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Endometriosis

ABSTRACT

Endometriosis is a disease in which endometrial glands and stroma implant and grow in areas outside the uterus. The disease has a multi-factorial etiology including genetic and environmental factors. Exposure to ovarian hormones appears to be essential. Estrogens stimulate ectopic endometrium growth and aberrations in estrogen signaling have been associated with the disease. Caucasians appear to be more likely to suffer from endometriosis than African Americans or Asians.  There is also an increased prevalence in taller women and those with a lower BMI. Genetic factors contribute to approximately 51% of endometriosis risk. Patients with an affected first-degree relative have an approximate 7 to 10-fold increased risk of developing endometriosis.

The prevalence of endometriosis ranges from 2-50% of reproductive aged women. The morbidity associated from endometriosis is great, as it can cause both chronic pelvic pain and infertility. In women with infertility, the prevalence ranges from 20-50%. Endometriosis is present in 71-87% of women with chronic pelvic pain.

A multidisciplinary approach is often required for the diagnosis and management of this disease. In women with pelvic pain and suspected endometriosis, first line treatment would be non-steroidal anti-inflammatory medications with/without oral contraceptives or progestins.

In the treatment of infertility associated with endometriosis, the age, history, health status, physical exam, and wishes concerning treatment should be weighed.  There are certain situations where surgery may be indicated for the treatment of infertility associated with endometriosis.  Controlled ovarian stimulation with IUI or IVF is often indicated.

 

DEFINITION

Endometriosis is a disease in which endometrial glands and stroma implant and grow in areas outside the uterus (Fig. 1).  The most common place to find implants is in the peritoneal cavity (involving the ovary, cul-de-sac, uterosacral, broad and round ligaments, fallopian tubes, colon, and appendix), but endometriosis lesions have occasionally been found in the pleural cavity, liver, kidney, gluteal muscles, bladder, abdominal scars, and even in men (1).

 

Figure 1.Common locations of endometriosis within the pelvis and abdomen. (Reprinted by permission from the New England Journal of Medicine 2001. Olive DL, Pritts EA. Treatment of Endometriosis. Vol. 345:267).

There are three typical types of endometriotic lesions: 1) superficial peritoneal and ovarian implants, 2) endometriomas (ovarian cysts that are lined with endometrioid mucosa), and 3) deep infiltrating endometriosis (complex nodules comprised of endometriotic tissue, adipose tissue, and fibromuscular tissue) (2,3).  The anatomical location and inflammatory response to these lesions are believed to account for the symptoms and signs associated with endometriosis.

ETIOLOGY

Classically, three theories exist to explain the etiology of endometriosis; 1) Sampson’s theory, 2) Meyer’s theory, and 3) Halban’s theory.  The most often quoted theory, and to date the one supported by the most evidence, is Sampson’s theory of transplantation and implantation.  This theory stemmed from observations made during surgeries in the 1920’s that many women shed endometrial debris through their fallopian tubes into the peritoneum during menstruation (4).  Not only has viable endometrial tissue been found in the fallopian tubes and peritoneal fluid of women, but in humans and other animals, endometrial tissue will grow if placed ectopically.  Further supporting this theory, endometriosis seems to occur most commonly in the gravitationally dependent parts of the pelvis (5).  Finally, the incidence of endometriosis is significantly increased in patients with mullerian anomalies or genital tract obstructions, both which increase the likelihood of retrograde flow (6).  One problem with this theory is that retrograde menstruation has been shown in 76-90% of menstruating women, which is much higher than the prevalence of endometriosis (7,8).  This discrepancy suggests that additional factors beyond the presence of ectopic tissue are needed to establish the disease, such as the amount of endometrial debris that reaches the peritoneal cavity, the immunocompetency of the woman to clear the debris, and the molecular abnormalities/properties inherent in the ectopic tissue.   Meyer’s theory (9) suggests that metaplasia of the coelomic epithelium is the origin of endometriosis.  This theory is logical, as cells from both the peritoneum and endometrium are derived from a common embryological precursor: the coelomic cell.  However, this has been a difficult theory to support scientifically.  If this postulate were correct, one would expect much higher rates of pleural endometriosis than are observed.   Halban’s theory is one that suggests that distant lesions are established by the hematogenous or lymphogenous spread of viable endometrial cells.  Although this metastatic theory explains rare endometriotic lesions in the brain or lung, it does not explain the gravitationally-dependent location of most foci of endometriosis.   Sampson’s transplantation and implantation theory is the most widely accepted, but most researchers agree that it grossly simplifies the disease process.  Whilst retrograde menstruation can transplant tissue fragments into the peritoneal cavity, the cells themselves must escape apoptosis (10), adhere to the underlying peritoneum (11,12), degrade the underlying extracellular matrix (13), generate a new vascular supply (14), and evade the immune surveillance system (15) in order to survive.   Clear molecular differences exist between endometriotic lesions and eutopic endometrium.  Secondary to the inflammation associated with endometriosis, there are increased levels of prostaglandins, chemokines, and cytokines, such as interleukin-1ß, intlerleukins-1, 6, 8 and tumor necrosis factor (TNF), which are thought to enhance the adhesion of endometriotic implants to the peritoneal surface.  These are associated with overproduction of prostaglandins, cytokines, and chemokines.  Proteolytic membrane metalloproteinases, also increased in endometriosis, promote implantation.  Angiogenesis and apoptosis are also altered in endometriosis in favor of survival of the implant.  Granulocytes, macrophages, and natural killer cells are attracted by the increased monocyte chemoattractant protein 1, interleukin-8, and RANTES (regulated upon activation normal T-cell expressed and secreted), present in endometriosis.  These inflammatory mediators accumulate in endometriotic tissue by autoregulatory positive feedback loops (16).   Erythrocyte breakdown from endometriotic lesions is internalized by macrophages and the non-protein-bound catalytic iron increases the production of reactive oxygen species, thereby perpetuating peritoneal damage and inflammation.  Studies have found no association between the oxidative stress and antioxidants such as Vitamins A and E (17), but the association between Vitamin D and endometriosis is more complex. Several studies suggest a  role for Vitamin D and its metabolites as local autocrine and paracrine agents involved in endometriosis etiology and pathology (18-20), but the exact mechanism has yet to be elucidated (21).   While steroidogenic factor 1 (SF1) is present in ectopic endometrial tissue, it is not found in the eutopic endometrium.  SF1 is involved in estradiol synthesis.  The increased amount of estradiol seen in the peritoneal fluid of patients with endometriosis increases local cyclo-oxygenase -2 (COX-2) activity, resulting in stimulation of prostaglandin E2 formation, which upregulates aromatase activity, resulting in additional estradiol to perpetuate the symptoms and lesions present in endometriosis (16).  There is elevated expression of both estrogen receptor α and estrogen receptor ß leading to a downregulation of progesterone receptors (2), ultimately causing the characteristic hormonal profile seen in endometriosis.  

DEMOGRAPHY

Endometriosis is diagnosed in women aged 12-80 and the average age at diagnosis is approximately 28. The disease has a multi-factorial etiology including genetic and environmental factors. Exposure to ovarian hormones appears to be essential with the vast majority of the cases found in women aged 20-50. Estrogens stimulate ectopic endometrium growth and aberrations in estrogen signaling have been associated with the disease (2, 22).  Caucasians appear to be more likely to suffer from endometriosis than African Americans or Asians (23).  There is also an increased prevalence in taller women and those with a lower BMI (24).  Genetic factors contribute to approximately 51% of endometriosis risk. Patients with an affected first-degree relative have an approximate 7 to 10-fold increased risk of developing endometriosis (25, 26).  Several at risk loci have been identified: WNT4, CDKN2B-AS1, and GREB1 (27,28).  WNT4 encodes for gene products that are important for the development of the female reproductive tract and for steroidogenesis.  The CDKN2B-AS1 gene is located in an enhancer region and includes a gene cluster that encodes tumor suppressor proteins such as p15, p16-NK4a, and p14ARF. GREB1 encodes an early-response gene in the estrogen receptor regulated pathway (27,28).  Risk factors for this disease include nulliparity, early menarche, and frequent or prolonged menses.  Protective factors include pregnancy, menopausal status, multiparity, and periods of lactation (29).  

PREVALENCE

The prevalence of endometriosis ranges from 2-50% of reproductive aged women.  Unfortunately, this number is widely disparate depending on the study.  In women with infertility, the prevalence ranges from 20-50% (30-33).  This may be due to the fact that endometriosis plays a causative role in infertility, but it also may be due to a diagnostic selection bias, as women with infertility often undergo laparoscopy as part of their clinical evaluation. Endometriosis is present in 71-87% of women with chronic pelvic pain (34-36).   Between the years 1965 and 1984, endometriosis increased from 10% to19% as the primary indication for hysterectomy in the USA.  Interestingly, this happened during a time in which a trend towards more conservative therapies as treatment modalities for endometriosis occurred (37).  This finding suggests a true increase in the incidence of the disease.  Varying theories have been proposed to explain this increase, including a delay in childbearing, declining use of oral contraceptives, and exposure to environmental toxins such as dioxin as a causative agent (38).  A recent study involving mice has demonstrated that bisphenol A (one of the highest volume chemicals produced worldwide) is able to elicit an endometriosis-like effect in mice who were prenatally exposed (39). Human studies, however, are equivocal (40-42).  Other studies have found higher phthalate concentrations in women with endometriosis than those without the disease (40, 42-48).

SYMPTOMS

Pain

The most common symptom for women who have endometriosis is pelvic pain, although the pathophysiologic mechanisms are not well understood and many women with endometriosis may not have this complaint.  The pain is most often cyclic, but may also be chronic in nature. The pain usually begins just before menses and continues throughout the duration of menstrual flow. Dysmenorrhea and deep dyspareunia are the most common pain complaints with 80% and 30% prevalence, respectively (49-51).  Dysuria, dyschezia, and intermenstural pelvic pain are less common and are associated with bladder or bowel lesions.  The pain may also be perceived in musculoskeletal regions, such as the flank, low back, or thighs.  The heterogeneity of the disease process makes it difficult to ascertain the exact etiology of the pain.   Actively bleeding lesions can certainly cause discomfort.  Pain may also be produced by the production of inflammatory mediators and neurologic stimulation.  It is likely that different types of lesions cause pain through differing pathways.  Early papular lesions contain higher levels of prostaglandins than older lesions.  These prostaglandins may activate afferent neuronal pathways.  Lesions deeply penetrating the peritoneum also have an increased propensity to cause pain, probably by direct irritation and invasion of pelvic nerves.  Endometriomas may cause a mass-effect, which can result in the perception of pain.  Adhesions and fibrosis cause pain by compromising the blood supply to certain neuronal plexi, or by placing small nerves on tension (52).  In addition to the perturbation of nerve fibers by the lesions, studies have demonstrated that neuronal fibers are present in endometriotic lesions found on the ovaries.  A nerve growth factor (a substance important for the maintenance of sensory nerves), S 100 (a neurotrophic factor), and PGP9.5 (an immunoreactive nerve fiber) have all been demonstrated in these lesions, which may contribute to the generation of pain (53).  Other studies have shown that there is an increased amount of nerve fibers present in the endometrium in patients with endometriosis (54-57). The significance of these observations remains to be determined, but it has been suggested that the presence of nerve fibers in the endometrium may be used to diagnose endometriosis.  With a chronic inflammatory environment in endometriosis, it is theorized that there may be a feedback cycle that promotes nociceptor sensitization with activation and neogenesis of sensory nerve fibers resulting in hyperalgesia (58).  Finally, endometriosis can be associated with increased pain perception secondary to abnormal modulation of nociceptive input and an increase in intensity of neuronal signaling to the brain (59-61).   Macrophages in the peritoneal fluid in women suffering from endometriosis are increased in concentration and activity (62).  As discussed previously, macrophages are part of an inflammatory cascade, which perpetuates the sensation of pain, as does the constant elevated production of prostaglandins.  

Infertility

The next most common symptom is that of infertility.  Women with moderate and severe endometriosis, particularly those in which the ovaries and oviducts are involved with adhesive disease, have decreased fertility rates.  It is theorized that this stems from the mechanical obstruction between the ovary and oviduct, with subsequent failure of gamete transport into the tubal ampulla.  Many women with moderate to severe endometriosis have undergone operative management for their disease ; as a result, they may have decreased amounts of functional ovarian tissue and thereby suffer from decreased fecundity.   Interestingly, women with only minimal or mild disease may also have decreased fertility when compared to those without clinical evidence of endometriosis.  It remains controversial whether endometriosis is the cause of this subfertility.  Some studies report that even minimal stage disease is associated with decreased fecundity, while other studies report no effect on fertility and pregnancy outcome (63, 64).  The monthly fecundity rate in normal couples is 15-20%, while the monthly fecundity rate in women with untreated endometriosis and infertility is 2-10% (65).  

In addition to the anatomic causes of infertility mentioned above, there are two other major theories that may explain the decreased monthly fecundity rates seen in women with endometriosis: 1) inflammation and 2) a locally altered hormonal profile. The altered cellular immunity results in an increased amount of inflammatory mediators, such as activated macrophages, RANTES  (regulated on activation, normal T-cell expressed and secreted), interleukins, and tumor necrosis factor in follicular fluid and in peritoneal fluid.  This results in a locally hostile, cytotoxic environment, characterized by oxidative stress, that negatively impacts most aspects of reproduction by causing altered folliculogenesis, oocyte degradation, structural DNA damage, decreased spermatozoal integrity, decreased fertilization potential, embryo fragmentation, impaired tubal function, and decreased endometrial receptivity.  The altered humoral immunity results in the presence of autoantibodies with the capability of binding to the endometrium and, possibly the oocyte, sperm, embryo, and tube.  The presence of elevated levels of steroidogenic factor-1, estrogen, and estrogen receptors in endometriotic cells ultimately leads to a down regulation of local progesterone receptors, and thus, an altered local reproductive hormonal profile (2,16, 66).   Altered folliculogenesis has been proposed as one of the contributing factors to infertility.  Characteristics of in vitro fertilization cycles in women with endometriosis include a slower follicular growth rate and reduced follicle size.  Changes in the cell-cycle kinetics of the granulosa cell that favor an increase in the amount of cells in the S phase and in cells undergoing apoptosis may play a role in this phenomenon.  There is a decreased amount of vascular endothelial growth factor present in follicular fluid, which is believed to be responsible for the decreased follicular health and vascularization seen in endometriosis.

