not found mutations in HOXA7, HOXA13, PBX1 (260), β-catenin (261), RXR-α, and RXR-γ genes (262). To date worldwide, excepting WNT4, none of these analyses have revealed a convincing association.

Several patients with congenital absence of the uterus and vagina have now been identified with mutations in the WNT4 gene (263-265). These patients all seem to have a variation of the classic presentation of congenital absence of the uterus known as Mayer-Rokitansky-Kuster-Hauser syndrome. In addition to müllerian aplasia, these patients have signs or biochemical evidence of androgen excess and either modified location of their ovaries (in two patients) or seemingly hypoplastic ovaries (in the third patient). Their phenotype is very similar to that of the WNT4 knockout female mouse: absence of the müllerian system associated with aberrant androgen overproduction and premature loss of follicles (266). Studies of these 3 patients have given further insight into the role of WNT4 in human reproductive development and steroidogenesis. Given the infrequency of these mutations in patients with congenital absence of the uterus and vagina, however, some have proposed that it is, in fact, a specific entity (264,265,267). A study of ovarian steroidogenesis and oocyte number in patients with müllerian agenesis undergoing IVF for transfer of embryos to a gestational carrier did not find impairment in either of these parameters (268). This further supports that WNT4 mutations are rare and a specific entity.

With the development of next generation sequencing and its ability to investigate genetically heterogeneous diseases, whole exome sequencing is utilized for diseases for which causative genes have not yet been identified. A recent whole exome sequencing and copy number variation case series in women with MRKH showed high frequency in loss of function variants of the OR4M2 and PDE1 1A genes and deletions in 15q11.2, 19 q13.31, 1pq36.21, 1q44, suggesting new candidate genes in the development of MRKH. (269)

One may question why, except in a rare phenotype that seems to be a different entity (i.e., patients with WNT4 mutations), no individuals with classic Mayer-Rokitansky-Kuster-Hauser syndrome have been found to harbor a mutation in a host of very likely candidate genes? Explanations might include: (1) the presence of mutations in yet-to-be-studied candidate genes; (2) multifactorial inheritance; or, (3) the presence of nonconventional genetic mechanisms. The latter seems to be an attractive explanation. In particular, this condition has the characteristics of disorders such as McCune-Albright Syndrome that are caused by somatic cell rather than germ-line mutations; somatic cell mutations occur at some point after fertilization in the dividing somatic cells of the embryo or in stable somatic cells later in life. They are almost never present in the germ cells. As a result the patient is usually a random occurrence within a family and neither inherits nor passes this condition on to the next generation. If this occurs during development (such as seen in McCune-Albright syndrome), the mutated somatic cells will migrate to various areas of the fetus; the phenotype always being consistent, but often with some variation dependent on the final location of the affected cells. The vast majority of patients with Mayer-Rokitansky-Kuster-Hauser syndrome are the only such affected member of a family. The consistent phenotypic findings in these patients all involve the loss of structural integrity (müllerian aplasia, renal agenesis, and bone defects) and some degree of variability exists with which specific system is involved. Patients with scoliosis, lobster claw defects and congenital amputations represent the extreme variation. Somatic cell mutations would easily explain each of these occurrences. The report of identical twins, one with isolated vaginal agenesis and the other with bilateral tibial longitudinal deficiency (congenital leg amputations) (270) makes a strong case that somatic cell mutations beginning in the initial embryo migrated to the bones in one twin and to the developing müllerian system of the other, after the process of identical twinning. Unfortunately, if, in fact, somatic cell mutations are etiologic for most cases of müllerian aplasia and involve genes that cause loss of structural integrity, the cells with the culprit mutations may no longer be present for analysis. They may have been in the original cells of the now absent uterus, vagina, kidney or bone. A recent comparative study of different tissues (blood, saliva, rudimentary uterus) in 5 pairs of discordant monozygotic twins found differences in copy number variations utilizing SNP microarray technology in the affected twin compared to non-affected twin in the following genes: MMP-14, LRP- 10, ECM, and neoangiogenesis genes. There were no differences between the mutation analyses in saliva, but similar differences in the blood and uterine tissue, mesodermal derivatives, suggesting a tissue specific mosaicism.(271)

For the transverse vaginal septum and imperforate hymen patients, molecular analysis has been essentially nonexistent.