Endometriosis has a multifactorial impact on the health and function of spermatozoa.  The increased generation of reactive oxygen species seen in women with endometriosis results in the loss of spermatozoal membrane integrity and enzyme inactivation, thereby decreasing the potential for fertilization.  Decreased sperm motility, mediated by glycoprotein-130 binding in sperm, is also a result of the negative impact endometriosis has on sperm function.  The endosalpinx in women with endometriosis tends to bind spermatozoa more tightly, resulting in a decreased number of available spermatozoa.  A decreased ability of spermatozoa to bind to the zona pellucida is evident in patients with endometriosis, resulting in impaired fertilization.  Taken together, it is clear that endometriosis has a negative impact on the spermatozoa (16).

There are a few proposed mechanisms by which endometriosis impairs implantation.  Explanations include the delayed histologic maturation or the biochemical alterations of the endometrium, both seen in endometriosis.  In another mechanism, αvβ 3 integrin, an adhesion factor which is normally increased during the optimum implantation window, is decreased or absent in patients with endometriosis (16). Evidence indicates that the eutopic endometrium of women who suffer from endometriosis is abnormal in its expression of implantation-associated proteins, e.g., complement protein (C3) (67), IL-6 (68), HOX A 10 and 11 (69), and b3 integrins (70).  The zona pellucida of the embryo is compromised by the cytotoxic effects of the reactive oxidative species, which may also lead to impaired implantation (16).

Other Symptoms and Associations

Other symptoms of endometriosis can include abnormal menstrual bleeding, diarrhea, constipation, and chronic fatigue.  There are elevated rates of autoimmune diseases, including hypothyroidism, rheumatoid arthritis, lupus erythematosus, Sjogren’s syndrome, and multiple sclerosis in patients with endometriosis.  Reports of allergies, asthma, chronic fatigue syndrome, and fibromyalgia are also more common in women with endometriosis than in women in the general US population (71).  

PHYSICAL EXAM FINDINGS

Unfortunately, due to the diffuse and often varying nature of endometriotic lesions, the physical examination is typically unrevealing.  The findings are variable and of limited precision in either localization or diagnosis of endometriosis.  However, tender nodules may be palpable along the uterosacral ligaments, rectovaginal septum, or within the cul-de-sac, especially if the examination is performed just before menses.  The astute clinician can appreciate pain or induration in the vicinity of otherwise non-palpable lesions, most commonly in the cul-de-sac, along the uterosacral ligaments, or rectovaginal septum.  The clinician may also appreciate uterine or adnexal fixation or a tender adnexal mass.  Because much disease is found in the dependent areas of the pelvis, it is critical to perform a systematic rectovaginal examination.  In that way, the practioner is assured of evaluating the cul-de-sac and uterosacral ligaments as well as the adnexa (72).

DIFFERENTIAL DIAGNOSIS

The differential diagnosis of endometriosis includes pelvic inflammatory disease, tubo-ovarian abscess, ectopic pregnancy, irritable bowel syndrome, interstitial cystitis, adenomyosis, pelvic adhesions, uterine fibroids, chronic or acute endometritis, ovarian neoplasms, musculoskeletal disease, gastrointestinal neoplasms, appendicitis, and diverticular disease.

DIAGNOSTIC MODALITIES

Laparoscopy with visualization of lesions is considered the gold standard for the diagnosis of endometriosis.  Dogma states that a biopsy of the lesion is the only way to truly confirm a disease diagnosis.  For those surgeons trained in advanced laparoscopy, direct visual recognition of endometriosis also allows treatment to be instituted immediately.  Laparoscopy is preferred over laparotomy as it provides visualization of the entire abdomen and pelvis at magnified views, has less morbidity than laparotomy, and carries a decreased risk of adhesion formation (73, 74).  One of the major limitations of diagnostic laparoscopy for pelvic pain is the assumption that those lesions seen (or not seen) directly correlate with the symptoms of pain. Attempts to develop noninvasive radiological imaging techniques as diagnostic tools for endometriosis have been compromised by lack of specificity.  Computed tomography was used initially to diagnose endometriomas in the setting of adnexal masses.  Unfortunately, not only was it difficult to distinguish between benign versus malignant masses, it was often impossible to distinguish adnexal structures and loops of bowel (56, 57).  Hence, this modality is of little value for this purpose.   Transvaginal ultrasound is considered the first-line imaging modality. The typical ultrasound appearance of an endometrioma is a homogeneously hypoechoic unilocular cystic mass with low-level internal echoes and a ground glass appearance. Occasionally, endometriomas may be multilocular, though they typically have fewer than five locules. The vaginal probe can further be used in the evaluation of endometriosis by helping to determine the mobility of pelvic organs. For example, if the ovary appears to be affixed to the uterus, the probe can be used to apply pressure to the ovary. If the ovary does not move with the applied pressure, one can conclude that there are adhesions preventing such movement (75).

The addition of cystoscopy, colonoscopy/ sigmoidoscopy, renal ultrasound, intravenous pyelogram, or barium enema should be considered if there is cyclic bowel/bladder dysfunction or back pain to rule out ureteral, bladder, or rectal involvement of deep lesions or other malignancyBecause of the broad range of symptoms that are included in endometriosis, a multi-disciplinary approach is often required for diagnosis and management (76-82).   At present, magnetic resonance imaging is the best imaging for identifying endometriosis, and it can identify implants as small as 3mm in size (83, 84).  It also has been shown to differentiate benign from malignant lesions, with excellent sensitivity and specificity, and is the method of choice for preoperative evaluation of any adnexal mass (85, 86).  Because of the large disparity in cost of an MRI versus a transvaginal ultrasound, physicians may resort to using an MRI in cases of ultrasonographically indeterminate pelvic masses (3).   Much research has been devoted to discovering a serum marker that will allow diagnosis and monitoring of treatment, to no avail.  Monoclonal antibodies raised against a high molecular weight ovarian cancer epithelial cell antigen, CA-125, have been used as a biochemical marker of endometriosis.  Moderate and severe endometriosis is associated with elevated levels of CA-125 in the peripheral blood (87).  Unfortunately, this marker is relatively non-specific, being increased in ovarian cancer and other gynecologic malignancies (fallopian tube carcinoma, germ cell tumors, adenocarcinoma of the cervix, sertoli-leydig cell tumors), benign gynecologic conditions (benign pelvic neoplasms, adenomyosis, pregnancy, pelvic inflammation), liver disease, colitis, colon cancer, diabetes, congestive heart failure, some autoimmune and rheumatologic disorders, ascites, breast cancer, lung cancer, and even during menstruation of women without any disease.  Although it may help define progression of disease in individual patients, its diagnostic utility is limited.   Studies have reported an increased density of nerve fibers exist in the eutopic endometrium of patients with endometriosis, but there is controversy as to whether aberrant innervation in the endometrium is a reflective of gynecologic pathology in general (53, 90, 91, 94, 95) or a specific feature of endometriosis (55, 92-97).  It was once thought that endometrial biopsies may prove to be useful in the diagnosis of endometriosis, but recent studies demonstrated the presence of neuronal markers in endometrial pipelle biopsies from women both with and without endometriosis (98).  

CLINICAL APPEARANCE

Grossly, peritoneal endometriosis can take on many visual appearances.  Classically, it was taught that endometriosis implants were blue-black “powder-burns” or “mulberry lesions” of the peritoneum.  More recently, several stages of implant development have been appreciated, each with a corresponding appearance.  Early, active lesions can appear as papular excrescences or vesicles, and can range in color from clear to pink, or bright red (98).  About a third of these lesions are in phase with the eutopic endometrium, and have a tendency to spontaneously grow and regress, suggesting a fluctuation of proliferation in association with the cyclic hormone production during the menstrual cycle (99).  Advanced, active lesions are associated with inflammation, fibrosis and hemorrhage, and take on a more classic appearance identifiable at surgery.  These lesions can express a myriad of colors, from black to brown, purple, red, or green.  These are due to the presence of heme degradation products as the foci undergo hemorrhage and fibrosis.  Dormant and healed lesions take on either a white or calcified appearance, and represent remnants of glands embedded in fibrous tissue (98).  The surface of peritoneum may also be puckered or contain windows (Allen-Masters windows). A specific manifestation is the endometrioma or chocolate cyst.  These ovarian cysts gained their moniker by the characteristic chocolate syrup appearance of their contents often seen at rupture.  They arise after implantation of ectopic endometrial tissue and subsequent invasion into the normal ovarian cortex.  The cell types present in these cysts include endometrial epithelium, both as glands and flattened cells, endometrial stroma, and hemosiderin-laden macrophages.  In some cases, ciliated cells, similar to those of oviduct epithelium, have been observed (56 100).   Under scanning electron microscopy, microscopic lesions have been found in the normal appearing peritoneum of women with and without endometriosis (101).  The clinical significance of these findings is presently unknown, but the existence and potential tissue activity in these occult lesions may contribute to the recurrence/occurrence of endometriosis or persistence/recurrence of symptoms in women even after successful ablation or excision of visible lesions.

Fig 2a

Fig 2b

Fig 2c
Figure 2. Intraoperative appearance of endometriosis. (a) The vesicular appearance of early, active lesions. (b) Peritoneal windows. (c) The characteristic blue-black appearance of more advanced, active implants.

 

HISTOLOGY

Although the histopathologic finding of ectopic endometrial glands and stroma is the sine qua non for establishing the diagnosis of disease, only about 50-70% of presumed endometriosis specimens fulfill these criteria.  Many specimens harbor fibrosis, chronic inflammation, and/or hemosiderin-laden macrophages.  Most pathologists and clinicians accept these latter findings as highly suggestive of disease status.  Eutopic endometrium and endometriotic implants are histologically similar, but as mentioned previously, they are functionally, biochemically, and hormonally different from each other.

CLASSIFICATION

The scheme most widely used to classify the extent of disease is the one derived from the American Society for Reproductive Medicine (ASRM), revised in 1996 (103)(Fig. 3 and 4).  This scheme designates disease extent based upon the total 3-dimensional volume of endometriosis.  Importance is placed upon the size, depth of invasion, bilaterality, ovarian involvement, extent of cul-de-sac involvement, as well as density of associated adhesions.  From this system, point scores are assigned and tallied, with scores of 1-15 representing minimal or mild disease, 16-40 moderate, and >40 severe.  It is important to note that this staging system was established to predict fertility outcomes and does not correlate with the more common symptom of pelvic pain.  Unfortunately, the classification system is not good at predicting  which women will suffer from infertility, as infertility may even occur in women with mild endometriosis.  Commonly, practitioners classify endometriosis as minimal (isolated small implants), mild (superficial implants <5cm total, only located on the ovaries and peritoneum, no adhesions), moderate (multiple superficial and invasive implants with or without adhesions), or severe (endometriomas), and the classification system by the ASRM is sometimes reserved for use in studies, where standardization of the classification is important.  Use of the ASRM classification system should be encouraged, however, because it best describes the full extent and location of endometriotic lesions. It provides clinicians caring for women with endometriosis with a detailed description of the location and extend of disease.

Figure 3. The American Fertility Society Revised Classification of Endometriosis. (Reprinted by permission from Fertil Steril 1985;43:351).

Figure 4. Examples and guidelines for use of the American Fertility Society Revised Classification of Endometriosis. (Reprinted by permission from Fertil Steril 1985;43:352).

TREATMENT

Treatment for endometriosis differs depending on the symptom being targeted and whether or not the patient is trying to become pregnant in the near future .  Treatment is either aimed at pain reduction, fertility restoration, or evaluation and treatment of a pelvic mass.  Unfortunately, there are few treatment options for those who desire both fertility in the near future and the treatment of pelvic pain. However, sometimes pregnancy will temporarily relieve the pain associated with endometriosis.  

Pain

Pain is the most commonly reported symptom of endometriosis, an estrogen-dependent condition characterized by chronic peritoneal and pelvic inflammation.  Since endometriosis is a chronic disease, it would be most beneficial to use agents that can be safely used long-term.  Dysmenorrhea is one of the most common complaints in women with endometriosis, so many of the hormonal agents aim to cause amenorrhea.  These treatments may also relieve deep dyspareunia, non-cyclic pelvic pain, and dyschezia.  Hormonal agents may have an additional effect on reducing the nerve fiber density present in endometriotic lesions, which is believed to be one of the factors involved in the origin of the pain caused by endometriosis (104).  Oral contraceptives, progestins, danazol, gestrinone, medroxyprogesterone acetate, and GnRH agonists are all supported by clinical trials showing approximately equal benefit over placebo (Fig. 5).  These hormonal agents create a hypoestrogenic (GnRH agonist), hyperandrogenic (danazol, gestrinone) or hyperprogestogenic (oral contraceptives, medroxyprogesterone acetate) state that suppresses endometrial cell proliferation.  Their side-effect profiles and costs lead one agent to be preferred over another.  However, once the agent is discontinued, the symptoms tend to recur.   A definitive diagnosis of endometriosis can only be made with surgery.  If there is sufficient clinical suspicion of endometriosis, it is reasonable to try empiric therapy with a single agent or a combination of agents.  Oftentimes, the pain will improve and surgery can be avoided.

The combined oral contraceptive pill (COCP) has been used for the treatment of endometriosis-associated pain for several years.  Its efficacy in reducing symptoms of pain is well-established and works by  decreasing retrograde menstruation, inducing a pseudo-pregnant state and causing decidualization and subsequent atrophy of eutopic and ectopic endometrium.  Advantages include a mild side-effect profile, several combinations from which to choose, and the choice of cyclic or continuous administration.  However, not all women are candidates for the use of COCPs.  COCPs are FDA approved for contraception, but they are used off-label for the treatment of endometriosis-associated pain.

There are many progestins (synthetic derivatives of progesterone) that have been used to treat endometriosis-associated pain.  Examples include norethindrone (norethisterone), medroxyprogesterone, and levonorgestrel.  There are several routes of administration.  They do not contain estrogen; thus, they are believed to be safer for use in women who have a contraindication to estrogen.   Norethindrone acetate is a progestin that is taken orally and is initiated at the dose of 5 mg/day and can be increased by increments of 2.5 mg to achieve amenorrhea or to a total dose of 20 mg/day.  Side effects include break-through bleeding and breast tenderness.  Favorable effects on bone-mineral density (short term) and lipid metabolism have been reported (105). Norethindrone acetate is FDA approved for the treatment of endometriosis-associated pain.   Medroxyprogesterone, a 17-hydroxy derivative of progesterone, taken orally, has moderate androgenic activity and minor effects on the lipid profile.  The dose may range from 15-50 mg/day.  Side effects may include breakthrough bleeding and depression/anxiety.  The intramuscular dose or subcutaneous dose of depot medroxyprogesterone acetate is 150 mg given every 3 months.  There is concern about the decrease in bone mineral density with long-term use of medroxyprogesterone.  However, studies have shown that after discontinuation of this medication, the bone mineral density profile improves. It takes an average of 7 months for menses to return.  The abnormal bleeding and lipid profiles are still concerns for long-term use (105).  Medroxyprogesterone is FDA approved for the treatment of endometriosis-associated pain.

The levonorgestrel-containing intrauterine device releases 20 µg/day and induces amenorrhea by causing the endometrium to become atrophic and inactive.  It has been shown to improve dysmenorrhea, relieve deep dyspareunia, and, as expected, reduce monthly blood loss.  Approximately one third of users will develop amenorrhea.  Reasons for discontinuation include irregular bleeding, pelvic pain, breast tenderness, and weight gain.  There is a 5% expulsion rate and a 1.5% infection rate associated with use of the intrauterine device.  Advantages of this system are that it doesn’t induce a systemic hypoestrogenic state and it is effective, without any further medical intervention, for five years.  It is currently being evaluated for postoperative use in women who undergo laparoscopy for endometriosis (105).  It is not FDA approved for the treatment of endometriosis-associated pain, but it is approved for the treatment of heavy menstrual bleeding.

For the relief of pain caused by the deep infiltrative type of endometriosis, a combination of different types of agents may be of benefit.  A study with continuous oral ethinyl estradiol 0.01 mg/day with cyproterone acetate 3 mg/day or only norethisterone 2.5 mg/day demonstrated that both regimens substantially decreased dysmenorrhea, non-cyclic pelvic pain, deep dyspareunia, and dyschezia, but both caused slight weight gain and undesirable changes in lipid profiles.  The group treated by norethisterone reported slightly better symptom improvement but also registered additional androgenic side effects (105).

GnRH agonists have been shown to decrease dysmenorrhea, dyspareunia, and non-cyclic pelvic pain by creating a hypoestrogenic environment.  Some agonists are given subcutaneously either once a month at a dose of 3.75 mg or once every three months at a dose of 11.25 mg.  The side effects of GnRH agonists include a reduction in bone mineral density and, therefore are not recommended for periods longer than 6 months.  Other side effects include hot flushes, emotional lability, vaginal dryness, insomnia, and loss of libido.  “Add-back therapy” with a low dose estrogen/progestin combination has been introduced to prevent the loss of bone mineral density and control the other side effects resulting from the hypoestrogenic environment, while continuing to control the symptoms of endometriosis (105).  GnRH agonists are FDA approved for the treatment of endometriosis-associated pain.

GnRH antagonists are newer compounds and avoid the undesirable “flare” caused by the agonists.  Unfortunately, they are not as well studied and must be administered subcutaneously at least once a week at a dose of 3 mg (105).  More recent studies have looked at oral formations of GnRH antagonists (106). These show promising results in terms of reduced dysmenorrhea and non-menstrual pelvic pain. However, much is still unknown regarding reversibility of adverse outcomes such as decreased bone mineral density and altered lipid profiles. Thus far, it appears that oral GnRH antagonists have less complete suppression of the hypothalamic-pituitary-ovarian axis compared to GnRH agonists. It may be inferred that hypoestrogenic side effects may be less with GnRH antagonists compared to GnRH agonists; however, studies showed incomplete suppression of ovulation, and pregnancy may still occur with the use of GnRH antagonists (106). Clinical trials are on-going, but currently GnRH antagonists are not FDA approved for the treatment of endometriosis-associated pain.

Danazol, an oral agent, induces an amenorrheic state by suppressing the hypothalamic-pituitary-ovarian axis, and is characterized by hyperandrogenemia and hypoestrogenemia.  The hormonal cyclicity of the menstrual cycle is interrupted, thereby disrupting steroidogenesis and estrogen production from the ovary that leads to the undesirable painful symptoms in endometriosis.  Its use is losing favor because of its undesirable side effects.  Weight gain, fluid retention, breast atrophy, acne, oily skin, hot flushes, hirsutism, and unfavorable changes in the lipid profile are among the side effects of danazol.  Alternative routes of delivery may result in a beneficial relief of symptoms, while significantly reducing the side effect profile.  Studies have looked at a danazol-releasing intrauterine device, vaginal ring, or vaginal capsules, but these preparations are not available at this time (105).  Danazol is FDA approved for the treatment of endometriosis-associated pain.

Gestrinone, an oral agent, is a 19-norsteroid derivative that was originally designed as an oral contraceptive.  It has the ability to block follicular development and estradiol production by causing both agonist and antagonist effects when bound to progesterone receptors.  Its ability to bind to androgen receptors, however, is responsible for its undesirable side effects: an unfavorable lipid profile, weight gain, hirsutism, seborrhea, and acne.  Like danazol, this medication is not frequently used (105).  Gestrinone is available in many countries for the treatment of endometriosis-associated pain, but is not approved for use in the US.

Aromatase inhibitors have recently become part of the armamentarium against endometriosis-associated pain.  Aromatase is the enzyme responsible for the conversion of androgens into estrogens, and is normally expressed in granulosa cells, skin fibroblasts, adipocytes, and syncytiotrophoblasts.  Although, steroidogenic factor-1 is found in endometriotic tissue, it is not found in eutopic endometrium.  Steroidogenic factor-1 activates aromatase gene transcription, thereby increasing the production of estrogen.  Because endometriosis is estrogen-dependent, and aromatase is responsible for estrogen production, aromatase inhibitors have therefore been employed to alleviate the painful symptoms caused by endometriosis.  Combination therapy of an aromatase inhibitor with a progestin, oral contraceptive agent, or GnRH agonist is recommended in the treatment of endometriosis in pre-menopausal women because of their ovarian stimulatory properties in this population and to prevent pregnancy.  Aromatase inhibitors have a tolerable side-effect profile and don’t reduce bone mineral density (105).  Aromatase inhibitors are not FDA approved for the treatment of endometriosis-associated pain, but clinical trials are ongoing.

Historically, there has been a well-established role for prostaglandin inhibitors, such as ibuprofen, in the treatment of endometriosis-associated pain.  Recently, a Cochrane Review evaluated non-steroidal anti-inflammatory use for the treatment of endometriosis (107).  It discovered two studies that met their inclusion criteria.  They found inconclusive evidence to show that non-steroidal anti-inflammatory agents are effective for the treatment of pain associated with endometriosis.  Despite this review, prostaglandin inhibitors are relatively safe, have a tolerable side-effect profile, and can generally be taken on a long-term basis by most patients, so they remain part of the first-line therapy for the treatment of endometriosis-associated pain.  However, their use is associated with undesirable and potentially severe gastrointestinal side-effects that may cause them not to be candidates for use in every patient.  Motrin (ibuprofen), a commonly used prostaglandin inhibitor is FDA approved for the treatment of endometriosis-associated pain.

Figure 5:  Options for the Treatment of Pain Associated with Endometriosis
Agent     Route       Side-Effects
Oral Contraceptive Agents Oral Mild nausea, vomiting
Progestins Oral, Injection, or Intrauterine Breakthrough bleeding, breast tenderness
Some have unfavorable effects on bone mineral density and lipid profile
Some have androgenic side-effects
GnRH Agonists Injection or Intranasal Symptoms of a hypoestrogenic state
(hot flushes, mood irritability, vaginal dryness, sleep disturbances, and decreased bone mineral density)
GnRH Antagonists* Oral Symptoms of hypoestrogenic state
  (hot flushes, decreased bone mineral density, unfavorable changes in the lipid profile)
Aromatase Inhibitors Oral Ovarian stimulation in pre-menopausal women
Danazol Oral Weight gain, fluid retention, breast atrophy, acne, oily skin, hot flushes, hirsutism
Unfavorable changes in the lipid profile
Gestrinone Oral Unfavorable changes in the lipid profile
Weight gain, hirsuitism, seborrhea, and acne
Prostaglandin Inhibitors Oral Unfavorable gastrointestinal side-effects

Figure 5.  Pharmacologic options in the treatment of pain associated with endometriosis.

*GnRH antagonists are not yet FDA approved, but may be a potential future treatment.

 

As previously mentioned, endometriosis is associated with a peritoneal and pelvic inflammatory cascade, and anti-inflammatory compounds may alleviate the pain associated with endometriosis.  Since the cyclo-oxygenase-2 pathway is upregulated, COX-2 inhibitors may have a role in treatment, but their main role in treatment of this disease is not well established. Other emerging therapies have targeted the molecular steps involved in endometriosis pathogenesis.  This includes agents that enhance cell-mediated immunity, agents that counteract tumor necrosis factor-α (TNF-α), anti-angiogenic agents, metalloproteinase inhibitors, hypocholesterolemic agents, and selective progesterone modulators.  A Cochrane Review examined the use of TNF-α inhibitors for the treatment of endometriosis-associated pain and found no benefit. (108).  Several antiangiogenic agents are in preclinical testing and show promise. Statin medications are also being evaluated as well as trichostatin A and valproic acid (109).  These agents are not FDA approved for the treatment of endometriosis-associated pain.

From the available evidence, it is clear that medical treatment is effective for endometriosis-associated pelvic pain.  In general, a non-steroidal anti-inflammatory agent alone or in combination with an oral contraceptive agent or a progestin derivative should be considered as first-line therapy.  GnRH analogues with add-back therapy and possibly aromatase inhibitors (with oral contraceptive agents, progestins, or a GnRH analog in premenopausal patients), should be regarded as second-line agents.  Danazol and gestrinone should be reserved for cases that have failed other medical treatments.   If a patient continues to have symptoms of pain despite medical therapy, or needs pain relief but desires pregnancy and thus must avoid hormonal compounds that interfere with ovulation, then conservative surgery should be performed with resection or ablation of lesions and lysis of adhesions.  In double-blind randomized control trials, laparoscopic laser treatment of pelvic pain associated with minimal-moderate endometriosis was found to decrease pain significantly(109, 111).  Follow up re-operation rates after initial surgical removal of lesions was 21%, 47%, and 55% at 2, 5, and 7 years of follow-up, respectively (112).  The highest predictor of re-operation was associated with  younger age of the patient.  The evidence shows that both ablation and resection of lesions are equally effective techniques.  Excision of ovarian endometriomas, however, is associated with better pain relief, lower recurrence, and higher pregnancy rates than cyst vaporization or coagulation (113).   There is no evidence supporting the performance of a uterosacral nerve ablation; however, if there is significant midline pain, presacral neurectomy may be of benefit (114).  It should be noted that presacral neurectomy requires excellent surgical skills, as there is significant risk of damaging the neurovascular plexus or causing retroperitoneal bleeding severe enough to require transfusion or re-operation.  Resection of rectovaginal lesions had an approximate 10% complication rate especially when a colorectal resection is performed (115, 116).  Good pain relief is usually achieved during the first year after bowel resection for deep endometriosis, but in a systematic literature review, pain recurrence was observed in one out of four patients and re-intervention was required in one out of five of these recurrences (117).   A debatable issue is the use of medical therapy, such as a GnRH agonist or medroxyprogesterone acetate, as a neoadjuvant or adjuvant to surgical management.  Preoperative medical therapy may be helpful to decrease the pelvic vascularity and size of the endometriotic implants, reducing intraoperative blood loss and surgical resection required.  However, by reducing the endometriotic load, the disease may be understaged, which may affect management.  Postoperative medical therapy may eradicate the residual implants.   A Cochrane Review, however, found insufficient evidence to show a benefit of hormonal suppression either before or after surgery when compared with surgery alone for long-term difference in pain relief from endometriosis (118).  There were two trials that compared pre-surgical medical therapy with surgery alone, but American Fertility Society scores were significantly improved in the medical treatment group in one study and not in the other.  Post-surgical hormonal suppression of endometriosis versus surgery alone (either no medical therapy or placebo) showed a modest reduction in pain after one year, but results were inconsistent and pain recurrence in both groups indicated no benefit beyond one year after treatment (119).  Further, there was no evidence that medical therapy pre- or post-surgery improves pregnancy rates.   There have been no trials comparing medical and surgical treatment to reduce endometriosis-associated pain.  The studies employing each treatment modality, though, have similar success rates.  With this in mind, it is reasonable to conclude that the treatments are equally effective.  With the development of newer medical therapies with better tolerated side-effect profiles, surgery may be avoided or delayed.  Ultimately, in women who no longer desire future childbearing, hysterectomy with bilateral salpingo-oophorectomy, often is considered as definitive therapy for the treatment.  Narcotics should never be considered for treatment of the chronic pain associated with endometriosis.  Referral to a multidisciplinary pain center may be of value. In the absence of other factors, if the pain continues to be present after surgery, a diagnosis of adenomyosis should be considered.