Contemporary Issues for Management

The diagnosis of CAUV is essentially clinical. The classic finding of absence of the vagina or a vaginal pouch (usually developed through prior coital attempts) associated with otherwise Tanner stage 5 breast and pubic hair development is unlikely anything else but CAUV. A search for associated physical findings of bony malformations (commonly scoliosis) and rarely inguinal hernias or scars from prior repair should be conducted. The inguinal hernias occur because the round ligaments can pull the unconnected uterine remnants and associated fallopian tubes and ovaries into the inguinal canals. The diagnosis of CAUV can be confirmed simply by a pelvic ultrasound study that demonstrates the presence of ovaries with follicular activity. The midline uterus will not be seen. Neither a karyotype nor laparoscopy is necessary for the diagnosis in the majority of CAUV patients. The prepubertal patient could be misdiagnosed with AIS. However, post-pubertal the clinical findings for CAUV and AIS are sufficiently different that diagnosis of each is usually straightforward. If in doubt, a serum total testosterone level is the least expensive method of resolving the confusion; levels within the female and male ranges will differentiate the two conditions. One must now always consider, however, the WNT4 mutation syndrome for which patients with müllerian aplasia may manifest symptoms or biochemical evidence of androgen excess and reduced ovarian reserve.

Although not currently recommended as first-line management, treatment of this condition has previously been surgical; a number of different surgical techniques have been utilized for creation of the vagina. In the United States, the McIndoe vaginoplasty has been the most commonly performed surgery for neovaginal creation. This is the classic procedure in which a skin graft is sewn around a mold and inserted into a newly dissected vaginal space. After a skin graft takes, the patient wears a vaginal mold for an extended period of time and until regular coitus to prevent scarring down of the neovagina. In other parts of the world and some areas of the US, the Vecchietti procedure is more commonly performed. In this procedure an olive shaped instrument is placed at the perineal dimple and pulled inward under tension by attached wires, sutures, or threads stretching the perineal skin in the direction of the normal vaginal axis. The tension cords were originally placed by abdominal surgery and in more recent years have been placed by laparoscopy (272-276). Another procedure, the Davydov procedure, was developed in Russia and is gaining popularity worldwide including the US (277,278). In this procedure, laparoscopic assistance is used to bring peritoneum from the pouch of Douglas into the space created for the neovagina. A purse-string suture is placed at the top and the neovagina is created. Results of both of these alternatives have been overall very encouraging (279-281).

The majority of patients, however, can avoid surgery altogether and should be encouraged to attempt creation of a neovagina first by the Ingram dilation technique (282,283). Experts have agreed that the nonsurgical approach should be the first line approach because it is successful in approximately 90% of patients, is less morbid than surgery, and is not associated with possible contracture (284,285). A vaginal dilator is held in place at the vaginal dimple with athletic underwear. The patient then sits on a bicycle seat of a stationary bicycle or a specially designed chair for regular periods of time. The size of the mold is increased over time and until a normal sized vagina is created or coitus can be initiated. With motivated patients and careful instructions and follow-up the majority of patients will succeed. When new patients are paired up with prior successful CAUV patients for support, this method rarely fails. Patient pairing is particularly helpful for the emotional support and personal advice that only women who have weathered the various challenges of this condition can provide.

The assisted reproductive technologies have provided these women a means of having their own biological children. The use of gestational carriers with IVF after oocyte retrieval and fertilization has made this possible. Given that the CAUV patient and her husband are the biological parents of these children, legal issues involving the gestational carriers are certainly better delineated and problems arising from them much less likely than were the initial uses of surrogacy.

Recent advances in uterine transplantation have led to the first live born infant and several additional pregnancies from a transplanted uterus to patients with MRKH. The Swedish team spent years of preparation and experimentation beginning with animal models understanding basics of the surgeries involved as well as immunosuppression. They developed separate teams for removing and implanting the uteri. In some parts of the world, including Sweden, the use of gestational carriers is banned. As a result, uterine transplantation is the only hope in these countries for having a biological child for these women with MRKH. Since this therapy remains highly experimental and fraught with both medical and ethical concerns regarding potential surgical complications as well as issues from immunosuppression, it should only be performed by teams as well prepared as the Swedish team, who has completed this remarkable feat.

Counseling patients with vaginal agenesis and other disorders of sexual development (DSD) requires special skills and sensitivities and is covered briefly at the end of this chapter.

The imperforate hymen and the transverse vaginal septum are surgically treated by one of a number of procedures described in most gynecologic textbooks. These procedures are usually straightforward. Occasionally the transverse vaginal septum is difficult and requires more involved surgery including an abdominal approach, a Z-plasty or skin graft. None-the-less, only an experienced surgeon should perform all of these procedures.