Figure 6. Treatment algorithm for pain associated with endometriosis

Infertility

The treatment of infertility, in the absence of pain, may involve expectant management, surgery, ovulation induction/stimulation with intrauterine insemination (IUI), or assisted reproductive techniques, such as in vitro fertilization (IVF).  Infertility clinics differ in their use of diagnostic laparoscopy in the evaluation of the etiology of infertility, at which time asymptomatic endometriosis may be diagnosed and excised or ablated.  Physicians that do not perform diagnostic laparoscopy as part of their evaluation for infertility would argue that there are significant anesthetic and surgical risks afforded by laparoscopy, ovarian reserve may be compromised by the incidental removal/destruction of normal ovarian tissue, surgery is a cause of adhesions, and that the practice of restoring normal tubal anatomy has fallen out of favor as the success of IVF continues to rise.   In women with stage I/II endometriosis, laparoscopic ablation of lesions may offer a small, but significant improvement in live birth rates.  A Cochrane Review evaluated two trials in which women were randomized to operative laparoscopy or diagnostic laparoscopy (120).  The two randomized trials  addressed the question of whether laparoscopic surgery improved outcomes in patients with otherwise unexplained infertility (121, 122).  When combining live birth rates and clinical on-going pregnancy rates after 20 weeks, the meta-analysis demonstrated an advantage of laparoscopic surgery when compared to diagnostic laparoscopy alone, with an odds-ratio (OR) of 1.64 [95% Confidence Interval (CI) 1.05-2.57].  However, the two studies had incompatible results, which should be taken into account in this interpretation.  One study (121) reported a large positive effect, while the other (122) reported a small negative effect.  The number needed to treat, resulting from this analysis, was 12 laparoscopies for one additional pregnancy.  The decision to perform surgery should, therefore, be balanced with the patient’s age, history, health status, and wishes, and should be entered into with informed consent about the risks and possible benefits.  For those that do undergo surgery, the Endometriosis Fertility Index (EFI), an intraoperative staging system, can be used to help predict future pregnancy rates. Based on the EFI, a patient may be better counseled regarding her likelihood of spontaneous conception and, if her prognosis is poor, more readily seek appropriate treatment (123). Suppressive medical therapy (such as in the treatment for pain) should not accompany surgery or be involved in the treatment of infertility, as it does not improve fertility and may increase the time to pregnancy.  Viable options for the medical treatment of endometriosis-related infertility are discussed.   Ovulation induction in women with stage I/II endometriosis confers a fertility benefit.  The three randomized trials in the literature addressing this issue, employing either GnRH agonists with follicle-stimulating hormone and luteinizing hormone, clomiphene citrate/IUI, or follicle-stimulating hormone/IUI, all showed increased pregnancy rates relative to the control arm, where the patient did not receive treatment (114).  Thus, both ovulation induction plus IUI and assisted reproductive technology have a place in the treatment of women with endometriosis-associated infertility.

If the patient is under the age of 35, initial treatment options include expectant management or controlled ovarian stimulation with clomiphene citrate or gonadotropins combined with IUI.  In a woman over the age of 35, because of the age-associated decrease in fecundity, controlled ovarian stimulation combined with intrauterine insemination or IVF are reasonable therapies.   In women with stage III/IV endometriosis, surgery is likely to be of benefit and is recommended by the ASRM Practice Committee, but there are no randomized controlled trials to document efficacy and some studies show a negative effect of surgery on pregnancy rates. (65, 124).   Advanced staged disease is often associated with a complex surgical history which, combined with an extensive amount of disease, causes the operation to be difficult, sometimes requiring advanced laparoscopic techniques or laparotomy.  These higher acuity surgeries have increased risk of serious complications.  In situations when the initial surgery fails to restore fertility, IVF, rather than another operation for infertility, is likely a better option.  However, randomized controlled trials are lacking.  If surgery is performed, then the subsequent treatment for infertility should be controlled ovarian stimulation (with gonadotropins) and IUI, or IVF, especially in women of advanced reproductive age (66).  If the plan is to go directly to IVF, then surgery may not show a benefit, as the slight possible benefit afforded by the surgery is greatly overshadowed by the benefits of IVF and indirect evidence suggests that surgery may not be beneficial in women with deep infiltrating disease (125).   There has been some debate about the management of endometriomas in stage IV disease in the absence of pain.  Only case series have been published, but the overall likelihood of pregnancy following endometrioma excision is around 50% (125); though this may be an overestimate with women achieving pregnancy through IVF.

The question then remains, should the endometriomas be removed prior to IVF?  A meta-analysis evaluated five studies that compared surgery versus no treatment for an endometrioma (121). In this analysis, there were no differences in the pregnancy rates or responses to controlled ovarian stimulation in the two groups, favoring against surgical resection.  There are no randomized controlled trials that evaluate this question.  Surgery for endometriomas should be performed to relieve pain and to confirm the diagnosis in situations when it is in question.   Overall, endometriosis is a common indication for IVF.  IVF allows the abnormal anatomy to be overcome by avoiding the tubes; the oocyte is retrieved directly from the ovaries, and the embryos are placed directly in the endometrial cavity, thereby bypassing the effects of the abnormal peritoneal environment.  Despite this, endometriosis is associated with overall lower chances of success.  This may be due to the negative impact of endometriosis on folliculogenesis and endometrial receptivity (126).  A meta-analysis demonstrated significantly lower pregnancy rates for women with endometriosis compared to women with tubal factor infertility controls.  Pregnancy rates were also significantly lower for women with severe endometriosis versus women with milder disease (127). However, the difference in pregnancy rates does not appear to be attributable to differing aneuploidy rates. A recent retrospective cohort study demonstrated no difference in aneuploidy rates between age-matched controls of women with endometriosis undergoing IVF compared to those without endometriosis (128).

Figure 7. Treatment algorithm for infertility associated with endometriosis.

Other Therapeutic Modalities

There have been many studies exploring complementary alternative medicine (CAM) for the treatment of symptoms associated with endometriosis.  Although these approaches are not FDA or USDA approved for the treatment of endometriosis, they may offer relief by suppressing cytokines and inflammatory pathways, inhibiting cyclooxygenase-2 (COX-2) pathways, acting as antioxidants, and alleviating pain by other mechanisms (129).  High frequency transcutaneous electrical nerve stimulation, acupuncture, vitamin B1, magnesium, reflexology, traditional Chinese medicine, herbal treatments, and homeopathy are examples of CAM that have been explored for use in endometriosis-associated pain.  Even though the risks associated with some of these modalities appear minimal, the standards of evidence-based medicine on aspects of safety and efficacy have not been applied to most studies involving CAM.  A promising note for CAM is that there is now a National Center for Complementary and Alternative Medicine (NCCAM) at the National Institutes of Health (NIH).  Hopefully, this will allow for proper evidence-based evaluation, so that their safety and efficacy can be compared with the more traditional approaches (129).  Another therapeutic modality that may be considered is patient self-help and support groups.  Information can be found at www.endometriosis.org (76).

ENDOMETRIOSIS-ASSOCIATED OVARIAN CANCER

Although it is not considered a pre-malignant condition, there are data to suggest that endometriosis has neoplastic potential (130).  Endometriosis shares several similarities with malignant diseases such as reduced apoptosis, invasion of endometrial cells into adjacent organs (bowel, bladder), increased angiogenesis, ability to spread distantly, etc. (131-133).  Women with endometriosis have twice the risk of developing epithelial ovarian cancer compared to controls, and a 4-fold increased risk if they also have subfertility (134).  The association is limited to clear cell (OR 3.05, 95% CI 2.43-3.84), endometroid (OR 2.04, 95% CI 1.67-2.48), and low-grade serous (OR 2.11, 95% CI 1.39-3.2) tumors (135).  The carcinogenic pathways, however, poorly understood.  There is thought that oxidative stress, inflammation and the estrogen dependent environment may play a role (136).   In women without malignant appearing features on ultrasound, 1-3% are found to have atypical endometriotic cells (125).  Generally, endometriosis associated malignancies arise from endometriomas.  Reactive oxygen species generated by catalytic iron from phagocytosed heme causes cell damage, DNA mutations, and genetic instability may be aided by the local ovarian environment of endometriomas (137-139).  This oxidative stress is thought to activate proto-oncogenes or disrupt tumor suppressor genes, such as ARID1A (140-142).   Clear cell and endometroid tumors also develop due to hormonally dependent and independent pathways.  Endometroid cell types are often estrogen receptor (ER) and progesterone (PR) receptor positive.  Clear cell tumors, however, generally have low receptor expression (143).  Endometroid histology is also thought to be estrogen-dependent based on the fact that there is a higher than expected rate of synchronous primary, estrogen-dependent endometrial endometroid carcinoma (144) concurrently with endometriosis-associated ovarian endometroid carcinoma. (145-147).  Clear cell carcinomas overexpress hepatocyte nuclear factor 1ß, which is a transcriptional factor that increases survival of endometrial cells during oxidative stress by inhibiting apoptosis, whereas endometroid cell types do not overexpress hepatocyte nuclear factor 1ß (137).   Knowing the increased risk of carcinoma in endometriosis patients, the question remains as to how to prevent progression.  There are limited data available regarding medical and surgical interventions for prevention.  It is known that oral contraceptives are associated with an 80% reduction in risk among women with endometriosis when used for greater than 10 years. Since endometroid subtypes are ER and PR positive, whereas clear cell subtypes are not, those at risk for the endometroid subtype are more likely to benefit from hormonal therapy while those more likely to develop the clear cell subtype would benefit from surgery.  In Western countries the endometroid subtype is more prevalent than clear cell, 10-20% versus 5-10%, respectively.  However, the opposite is true in Asian countries.  This could suggest a population based treatment approach (144-146).

CONCLUSION

Endometriosis is an enigmatic disease, the pathophysiology of which we are just beginning to understand.  Symptomatic women with endometriosis, suffering from infertility or pain, are often difficult patients to treat because we have few treatment options to offer.  The therapies themselves are imperfect, with none that permanently eradicate the disease.  A multidisciplinary approach is often required for the diagnosis and management of this disease.   In deciding how to treat women with pelvic pain or infertility, we must consider the best available evidence to form our decisions.  In women with pelvic pain and suspected endometriosis, first line treatment would be non-steroidal anti-inflammatory medications with/without oral contraceptives or progestins.  Continuous combined oral contraceptives have been shown to provide significant pain reduction from baseline over cyclic combined oral contraceptives(148).  If these conservative approaches fail, three alternative treatments would be empiric: GnRH agonist therapy with estrogen and progestin add-back therapy, aromatase inhibitors, or operative laparoscopy.  The laparoscopy should include lysis of adhesions and excision or ablation of endometriosis.  Surgery for pain may be followed by medical treatment with GnRH agonist and add-back therapy (Fig. 6).  It is important to note that at no time are narcotics advocated for the treatment of endometriosis-associated pelvic pain except immediately pre-operatively or post-operatively.  If the above measures fail, patients should be enrolled into a multi-disciplinary chronic pelvic pain treatment group.  This should include physicians from many subspecialties, including psychiatry, anesthesiology, gynecology, and often gastroenterology and urology.

 

     

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Adrenal Insufficiency

ABSTRACT

Adrenal insufficiency is a serious pathologic condition characterized by decreased production or action of glucocorticoids and/or mineralocorticoids and adrenal androgens. This life-threatening disorder may be classified as primary, secondary or tertiary, resulting from diseases affecting the adrenal cortex, the anterior pituitary gland or the hypothalamus, respectively. The clinical manifestations of adrenal insufficiency include anorexia, abdominal pain, weakness, weight loss, fatigue, hypotension, salt craving and hyperpigmentation of the skin in case of primary adrenal insufficiency. The diagnosis of adrenal insufficiency can be confirmed by demonstrating inappropriately low cortisol secretion, determining whether the cortisol deficiency is secondary or primary, and defining the cause of the disorder. Treatment with glucocorticoid and/or mineralocorticoid replacement should be initiated when glucocorticoid and or mineralocorticoid deficiency is suspected. This chapter will provide an overview of the epidemiology, etiology, pathophysiology, clinical manifestations, diagnosis and treatment of adrenal insufficiency. Finally, special conditions of adrenal insufficiency, including critical illness, pregnancy, infancy and childhood will also be discussed.  For complete coverage of this and related areas of Endocrinology, please visit our free online textbook, WWW.ENDOTEXT.ORG.

 

 

INTRODUCTION

Adrenal insufficiency is a disorder first described by Thomas Addison in 1855, which is characterized by deficient production or action of glucocorticoids and/or mineralocorticoids and adrenal androgens. This life-threatening disease may result from disorders affecting the adrenal cortex (primary), the anterior pituitary gland (secondary), or the hypothalamus (tertiary) (Figure 1) (1-3). The clinical symptoms of adrenal insufficiency include weakness, fatigue, anorexia, abdominal pain, weight loss, orthostatic hypotension, salt craving, and characteristic hyperpigmentation of the skin occurring with primary adrenocortical failure (4, 5). Regardless of etiology, adrenal insufficiency was an invariably fatal disorder, until the synthesis of cortisone by Kendall, Sarett, and Reichstein (6-9) in 1949, and the introduction of substitution therapy with life-saving synthetic glucocorticoids subsequently. However, despite this progress, there are still numerous challenges regarding the diagnosis and treatment of patients with adrenal insufficiency.

Figure 1: Types of adrenal insufficiency. CRH: corticotropin-releasing hormone, ACTH: adrenocorticotropic hormone.

Figure 1: Types of adrenal insufficiency. CRH: corticotropin-releasing hormone, ACTH: adrenocorticotropic hormone.

EPIDEMIOLOGY

The prevalence of chronic primary adrenal insufficiency in Europe has been doubled from 40–70 cases per million population in the 1960s (10, 11) to 93–144 cases per million population by the end of the last century and in recent years (12-16). The currently estimated incidence of this disorder is 4.4–6 new cases per million population per year (15). Primary adrenal insufficiency affects more frequently women, and clinical manifestations can present at any age, although most often between 30 and 50 years (12).

 

Secondary adrenal insufficiency occurs more frequently than primary adrenal insufficiency (1). Its estimated prevalence is 150–280 per million and is more common in women than men (14, 17-20). Affected patients are mostly diagnosed in the sixth decade of life (18, 19).

 

The most common cause of tertiary adrenal insufficiency is chronic exogenous administration of synthetic glucocorticoids, which causes prolonged suppression of hypothalamic corticotropin-releasing hormone (CRH) secretion through negative feedback mechanisms (21).

 

CAUSES OF ADRENAL INSUFFICIENCY

Causes of Primary Adrenal Insufficiency

The etiology of primary adrenal insufficiency has changed over time. Prior to 1920, the most common cause of primary adrenal insufficiency was tuberculosis, while since 1950, the majority of cases (80-90%) have been ascribed to autoimmune adrenalitis, which can be isolated (40%) or in the context of an autoimmune polyendocrinopathy syndrome (60%) (1, 2, 22-24).

 

Autoimmune adrenalitis (Addison’s disease): This condition is characterized by destruction of the adrenal cortex by cell-mediated immune mechanisms. Antibodies that react against steroid 21-hydroxylase are detected in approximately 90% of patients with autoimmune Addison’s disease (16), but only rarely in patients with other causes of adrenal insufficiency or normal subjects (25). Considerable progress has been made in identifying genetic factors that predispose to the development of autoimmune adrenal insufficiency (2). In addition to the major histocompatibility complex (MHC) haplotypes DR3-DQ2 and DR4-DQ8, other genetic factors, such as protein tyrosine phosphatase non-receptor type 22 (PTPN22), cytotoxic T lymphocyte antigen 4 (CTLA-4), and the major histocompatibility complex class II transactivator (CIITA) have been associated with this condition (23-29).

 

Primary adrenal insufficiency may also present as part of autoimmune polyendocrinopathy syndromes. Patients with autoimmune polyendocrinopathy syndrome type 1 (APS1) or APECED (Autoimmune Polyendocrinopathy, Candidiasis, Ectodermal Dystrophy) syndrome may present with chronic mucocutaneous candidiasis, adrenal insufficiency, hypoparathyroidism, hypoplasia of the dental enamel and nail dystrophy, while type 1 Diabetes Mellitus (T1DM) or pernicious anemia, may develop later in life (30, 31). Clinical manifestations of autoimmune polyendocrinopathy syndrome type 2 (APS2) include autoimmune adrenal insufficiency, autoimmune thyroid disease and/or T1DM, whereas autoimmune polyendocrinopathy syndrome type 4 (APS4) is characterized by autoimmune adrenal insufficiency and one or more other autoimmune diseases, such as atrophic gastritis, hypogonadism, pernicious anemia, celiac disease, myasthenia gravis, vitiligo, alopecia and hypophysitis, but without any autoimmune disorders of APS1 or APS2 (23, 24, 26, 31-33).

 

Adrenoleukodystrophy: This is an X-linked recessive disorder affecting 1 in 20.000 males (2). The molecular basis of this condition has been ascribed to mutations in the ABCD1 gene, which result in defective beta oxidation of very long chain fatty acids (VLCFAs) within peroxisomes. The abnormally high concentrations of VLCFAs in affected organs, including the adrenal cortex, result in the clinical manifestations of this disorder, which include neurological impairment due to white-matter demyelination and primary adrenal insufficiency, with the latter presenting in infancy or childhood (1-3, 34).

 

Hemorrhagic infarction: Bilateral adrenal infarction caused by hemorrhage or adrenal vein thrombosis may also lead to adrenal insufficiency (35, 36). The diagnosis is usually made in critically ill patients in whom a computed tomography (CT) scan of the abdomen shows bilateral adrenal enlargement. Several coagulopathies and the heparin-induced thrombocytopenia syndrome have been associated with adrenal vein thrombosis and hemorrhage, while the primary antiphospholipid syndrome has been recognized as a major cause of adrenal hemorrhage (37). Adrenal hemorrhage has been mostly associated with meningococcemia (Waterhouse-Friderichsen syndrome) and Pseudomonas aeruginosa infection (38).

 

Infectious adrenalitis: Many infectious agents may attack the adrenal gland and result in adrenal insufficiency, including tuberculosis (tuberculous adrenalitis), disseminated fungal infections and HIV-associated infections, such as adrenalitis due to cytomegalovirus and mycobacterium avium complex (39-41).

 

Drug-induced adrenal insufficiency : Drugs that may cause adrenal insufficiency by inhibiting cortisol biosynthesis, particularly in individuals with limited pituitary and/or adrenal reserve, include aminoglutethimide (antiepileptic), etomidate (anesthetic-sedative) (42, 43), ketoconazole (antimycotic) (44) and metyrapone (45). Drugs that accelerate the metabolism of cortisol and most synthetic glucocorticoids by inducing hepatic mixed-function oxygenase enzymes, such as phenytoin, barbiturates, and rifampicin can also cause adrenal insufficiency in patients with limited pituitary or adrenal reserve, as well as those who are on replacement therapy with glucocorticoids (46). Furthermore, some of novel tyrosine kinase-targeting drugs (e.g. sunitinib) have been shown in animal studies to cause adrenal dysfunction and hemorrhage (47).

 

Other causes of primary adrenal insufficiency are listed in Table 1.

 

Table 1.  Causes of Primary Adrenal Insufficiency

Disease Pathogenetic Mechanism
Autoimmune adrenalitis  
Isolated

Associations with HLA-DR3-DQ2, HLADR4-DQ8, MICA, CTLA-4, PTPN22,

CIITA, CLEC16A, Vitamin D receptor

APS type 1 (APECED) AIRE gene mutations
APS type 2

Associations with HLA-DR3, HLA-DR4,

CTLA-4

APS type 4 Associations with HLA-DR3, CTLA-4
   
Infectious adrenalitis  
Tuberculous adrenalitis Tuberculosis
AIDS HIV-1, cytomegalovirus
Fungal adrenalitis

Histoplasmosis, cryptococcosis,

coccidiodomycosis

Syphilis Treponema pallidum
African Trypanosomiasis Trypanosoma brucei
   
Bilateral adrenal hemorrhage

Meningococcal sepsis (Waterhouse-

Friderichsen syndrome), primary

antiphospholipid syndrome

   
Bilateral adrenal metastases

Primarily lung, stomach, breast and colon

cancer

   
Bilateral adrenal infiltration

Primary adrenal lymphoma, amyloidosis,

haemochromatosis

   
Bilateral adrenalectomy

Unresolved Cushing’s syndrome,

bilateral adrenal masses, bilateral pheochromocytoma

   
Drug-induced adrenal insufficiency  

Anticoagulants (heparin, warfarin),

tyrosine kinase inhibitors (sunitinib)

Hemorrhage
Aminoglutethimide Inhibition of P450 aromatase (CYP19A1)
Trilostane

Inhibition of 3β-hydroxysteroid

dehydrogenase type 2 (HSD3B2)

Ketoconazole, fluconazole, etomidate

Inhibition of mitochondrial cytochrome

P450-dependent enzymes (e.g. CYP11A1,

CYP11B1)

Phenobarbital

Induction of P450-cytochrome enzymes

(CYP2B1, CYP2B2), which enhance

cortisol metabolism

Phenytoin, rifampin, troglitazone

Induction of P450-cytochrome enzymes

(primarily CYP3A4), which enhance

cortisol metabolism

   
Genetic disorders  

Adrenoleukodystrophy or

adrenomyeloneuropathy

ABCD1 and ABCD2 gene mutations
Congenital adrenal hyperplasia  
     21-Hydroxylase deficiency CYP21A2 gene mutations
     11β-Hydroxylase deficiency CYP11B1 gene mutations

      3β-hydroxysteroid dehydrogenase

type 2 deficiency

HSD3B2 gene mutations
     17α-Hydroxylase deficiency CYP17A1 gene mutations
     P450 Oxidoreductase deficiency POR gene mutations
     P450 side-chain cleavage deficiency CYP11A1 gene mutations
     Congenital lipoid adrenal hyperplasia StAR gene mutations
Smith-Lemli-Opitz syndrome DHCR7 gene mutations
Adrenal hypoplasia congenita  
     X-linked NR0B1 gene mutations
     Xp21 contiguous gene syndrome

Deletion of the Duchenne muscular

dystrophy, glycerol kinase and NR0B1

genes

     SF-1 linked NR5A1 gene mutations
IMAGe syndrome CDKN1C gene mutations
Kearns-Sayre syndrome Mitochondrial DNA deletions
Wolman’s disease LIPA gene mutations

Sitosterolaimia (also known as

phytosterolemia)

ABCG5 and ABCG8 gene mutations

Familial glucocorticoid deficiency

(FGD, or ACTH insensitivity syndromes)

 
     Type 1 MC2R gene mutations
     Type 2 MRAP gene mutations
     Variant of FGD MCM4 gene mutations
      FGC - Deficiency of mitochondrial ROS        detoxification NNT, TXNRD2, GPX1, PRDX3 gene mutations

Primary Generalized Glucocorticoid

Resistance or Chrousos syndrome

NR3C1 gene mutations
Sphingosine-1-phosphate lyase 1 deficiency SPGL1 gene mutations
Infantile Refsum disease PHYH, PEX7 gene mutations
Zellweger syndrome PEX1 and other PEX gene mutations
Triple A syndrome (Allgrove’s syndrome) AAAS gene mutations

 

Modified from References (48, 49)

 

Causes of Secondary and Tertiary Adrenal Insufficiency

Secondary adrenal insufficiency may be caused by any disease process that affects the anterior pituitary and interferes with ACTH secretion. The ACTH deficiency may be isolated or occur in association with other pituitary hormone deficits.

 

Tertiary adrenal insufficiency can be caused by any process that involves the hypothalamus and interferes with CRH secretion. The most common cause of tertiary adrenal insufficiency is chronic administration of synthetic glucocorticoids that suppress the hypothalamic-pituitary-adrenal (HPA) axis (50).

 

Other causes of secondary and tertiary adrenal insufficiency are listed in Tables 2 and 3 respectively.

 

Table 2.  Causes of Secondary Adrenal Insufficiency.

Disease Pathogenetic Mechanism
Space occupying lesions or trauma  

Pituitary tumors (adenomas, cysts,

craniopharyngiomas, ependymomas,

meningiomas, rarely carcinomas) or

trauma (pituitary stalk lesions)

Decreased ACTH secretion

Pituitary surgery or irradiation for pituitary

tumors, tumors outside the HPA axis or

leukemia

Decreased ACTH secretion

Infections or Infiltrative processes

(lymphocytic hypophysitis,

hemochromatosis, tuberculosis, meningitis,

sarcoidosis, actinomycosis, histiocytosis X,

Wegener’s granulomatosis)

Decreased ACTH secretion
Pituitary apoplexy Decreased ACTH secretion

Sheehan’s syndrome (peripartum pituitary

apoplexy and necrosis)

Decreased ACTH secretion
   
Genetic disorders  

Transcription factors involved in pituitary

development

 
     HESX homeobox 1 HESX1 gene mutations
     Orthodentical homeobox 2 OTX2 gene mutations
     LIM homeobox 4 LHX4 gene mutations
     PROP paired-like homeobox 1 PROP1 gene mutations
     SRY (sex-determining region Y) – box 3 SOX3 gene mutations
     T-box 19 TBX19 gene mutations

Congenital Proopiomelanocortin (POMC)

deficiency

POMC gene mutations
Prader-Willi Syndrome (PWS)

Deletion or silencing of genes in the

imprinting center for PWS

 

Modified from Reference (48)

 

 

Table 3.  Causes of Tertiary Adrenal Insufficiency.

Disease Pathogenetic Mechanism
Space occupying lesions or trauma  

Hypothalamic tumors

(craniopharyngiomas or metastasis from

lung, breast cancer)

Decreased CRH secretion

Hypothalamic surgery or irradiation for

central nervous system or nasopharyngeal

tumors

Decreased CRH secretion

Infections or Infiltrative processes

(lymphocytic hypophysitis,

hemochromatosis, tuberculosis, meningitis,

sarcoidosis, actinomycosis, histiocytosis X,

Wegener’s granulomatosis)

Decreased CRH secretion
Trauma, injury (fracture of skull base) Decreased CRH secretion
   
Drug-induced adrenal insufficiency  

Glucocorticoid therapy (systemic or topical) or endogenous glucocorticoid

hypersecretion (Cushing’s syndrome)

Decreased CRH and ACTH secretion
Mifepristone

Tissue resistance to glucocorticoids

through impairment of glucocorticoid

signal transduction

Antipsychotics (chlorpromazine),

antidepressants (imipramine)

Inhibition of glucocorticoid-induced gene

transcription

 

Modified from Reference (48)

 

PATHOPHYSIOLOGIC MECHANISMS OF ADRENAL INSUFFICIENCY

Pathophysiology of Primary Adrenal Insufficiency

In primary adrenal insufficiency, although the above mentioned causes lead to gradual destruction of the adrenal cortex, the symptoms and signs of the disease appear when the loss of adrenocortical tissue is higher than 90% (37). At the molecular and cellular level, a viral infection, even subclinical, or an excessive tissue response to inflammatory signals may potentially lead to apoptosis or necrosis of adrenocortical cells. Cellular components, such as 21OH-derived peptides, trigger the activation of local dendritic cells, which then transport and present these antigens to CD4+ Th1 cells. Upon activation, CD4+ Th1 cells help the committed clonal expansion of cytotoxic lymphocytes and autoreactive B cells releasing antibodies against 21-hydroxylase and possibly other antibodies. The gradual destruction of adrenocortical tissue seems to be mediated by four distinct and complementary molecular mechanisms: (a) direct cytotoxicity by lymphocytes that induce apoptosis; (b) direct cytotoxic actions by IFN-γ and lymphotoxin-α released by activated CD4+ Th1 cells; (c) cellular cytotoxicity by autoantibodies or by autoantibody-mediated activation of the complement system; and (d) cytotoxic effects of inflammatory cytokines (IL-1β, TNF-α) and free radicals (superoxide, NO) secreted by monocytes/macrophages or by the adrenal cells (51).

 

In the initial phase of chronic gradual destruction, the adrenal reserve is decreased and although the basal steroid secretion is normal, the secretion in response to stress is suboptimal. Consequently, any major or even minor stressor can precipitate an acute adrenal crisis. With further loss of adrenocortical tissue, even basal steroid secretion is decreased, leading to the clinical manifestations of the disease. Low plasma cortisol concentrations result in the increase of production and secretion of ACTH due to decreased negative feedback inhibition (37). The elevated plasma ACTH concentrations are responsible for the well-recognized hyperpigmentation observed in these patients.