CHRONIC ANOVULATION

Polycystic ovarian syndrome (PCOS) and a number of other endocrine abnormalities may result in chronic anovulation and may present as delayed menarche as reported in the MCG series (83). Although most patients with PCOS present in adolescence with menstrual irregularity, occasionally a patient will present with primary amenorrhea. If patients are androgenzied and have not menstruated they should be evaluated by at least age 14 years as covered above. These patients may not have their first menses until given a progestin challenge. While most of them have classic PCOS, other endocrinopathies and hypothalamic dysfunction need to be ruled out. The contemporary management of PCOS and its associated gynecologic and metabolic disorders includes evaluation for diabetes and hyperlipidemias and consideration for treatment of it as an insulin resistant state in addition to the classic management considerations of ovarian suppression, endometrial protection, as well as androgen targeted treatments. This topic is covered in greater detail elsewhere in this text.

DISORDERS OF SEXUAL DEVELOPMENT

Patients with androgen insensitivity present at puberty with normal onset of breast development, absent pubic hair, and delayed menarche. These 46,XY women have been found to harbor mutations in their androgen receptor genes that render their androgen receptors nonfunctional. Despite normal testes development and normal male testosterone production, they are unable to convert the testosterone signal into the end organ events of masculinization of the external genitalia in-utero or at puberty. They present with a normal female phenotype and a small blind vaginal pouch. At puberty, their androgens are converted to estrogens with normal breast development. They are usually taller than predicted by mid-parental height for females because of the presence of the Y chromosome and its associated statural genes. The presence of the Y chromosome places them at risk for developing malignancies of their gonads and dictates removal. Unlike gonadal dysgenesis patients, the risk does not increase until after puberty; additionally, these tumors are usually seminomas rather than the gonadoblastomas or germ cell tumors. Unless the testes are located within the inguinal canals, they are usually left in place until after breast development is complete.

Molecular Findings

Androgen insensitivity syndrome has been extensively studied by molecular analysis (286,287). A number of intriguing and frustrating findings have been made. First, mutations have been found in virtually every portion of the androgen receptor (AR) gene (288). Mutations in the hormone binding region of the AR gene have explained those classic patients previously determined to have nonfunctional androgen receptors. Mutations in the DNA binding domain helped explain why other AIS patients with the same classic phenotype had normally binding androgen receptors. Second, many families studied have mutations unique to their specific family (286). Until gene sequencing is routine, this precludes studying patients with a suspicious AIS phenotype for a specific AR mutation. Third, identification of mutations in this gene has widened the spectrum of incomplete AIS phenotypes to include phenotypic females with genital ambiguity, phenotypic males separately with undermasculinization (289), gynecomastia, breast cancer (290), prostatic cancer, or azoospermia/severe oligospermia (291). Fourth, individuals with the same mutations have exhibited varying phenotypes (288,292,293). Finally, clinical correlations have been made between specific mutations and the ability to masculinize further with exogenous androgens for those individuals with a male sex of rearing and not presenting as delayed female puberty (294,295).

Contemporary Issues for Management

For the classic patient with AIS who presents with delayed menarche, absent pubic hair, and a vaginal pouch, an expedient evaluation and diagnosis is necessary. Unlike the CAUV patients, once the diagnosis of AIS is suspected, chromosomal analysis is necessary to document a 46,XY karyotype. It is necessary to remove the gonads in patients with AIS (296). This can be done after puberty to allow spontaneous breast development. Support for this includes the fact that the earliest reported malignancy in patients with AIS is 14 years of age.

No doubt, one of the most critical issues related to this syndrome is counseling. No longer is it possible or advisable to hide the presence of the 46,XY finding from these patients. However, a multidisciplinary and well thought out approach and close follow-up is needed for such counseling. The psychosexual transition during adolescence is difficult and patients with intersex disorders/disorders of sexual development will face an even more difficult transition. Patients and their family require support and should be actively involved in the decision processes. Links to a variety of support groups for specific disorders can be found on the Disorders of Sexual Development website (296,297).

Many of these patients have a vaginal pouch, the embryonic remnant of the prostatic utricle. For them, coital attempts will enlarge the vagina and surgery is not needed. For others a similar, although somewhat different, approach can be utilized as was described for the patients with CAUV. Furthermore, once gonadectomy is performed, estrogen replacement therapy is essential for all of the obvious reasons.

 

 

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