 

Pathophysiology of Secondary and Tertiary Adrenal Insufficiency

In secondary or tertiary adrenal insufficiency, the resultant ACTH deficiency leads to decreased secretion of cortisol and adrenal androgens, while mineralocorticoid production remains normal. In the early stages, basal ACTH secretion is normal, while stress-induced ACTH secretion is impaired (37). With further loss of basal ACTH secretion, there is atrophy of zonae fasciculata and reticularis of the adrenal cortex. Therefore, basal cortisol secretion is decreased, but aldosterone secretion by the zona glomerulosa is preserved.

 

CLINICAL MANIFESTATIONS OF ADRENAL INSUFFICIENCY

The clinical manifestations of adrenal insufficiency depend upon the extent of loss of adrenal function and whether mineralocorticoid production is preserved. The onset of adrenal insufficiency is often gradual and may go undetected until an illness or other stress precipitates an adrenal crisis (50, 52).

 

Adrenal Crisis: Adrenal crisis or acute adrenal insufficiency may complicate the course of chronic primary adrenal insufficiency, and may be precipitated by a serious infection, acute stress, bilateral adrenal infarction or hemorrhage. It is rare in patients with secondary or tertiary adrenal insufficiency. The main clinical manifestation of adrenal crisis is shock, but patients may also have nonspecific symptoms, such as anorexia, nausea, vomiting, abdominal pain, weakness, fatigue, lethargy, confusion or coma. Hypoglycemia is rare in acute adrenal insufficiency, but more common in secondary adrenal insufficiency.

 

The major factor precipitating an adrenal crisis is mineralocorticoid deficiency and the main clinical problem is hypotension. Adrenal crisis can occur in patients receiving appropriate doses of glucocorticoid if their mineralocorticoid requirements are not met (53), whereas patients with secondary adrenal insufficiency and normal aldosterone secretion rarely present in adrenal crisis. However, glucocorticoid deficiency may also contribute to hypotension by decreasing vascular responsiveness to angiotensin II, norepinephrine and other vasoconstrictive hormones, reducing the synthesis of renin substrate, and increasing the production and effects of prostacyclin and other vasodilatory hormones (54, 55).

 

Chronic Primary Adrenal Insufficiency: The clinical manifestations of chronic primary adrenal insufficiency are owing to deficient concentrations of all adrenocortical hormones (mineralocorticoids, glucocorticoids, adrenal androgens) and include general malaise, fatigue, weakness, anorexia, weight loss, nausea, vomiting, abdominal pain or diarrhea, which may alternate with constipation, hypotension, electrolyte abnormalities (hyponatremia, hyperkalemia, metabolic acidosis), hyperpigmentation, autoimmune manifestations (vitiligo), decreased axillary and pubic hair, and loss of libido and amenorrhea in women (50, 52). The onset of chronic adrenal insufficiency is often insidious and the diagnosis may be difficult in the early stages of the disease.

 

Secondary or Tertiary Adrenal Insufficiency: The clinical features of secondary or tertiary adrenal insufficiency are similar to those of primary adrenal insufficiency. However, hyperpigmentation of the skin does not occur, because the secretion of ACTH is not increased. Also, since the production of mineralocorticoids by the zona glomerulosa is mostly preserved, dehydration and hyperkalemia are not present, and hypotension is less prominent. Hyponatremia and increased intravascular volume may be the result of “inappropriate” increase in vasopressin secretion. Hypoglycemia is more common in secondary adrenal insufficiency possibly due to concomitant growth hormone insufficiency and in isolated ACTH deficiency. Clinical manifestations of a pituitary or hypothalamic tumor, such as symptoms and signs of deficiency of other anterior pituitary hormones, headache or visual field defects, may also be present (50, 52).

 

DIAGNOSIS OF ADRENAL INSUFFICIENCY

The clinical diagnosis of adrenal insufficiency can be confirmed by demonstrating inappropriately low cortisol secretion, determining whether the cortisol deficiency is secondary or primary and, hence, dependent or independent of ACTH deficiency, and detecting the cause of the disorder (50, 52).

 

Basal morning serum cortisol concentrations: The diagnosis of adrenal insufficiency depends upon the demonstration of inappropriately low cortisol secretion. Serum cortisol concentrations are normally highest in the early morning hours (06:00h – 08:00h), ranging between 10 – 20 mcg/dL (275 – 555 nmol/L) than at other times of the day. Serum cortisol concentrations determined at 08:00h of less than 3 µg/dL (80 nmol/L) are strongly suggestive of adrenal insufficiency (56), while values below 10 µg/dL (275 nmol/L) make the diagnosis likely. Simultaneous measurements of cortisol and ACTH concentrations confirm in most cases the diagnosis of primary adrenal insufficiency.

 

Morning salivary cortisol concentrations: Adrenal insufficiency is excluded when salivary cortisol concentration at 08:00h is higher than 5.8 ng/mL (16 nmol/L), whereas the diagnosis is more possible for values lower than 1.8 ng/mL (5 nmol/L).

 

Urinary free Cortisol (UFC): Basal urinary cortisol and 17-hydroxycorticosteroid excretion is low in patients with severe adrenal insufficiency, but may be low-normal in patients with partial adrenal insufficiency. Generally, baseline urinary measurements are not recommended for the diagnosis of adrenal insufficiency.

 

Basal plasma ACTH, renin and aldosterone concentrations: Basal plasma ACTH concentration at 08:00h, when determined simultaneously with the measurement of basal serum cortisol concentration, may both confirm the diagnosis of adrenal insufficiency and establish the cause (57). The normal values of basal 08:00h plasma ACTH concentrations range between 20-52 pg/mL (4.5-12 pmol/L). In primary adrenal insufficiency, the 08:00h plasma ACTH concentration is elevated, and is coupled with increased concentration or activity of plasma renin, low aldosterone concentrations, hyperkalemia and hyponatremia. In the cases of secondary or tertiary adrenal insufficiency, plasma ACTH concentrations are low or low normal, associated with normal values of plasma concentrations of renin and aldosterone.

           

Standard dose ACTH stimulation test: Adrenal insufficiency is usually diagnosed by the standard-dose ACTH test, which determines the ability of the adrenal glands to respond to 250 mcg intravenous or intramuscular administration of ACTH(1-24) by measurement of serum cortisol concentrations at 0, 30 and 60 min following stimulation. The test is defined as normal if peak cortisol concentration is higher than 18–20 mcg/dL (500–550 nmol/L), thereby excluding the diagnosis of primary adrenal insufficiency and almost all cases of secondary adrenal insufficiency. However, if secondary adrenal insufficiency is of recent onset, the adrenal glands will have not yet atrophied, and will still be capable of responding to ACTH stimulation normally. In these cases, a low-dose ACTH stimulation test or an insulin-induced hypoglycemia test may be required to confirm the diagnosis (58-60).

 

Low-Dose ACTH stimulation test: This test theoretically provides a more sensitive index of adrenocortical responsiveness because it results in physiologic plasma ACTH concentrations. This test should be performed at 14:00h, when the endogenous secretion of ACTH is at its lowest. The results might not be valid if it is performed at another time. At 14:00h, a blood sample is collected for determination of basal cortisol concentrations. The low dose of ACTH(1-24) (500 nanograms ACTH(1-24)/1.73 m2  ) is then administered as an intravenous bolus. In normal subjects, this dose results in a peak plasma ACTH concentration about twice that of insulin-induced hypoglycemia (60). Subsequently, blood samples are collected at +10 min, +15 min, +20 min, +25 min, +30 min, +35 min, +40 min and +45 min after stimulation for determination of serum cortisol concentrations (51). A value of 18 µg/dL (500 nmol/L) or more at any time during the test is indicative of normal adrenal function. The advantage of this test is that it can detect partial adrenal insufficiency that may be missed by the standard-dose test (58-62). The low-dose test is also preferred in patients with secondary or tertiary adrenal insufficiency (63-66).

 

Prolonged ACTH Stimulation Tests: Prolonged stimulation with exogenous administration of ACTH helps differentiate between primary and secondary or tertiary adrenal insufficiency. In secondary or tertiary adrenal insufficiency, the adrenal glands display cortisol secretory capacity following prolonged stimulation with ACTH, whereas in primary adrenal insufficiency, the adrenal glands are partially or completely destroyed and do not respond to ACTH. The prolonged ACTH test consists of the intravenous administration of 250 μg of ACTH as an infusion over eight hours (8-hour test) or over 24 hours on two (or three) consecutive days (two-day test), and the measurement of serum cortisol, and 24-hour urinary cortisol and 17-hydroxycorticoid (17-OHCS) concentrations before and after the infusion (67).

 

Insulin-induced hypoglycemia test: This test provides an alternative choice for confirmation of the diagnosis when secondary adrenal insufficiency is suspected. The insulin tolerance test helps in the investigation of the integrity of the HPA axis and has the ability to assess growth hormone reserve. Insulin, at a dose of 0.1-0.15 U/kg, is administered to induce hypoglycemia, and measurements of cortisol concentrations are determined at 30 min intervals for at least 120 min (68, 69). This test is contraindicated in patients with cardiovascular disease or a history of seizures, and requires a high degree of supervision.

 

Corticotropin-releasing hormone (CRH) test: This test is used to differentiate between secondary and tertiary adrenal insufficiency. It consists of intravenous administration of CRH (1 mcg/kg up to a maximum of 100 mcg) and determination of serum cortisol and plasma ACTH concentrations at 0, 15, 30, 45, 60, 90 and 120 min following stimulation. Patients with secondary adrenal insufficiency demonstrate little or no ACTH response, whereas patients with tertiary adrenal insufficiency show an exaggerated and prolonged response of ACTH to CRH stimulation, which is not followed by an appropriate cortisol response (70, 71).

 

Autoantibody screen: Adrenocortical antibodies or antibodies against 21-hydroxylase can be detected in more than 90% of patients with recent onset autoimmune adrenalitis. Furthermore, antibodies that react against other enzymes involved in the steroidogenesis (P450scc, P450c17) and anti-steroid-producing cell antibodies are present in some patients (1, 3, 22-26, 33, 72-74).

           

Very long chain fatty acids: To exclude adrenoleukodystrophy, plasma very long chain fatty acids should be determined in male patients with isolated Addison’s disease and negative autoantibodies (34).

 

Imaging: Patients without any associated autoimmune disease and negative autoantibody screen should undergo a computed tomography (CT) scan of the adrenal glands. In cases of tuberculous adrenalitis, the CT scan shows initially hyperplasia of the adrenal glands and subsequently spotty calcifications during the late stages of the disease. Bilateral adrenal lymphoma, adrenal metastases or adrenal infiltration (sarcoidosis, amyloidosis, hemochromatosis) may also be detected by CT scan. If central adrenal insufficiency is suspected, a magnetic resonance imaging (MRI) scan of the hypothalamus and pituitary gland should be performed. This may detect any potential disease process, such as craniopharyngiomas, pituitary adenomas, meningiomas, metastases and infiltration by Langerhans cell histiocytosis, sarcoidosis or other granulomatous diseases (75, 76). It should be noted that imaging is not required when adrenal cortex autoantibodies are detected.

 

TREATMENT OF ADRENAL INSUFFICIENCY

Adrenal insufficiency is one of the most life-threatening disorders. Treatment should be administered to the patients as soon as the diagnosis is established, or even sooner if an adrenal crisis occurs (77, 78).

 

Treatment of Chronic Adrenal Insufficiency: One of the most important aspects of the management of chronic primary adrenal insufficiency is patient and family education. Patients should understand the reason for life-long replacement therapy, the need to increase the dose of glucocorticoid during minor or major stress and to inject hydrocortisone, methylprednisolone or dexamethasone in emergencies.

Emergency precautions: Patients should wear a medical alert (Medic Alert) bracelet or necklace and carry the Emergency Medical Information Card, which should provide information on the diagnosis, the medications and daily doses, and the physician involved in the patient’s management. Patients should also have supplies of dexamethasone sodium phosphate and should be educated about how and when to administer them.

Glucocorticoid replacement therapy: Patients with adrenal insufficiency should be treated with hydrocortisone, the natural glucocorticoid, or cortisone acetate if hydrocortisone is not available. The hydrocortisone daily dose is 10-12 mg per meter square body surface area and can be administered in two to three divided doses with one half to two thirds of the total daily dose being given in the morning (1-5, 77, 79-85). Small reductions of bone mineral density (BMD) probably due to higher than recommended doses (86), as well as impaired quality of life (87, 88) were observed in patients treated with hydrocortisone. A longer-acting synthetic glucocorticoid, such as prednisone, prednisolone or dexamethasone, should be avoided because their longer duration of action may produce manifestations of chronic glucocorticoid excess, such as loss of lean body mass and bone density, and gain of visceral fat (89). Recently, preparations of hydrocortisone that lead to both delayed and sustained release of this compound have been developed and are under clinical investigation (90, 91). These formulations maintain stable cortisol concentrations during 24 hours and physiologic circadian rhythmicity with the cortisol peak occurring during the early morning after oral intake of the preparation at bed-time. Furthermore, a novel once-daily (OD) dual-release hydrocortisone tablet has been developed to maintain more physiologic circadian-based serum cortisol concentrations. Compared to the conventional treatment, the OD dual-release hydrocortisone improved glucose metabolism, cardiovascular risk factors and quality of life (92). Regardless of the type of the formulation used, glucocorticoid replacement should be monitored clinically, evaluating weight gain/loss, arterial blood pressure, annualized growth velocity and presence of Cushing features (93).

Glucocorticoid replacement during minor illness or surgery: During minor illness or surgical procedures, glucocorticoids should be given at a dosage up to three times the usual maintenance dosage for up to three days. Depending on the nature and severity of the illness, additional treatment may be required.

Glucocorticoid replacement during major illness or surgery: During major illness or surgery, high doses of glucocorticoid analogues (10 times the daily production rate) are required to avoid an adrenal crisis. A continuous infusion of 10 mg of hydrocortisone per hour or the equivalent amount of dexamethasone or prednisolone eliminates the possibility of glucocorticoid deficiency. This dose can be halved the second postoperative day, and the maintenance dose can be resumed at the third postoperative day.

Mineralocorticoid replacement therapy: Mineralocorticoid replacement therapy is required to prevent intravascular volume depletion, hyponatremia and hyperkalemia. For these purposes, fludrocortisone (9-alpha-fluorohydrocortisone) in a dose of 0.05 - 0.2 mg daily should be taken in the morning. The dose of fludrocortisone is titrated individually based on the findings of clinical examination (mainly the body weight and arterial blood pressure) and the levels of plasma renin activity. Patients receiving prednisone or dexamethasone may require higher doses of fludrocortisone to lower their plasma renin activity to the upper normal range, while patients receiving hydrocortisone, which has some mineralocorticoid activity, may require lower doses. The mineralocorticoid dose may have to be increased in the summer, particularly if patients are exposed to temperatures above 29ºC (85ºF) (77, 79-85). If patients receiving mineralocorticoid replacement develop hypertension, the dose of fludrocortisone should be reduced accordingly (93). In case of uncontrolled blood pressure, patients should be encouraged to continue fludrocortisone and initiate antihypertensive therapy, such as angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, or dihydropyridine calcium blockers (93, 94).

Androgen replacement: In women, the adrenal cortex is the primary source of androgen in the form of dehydroepiandrosterone and dehydroepiandrosterone sulfate. Treatment with DHEA enhances mood and general well being both in adult patients and in children and adolescents with adrenal insufficiency (80-85, 87, 95-101). A single oral morning dose of DHEA of 25-50 mg may be sufficient to maintain normal serum androgen concentrations in premenopausal women with primary adrenal insufficiency, who present with decreased libido, anxiety, depression, and low energy levels (93). If symptoms are still present during a period of 6 months, patients are advised to discontinue DHEA replacement (93). Naturally, women should be encouraged to report any side effects of androgen therapy. Finally, DHEA replacement should be monitored by determining serum DHEA concentrations in the morning before patient receives her daily DHEA dose (93).

 

Treatment of adrenal crisis: The aim of initial management in adrenal crisis is to treat hypotension, hyponatremia and hyperkalemia, and to reverse glucocorticoid deficiency. Treatment should be started with immediate administration of 100 mg hydrocortisone i.v. and rapid rehydration with normal saline infusion under continuous cardiac monitoring, followed by 100–200 mg hydrocortisone in glucose 5% per 24-hour continuous iv infusion; alternatively, hydrocortisone could be administered iv or im at a dose of 50-100 mg every 6 hours depending on body surface area and age (80). With daily hydrocortisone doses of 50 mg or more, mineralocorticoids in patients with primary adrenal insufficiency can be discontinued or reduced because this dose is equivalent to 0.1 mg fludrocortisone (79). Once the patient’s condition is stable and the diagnosis has been confirmed, parenteral glucocorticoid therapy should be tapered over 3-4 days and converted to an oral maintenance dose (1-3, 77, 79-85). Patients with primary adrenal insufficiency require life-long glucocorticoid and mineralocorticoid replacement therapy.

           

Treatment of chronic secondary and tertiary adrenal insufficiency: In chronic secondary or tertiary adrenal insufficiency, glucocorticoid replacement is similar to that in primary adrenal insufficiency, however, measurement of plasma ACTH concentrations cannot be used to titrate the optimal glucocorticoid dose. Mineralocorticoid replacement is rarely required, while replacement of other anterior pituitary deficits might be necessary.

 

ADRENAL INSUFFICIENCY IN CRITICALLY ILL PATIENTS

Clinical manifestations of adrenal insufficiency are common in critically ill patients, specifically in patients with severe pneumonia, adult respiratory distress syndrome, sepsis, trauma, HIV infection or after treatment with etomidate (2, 102-106).

 

The molecular pathogenetic mechanisms underlying adrenal insufficiency in critical illness have not been fully elucidated. However, it seems that both inadequate cortisol secretion and impaired glucocorticoid receptor signaling are convincingly involved. Indeed, proinflammatory cytokines may compete with ACTH on its receptor (107) and/or induce tissue resistance to glucocorticoids (108-110). Moreover, the widely used medications during the treatment of sepsis may impair both glucocorticoid production and glucocorticoid signaling. Furthermore, other neuropeptides, signaling molecules, components of oxidative stress and the impaired adrenal blood flow contribute to adrenal insufficiency.

 

To provide recommendations on the diagnosis and management of adrenal insufficiency in critically ill patients, the American College of Critical Care Medicine suggested that the diagnosis is best made by a delta total serum cortisol of < 9 mcg/dL following ACTH (250 microg) administration or a random total cortisol of < 10 mcg/dL. Hydrocortisone at a dose of 200 mg/day in four divided doses or as a continuous infusion at a dose of 240 mg/day (10 mg/hr) for at least 7 days is recommended for patients with septic shock. Methylprednisolone at a dose of 1 mg/kg/day for at least 14 days is recommended in patients diagnosed with severe early acute respiratory distress syndrome. The role of glucocorticoid therapy in other critically ill patients remains to be further elucidated (111).

 

ADRENAL INSUFFICIENCY DURING PREGNANCY

Although adrenal insufficiency is relatively rare in pregnancy, it may be associated with significant maternal and/or fetal morbidity and mortality if it remains undiagnosed or untreated (112, 113). Symptoms are usually “nonspecific”, such as nausea, vomiting and fatigue, making the diagnosis of adrenal insufficiency challenging. The current diagnostic tests are serum cortisol concentrations and the cosyntropin stimulation test (93, 113). However, it should be emphasized that the peak cortisol response following ACTH stimulation is higher in pregnant than in non-pregnant women during the second and third trimesters, as a result of physiologic pregnancy-associated hypercortisolism and elevations of cortisol-binding globulin (114). Regarding glucocorticoid replacement during pregnancy, hydrocortisone, cortisone acetate, prednisolone or prednisone can be administered; in contrast,, fluorinated glucocorticoids such as dexamethasone should be avoided because they cross the placenta at higher rates (93). Mineralocorticoid replacement is usually more complicated to assess during pregnancy because of the “nonspecific” symptoms often observed in physiologic pregnancy (93). A hydrocortisone stress dose (bolus intravascular injection of 50-100 mg hydrocortisone followed by continuous infusion of 100-200 mg hydrocortisone/24h) should be administered at the beginning of active labor (93, 115, 116).

 

ADRENAL INSUFFICIENCY IN INFANCY AND CHILDHOOD

Children with primary adrenal insufficiency should be treated with hydrocortisone phosphate at a daily dose of 10-12 mg per meter square body surface area divided into two or three doses (93). Alternatively, cortisone acetate can be administered with safety also as two to three daily doses. Intermediate-acting or long-acting glucocorticoid analogues, such as prednisolone/prednisolone or dexamethasone respectively, are not recommended due to undesirable chronic side effects, such as glucose intolerance or osteopenia/osteoporosis. The hydrocortisone daily dose should be adjusted according to the increasing body surface area of the child. Caution should be paid to decreased growth velocity, excessive weight gain or other clinical manifestations suggestive of iatrogenic Cushing syndrome. Children with primary adrenal insufficiency also require fludrocortisone at a daily dose of 50-300 μg (93). During the first 6 months, infants require supplementation of sodium chloride at a dose of 1-2 g/day administered in multiple feedings, because the infant kidney is physiologically resistant to mineralocorticoids and the infant milk (breast milk or formula) has relatively low sodium content (93, 117).

 

 

 

 

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TSH Receptor Mutations and Diseases

ABSTRACT

The thyroid-stimulating hormone (TSH) receptor (TSHR) is a member of the glycoprotein hormone receptors (GPHRs), a sub-group of class A G protein-coupled receptors (GPCRs). TSHR and its ligand thyrotropin are of essential importance for growth and function of the thyroid gland. The TSHR activates different G-protein subtypes and signaling pathways, of which Gs and Gq induced signaling are of highest importance in the thyroid gland. A proper interplay between TSH and TSHR is pivotal for thyroid growth and regulated production and release of thyroid hormones (TH). Autoimmune (antibody binding) or non-autoimmune (occurrence of mutants) TSHR dysfunctions are the underlying cause of several pathologies, including rare cancer-development. The sequential processes of TSHR binding, signal transduction across the cell-membrane and activation of intracellular effectors involve elaborate specific structural properties of the receptor and several interacting proteins. In consequence, different pathogenic mutations at TSHR or TSH may have diverse impact on particular molecular functions, but finally result in either hypo- or hyperthyroid states accompanied or not by various growth anomalies. We here summarize current knowledge regarding naturally occurring TSHR mutations, associated diseases and related molecular pathogenic mechanisms at the level of TSHR structure and function. For complete coverage of this and related areas in Endocrinology, visit our free web-books, www.endotext.org and www.thyroidmanager.org

 

 

GAIN-OF-FUNCTION MUTATIONS

On a theoretical basis, for a hormone receptor, “gain-of-function” may have several meanings: (i) activation in the absence of ligand (constitutively); (ii) increased sensitivity to its normal agonist; (iii) increased, or de novo sensitivity to an allosteric modulator; (iv) broadening of its specificity. When the receptor is part of a chemostat, as is the case for the TSHR, the first situation is expected to cause tissue autonomy, whereas the second would simply cause adjustment of TSH to a lower value. In the third and fourth cases, inappropriate stimulation of the target will occur because the illegitimate agonists or modulators are not expected to be subject to the normal negative feedback. If a gain-of-function mutation of the first category occurs in a single cell normally expressing the receptor (somatic mutation), it will become symptomatic only if the regulatory cascade controlled by the receptor is mitogenic in this particular cell type or, during development, if the mutation affects a progenitor contributing significantly to the final organ. Autonomous activity of the receptor will cause clonal expansion of the mutated cell. If the regulatory cascade also positively controls function, the resulting tumor may progressively take over the function of the normal tissue and ultimately result in autonomous hyperfunction. If the mutation is present in all cells of an organism (germline mutation) autonomy will be displayed by the whole organ.

From what we know of thyroid cell physiology it is easy to predict the phenotypes associated with gain-of-function of the cAMP-dependent regulatory cascade. Two observations provide pertinent models of this situation. Transgenic mice made to express ectopically the adenosine A2a receptor in their thyroid display severe hyperthyroidism associated with thyroid hyperplasia (1). As the A2a adenosine receptor is coupled to Gs and displays constitutive activity due to its continuous stimulation by ambient adenosine (2), this model mimics closely the situation expected for a gain-of-function germline mutation of the TSHR. Patients with the McCune-Albright syndrome are mosaïc for mutations in the Gs protein (Gsp mutations) leading to the constitutive stimulation of adenylyl-cyclase (3). Hyperfunctioning thyroid adenomas develop in these patients from cells harboring the mutation, making them a model for gain-of-function somatic mutations of the TSHR. A transgenic model in which Gsp mutations are targeted for expression in the mouse thyroid has been constructed. Though with a less dramatic phenotype this represents also a pertinent model for a gain-of-function of the cAMP regulatory cascade (4).

Since the TSHR is capable of activating both Gs and Gq (though with lower potency) the question arises whether mutations with a different effect on the two cascades would be associated with different phenotypes. Studies in mice (5) and rare patients (6) suggests that activation of Gq may be required to observe goitrogenesis in patients with non-autoimmune familial hyperthyroidism. However, when tested in transfected non-thyroid cells, all identified gain-of-function mutations of the TSHR stimulate constitutively Gs, with only a minority capable of stimulating both Gs and Gq (7,8). Also, thyroid adenomas or multinodular goitre are frequent in McCune Albright syndrome, which is characterized by pure Gs stimulation (9).

Familial non-autoimmune hyperthyroidism or hereditary toxic thyroid hyperplasia

 

The major cause of hyperthyroidism in adults is Graves' disease in which an autoimmune reaction is mounted against the thyroid gland and auto-antibodies are produced that recognize and stimulate the TSHR (10,11). This may explain why the initial description by the group of Leclère of a family showing segregation of thyrotoxicosis as an autosomal dominant trait in the absence of signs of autoimmunity was met with skepticism (12). Re-investigation of this family together with another family from Reims identified two mutations of the TSHR gene, which segregated in perfect linkage with the disease (13). A series of additional families have been studied since (14-28) [Figure 1 and Table 1, For a completed list of naturally occurring TSHR single amino acid substitutions with their functional characteristics, see the glycoprotein hormone receptor information resource “SSFA” (29,30) available under: http://www.ssfa-gphr.de]. The functional characteristics of these mutant receptors confirm that they are constitutively stimulated (see below). This nosological entity, hereditary toxic thyroid hyperplasia (HTTH), sometimes called Leclère’s disease, is characterized by the following clinical characteristics: autosomal dominant transmission; hyperthyroidism with a variable age of onset (from infancy to adulthood, even within a given family); hyperplastic goiter of variable size, but with a steady growth; absence of clinical or biological stigmata of auto-immunity. An observation common to most cases is the need for drastic ablative therapy (surgery or radioiodine) in order to control the disease, once the patient has become hyperthyroid (13,31). The autonomous nature of the thyroid tissue from these patients has been elegantly demonstrated by grafting in nude mice (32). Contrary to tissue from Graves' disease patients, HTTH cells continue to grow in the absence of stimulation by TSH or TSAb.

The prevalence of hereditary toxic thyroid hyperplasia is difficult to estimate. It is likely that many cases are still mistaken for Graves' disease. This may be explained by the high frequency of thyroid auto-antibodies (anti-thyroglobulin, anti-thyroperoxidase) in the general population. It is expected that wider knowledge of the existence of the disease will lead to better diagnosis. This is not a purely academic problem, since presymptomatic diagnosis in children of affected families may prevent the developmental or psychological complications associated with infantile or juvenile hyperthyroidism (for a review, see (33)). A country-wide screening for the condition has been performed in Denmark. It was found in one out of 121 patients with juvenile thyrotoxicosis (0.8%; 95% CI: 0.02-4.6%), which corresponds to one in 17 patients with presumed non-autoimmune juvenile thyrotoxicosis (6%; 95% CI:0.15-28.69) (34).

 Sporadic toxic thyroid hyperplasia

Cases with toxic thyroid hyperplasia have been described in children born from unaffected parents (35-39). Conspicuously, congenital hyperthyroidism was present in most of the cases and required aggressive treatment. Mutations of one TSHR allele were identified in the children, but were absent in the parents. As paternity was confirmed by mini- or microsatellite testing, these cases qualify as true neo-mutations. When comparing the amino acid substitutions implicated in hereditary and sporadic cases, for the majority, they do not overlap (see Table 1). Whereas most of the sporadic cases harbor mutations that are also found in toxic adenomas, most of the hereditary cases have "private" mutations. Although there may be exceptions, the analysis of the functional characteristics of the individual mutant receptors in COS cells, and the clinical course of individual patients, suggest an explanation for this observation: "sporadic" and somatic mutations seem to have a stronger activating effect than "hereditary" mutations (40). From their severe phenotypes, it is likely that newborns with neo-mutations would not have survived, if not treated efficiently. On the contrary, from inspection of the available pedigrees, it seems that the milder phenotype of patients with “hereditary" mutations has only limited effect on reproductive fitness. The fact that "hereditary" mutations are rarely observed in toxic adenomas is compatible with the suggestion that they would cause extremely slow tissue growth and, accordingly, would rarely cause thyrotoxicosis if somatic. There is, however, no a priori reason for neo-mutations to cause stronger gain-of-function than hereditary mutations. Accordingly, an activating mutation of the TSHR gene has been found in a six month child with subclinical hyperthyroidism presenting with weight loss as the initial symptom (41).

 

 Somatic mutations: autonomous toxic adenomas

 

  Soon after mutations of Gαs had been found in adenomas of the pituitary somatotrophs (42), similar mutations (also called Gsp mutations) were found in some toxic thyroid adenomas and follicular carcinomas (43-46). The mutated residues (Arg201, Glu227) are homologous to those found mutated in the ras proto-oncogenes: i.e. the mutations decrease the endogenous GTPase activity of the G protein, resulting in a constitutively active molecule. Toxic adenomas were found to be a fruitful source of somatic mutations activating the TSHR, probably because the phenotype is very conspicuous and easy to diagnose (47). Whereas mutations are distributed all along the serpentine portion of the receptor and even in the extracellular amino-terminal domain (48-54), there is clearly a hotspot at the cytoplasmic end of the sixth transmembrane segment (see Figure 1). The clustering reflects the pivotal role of this portion in the activation mechanism observed in the TSHR and in class A GPCRs generally [e.g. (55-61)].

Despite some dispute about the prevalence of TSHR mutations in toxic adenomas (which may be due to different origin of patients (62,63) or different sensitivity of the methodology) the current consensus is that activating mutations of the TSHR are the major cause of solitary toxic adenomas and account for about 60 to 80% of cases (15,7,64-66). Contrary to initial suggestions (63), the same percentage of activating TSHR mutations is observed in Japan, an iodine-sufficient country with low prevalence of toxic adenomas (67). In some patients with a multinodular goiter and two zones of autonomy at scintigraphy, a different mutation of the TSHR was identified in each nodule (68,36,69,70). This indicates that the pathophysiological mechanism responsible for solitary toxic adenomas can be at work on a background of multinodular goiter.

Table 1

 

CODONS Substitution

Somatic

mutation

Germline

neo- mutation

Germline

familial

Cancer

Stimulation

of basal cAMP

Stimulation of IP/Ca
Ser 281 Asn +       + -
    Thr +       + -
    Ile   +     + -
Asp 403 deletion +       + nd
Asn 406 Ser     +   + nd
Ser 425 Ile +       + -
Ala 428 Val   +     nd nd
Gly 431 Ser     +   + +
Met 453 Thr + +   + + -
Met 463 Val     +   + -
Ala 485 Val     +   + -
Ile 486 Phe +       + +
    Met +       + ±
    Asn   +     + -
Ser 505 Arg     +   + -
    Asn   +     + -
Val 509 Ala     +   + -
Leu 512 Arg +       + nd
    Gln +       + nd
Ile 568 Thr + +     + ±
    Phe +       + ±
Glu 575 Lys     +   + nd
Ala 593 Asn +       + nd
Val 597 Leu   +     + nd
    Phe         + nd
Y613-F631 deletion           -
Tyr 601 Asn +       + -
Asp 617 Tyr     +   + ±
Asp 619 Gly +       + -
Thr 620 Ile +     + + nd
Ala 623 Ile +       + ±
    Val +   +   + -
    Ser +     + + -
    Phe +       + nd
Met 626 Ile     +   + nd
Ala 627 Val +       + nd
Leu 629 Phe +   +   + -
Ile 630 Leu +       + -
Phe 631 Leu +       + -
    Cys + +     + -
    Ile       + + -
Thr 632 Ile + +     + -
Thr 632 Ala +     + + nd
Asp 633 Tyr +     + + -
    Glu +       + -
    His +     + + -
    Ala +       + -
Ile 635 Val +       + nd
Cys 636 Trp     +   + -
    Arg   +     + ±
Pro 639 Ser +   +   + +
Ile 640 Lys +       + nd
Asn 650 Tyr     +   + -
Val 656 Phe +       + -
Del 658-661   +         -
Asn 670 Ser     +   + -
Cys 672 Thr     +   + -
Leu 677 Val       + + nd

 

Table 1: Gain-of-function TSHR mutations. The nature of the mutations is indicated with their origins (somatic, germline sporadic, germline familial, cancer) and effects on intracellular regulatory cascades. nd - not determined; “-“ decreased functional property; “+” enhanced; “+/-“ similar to wild type.

 

In agreement with this notion, activating mutations of the TSHR have been identified in hyperfunctioning areas of multinodular goiter (70,19,65,23). The independent occurrence of two activating mutations in a patient may seem highly improbable at first. However, the multiplicity of the possible targets for activating mutations within the TSHR makes this less unlikely. It is also possible that a mutagenic environment is created in glands exposed to a chronic stimulation by TSH, resulting in H2O2 generation (71,72). Finally the involvement of TSHR mutations in thyroid cancers has been implicated in a limited number of follicular thyroid carcinoma (73-81).

 

Figure 1

Legend to figure 1: Structural model of the TSHR with interacting proteins and highlighted positions for gain-of-function mutations. Left: The model shows different parts of the receptor for which homologous structural information is available. The leucine-rich repeat domain (LRRD) and the hinge region are both harboring determinants for hormone (TSH model (surface) based on the FSH structure (82)) and antibody binding. The hinge region (colored pink) structurally links the LRRD with the serpentine domain made of transmembrane helices (H) 1-7 connected by intracellular (I1-3) and extracellular (E1-3) loops. Three cysteine bridges (yellow spheres) between the C-terminal LRRD and the C-terminus of the hinge region are indicated that are required for correct receptor arrangement and function. Wild type positions of constitutively activating mutations are indicated by side-chain representation (red sticks). Right: The known activating mutations (Table 1) are distributed over the entire serpentine portion of the receptor structure with clustering in the central core and specifically in helix 6. In contrast to other glycoprotein-hormone receptors (GPHRs), naturally occurring activating mutations were also identified in the extracellular loops and in the hinge region (Ser281).

 

Structure-function relationships of the TSHR

An important observation has been that the wild-type TSHR itself displays significant constitutive activity [(83,47) and review (84)]. This characteristic is not exceptional amongst GPCRs (e.g. (85)), but interestingly, it is not shared, at least to the same level, by its close relatives, the human luteinizing hormone/chorionic gonadotropin (LH/CG) receptor (LHCGR) and the human follitropin (FSH) receptor (FSHR). Compared to the TSHR, the LHCGR displays minimal basal activity and the human FSH receptor is totally silent (86). Together with the observation that mutations in residues distributed over most of the serpentine portion of the TSHR are equally effective in activating it (which does not seem to be a general characteristic in all GPCRs) this suggests that the unliganded TSHR might be less constrained than other GPCRs. As a consequence, being already “noisy” it would be more prone to further destabilization by a wide variety of mutations affecting multiple structural elements (Figure 1).

The effect of activating mutations must accordingly be interpreted in terms of “increase in constitutive activity”. Most constitutively active mutant receptors (also referred to as “CAMs”) found in toxic adenomas and/or toxic thyroid hyperplasia share common characteristics: i) they increase the constitutive activity of the receptor toward stimulation of adenylyl cyclase; ii) with a few notable exceptions (see Table 1 and below) (48), they do not display constitutive activity toward the inositol phosphate/diacylglycerol pathway; iii) their expression at the cell surface is decreased (from slightly to severely); iv) most, but not all of them keep responding to TSH for stimulation of cAMP and inositol phosphate generation, with a tendency to do so at decreased median effective concentrations; and v) they bind 125I-labeled bovine TSH with an apparent affinity higher than that of the wild-type receptor. Of note, CAMs with mutations at Ser281 (to Ile) (37) in the extracellular N-terminal part, at Ile486 (to Phe or Met) (48) and Ile568 (to Thr) (48) in the first and second extracellular loops, respectively, and at both Asp633 (to His) (7) and Pro639 (to Ser) (69) in transmembrane helix 6 are exceptional in that in addition to stimulating adenylyl cyclase, they cause constitutive activation of the inositol phosphate pathway. The constitutive activity of these mutants is interesting as it points to positions and structural fragments of the wild type receptor which may be of high relevance for its physiological coupling to both Gs and Gq (Figure 1 and Table 1).

No direct relationship is found between the level of cAMP achieved by different mutants in transfected COS cells and their level of expression at the cell membrane (87), which means that individual mutants have widely different “specific constitutive activity” (measured as the stimulation of cAMP accumulation/receptor number at the cell surface). Although this specific activity may tell us something about the mechanisms of receptor activation, it is not a measure of the actual phenotypic effect of the mutation in vivo. Indeed, one of the relatively mild mutations, observed up to now only in a family with HTTH (Cys672Tyr) (13), is among the strongest according to this criterion.

Differences between the effects of the mutants in transfected COS or HEK293 cells and thyrocytes in vivo render these correlations a difficult exercise. Indeed, most of the activating mutations of the TSHR have been studied by transient expression in COS or HEK293T cells and there is no guarantee that the mutants will function in an identical way in these artificial systems as they do in thyrocytes (88). In thyrocytes, a better relation has been observed between adenylylcyclase stimulation and differentiation than with growth (88). However, the built-in amplification associated with transfection of constructs in COS or HEK 293T cells has the advantage of allowing detection of even slight increases in constitutive activity of certain TSHR mutants.

 

According to a current model of GPCR activation, the receptor would exist under at least two interconverting conformations: R (silent conformations) and R* (active forms) (89,55,90,91) (Figure 2). The unliganded wild type receptor would shuttle between both forms, the equilibrium being in favor of R (90). Binding of the agonistic ligand is believed to stabilize the R* conformation.

 

Figure 2

Legend to figure 2: Left. Schematic representation of the equilibria between inactive (R) and active (R*) conformations of TSHR. The triangles indicate the equilibrium point of the wild type receptor (pink) and hypothetical mutants with increasing constitutive activity (brown, red). The situation of a receptor which would be devoid of basal activity is also indicated (blue triangle). Note that the wild type receptor (pink) has basal activity. Right. The concentration action curves corresponding to the hypothetical mutants and wild type receptors are indicated with the same color code.

 

The resulting R-to-R* transition was supposed to involve a conformational change that modifies the relative position of transmembrane helices to each other, which in turn would translate into conformational changes in the cytoplasmic crevice between the intracellular loops and transmembrane helices interacting with the hetero-trimeric G-protein. This model is strongly supported by solved crystal structures of active GPCR conformations (59,61) reviewed in (92)]. They reveal that receptor activation and signal transduction is characterized by specific movements of transmembrane helices (TMHs) 5, 6 and 7 leading to modification of their distances relative to each other. Helix 6 is a major player in this process, its cytoplasmic end moves away from that of TMH3 by turning around a pivotal helix-kink. The result is an “opening” of the cytoplasmic crevice of the receptor allowing interaction with the G protein (59). (A more detailed description of the activation mechanics, adapted to a model of the TSHR, is given in the legend to Figure 3.) This essential function of TMH6 for determination of an active state (59,61) might explain, why most activating TSHR mutations were found in this particular helix (Figure 1). This conclusion is in accordance with the early concept that the silent form of GPCRs would be submitted to structural constraints requiring the wild-type primary structure of the helix 6 and the connected third intracellular loop (93,90,91), and explains why these constraints could be released by a wide spectrum of amino acid substitutions in this segment as observed also for the TSHR (60).

 

Figure 3

Legend to figure 3: Model of the TSHR structure in complex with TSH and G-protein and illustration of the putative activation mechanism. The receptor is displayed as a backbone cartoon in complex with the hetero-dimeric hormone and a hetero-trimeric G-protein molecule (surface representations). For the serpentine portion of the receptor, the model is based on the solved structure of the β2-adrenergic/Gs crystal (61). The ectodomain (in complex with TSH) and the hinge region were modeled based on a determined and homologous FSHR-FSH structure (82). A selection of residues affected by known activating mutations are shown as red sticks and identified by their position in the primary structure of the protein (see Figure 1). Their positions tentatively illustrate the “path” followed by the activation signal, from outside the membrane (in the ectodomain) to the cytoplasmic surface of the receptor, via the transmembrane helices. Briefly, the hormone binds to both the Leucine rich repeat domain (LRRD) and the hinge region (e.g. sulfated tyrosine 385 (sTyr)). This initial signal is transduced into a conformational change of a module (intramolecular agonist) constituted by the “hinge” region of the ectodomain and the exoloops (E1-3) of the serpentine domain. In favor of this model, several residues belonging to this module (Ser281 in the ectodomain; Asp403 and Asn406 at the ectodomain-serpentine domain border; Ile486, Ile568, Val656 in the exoloops) can activate the receptor constitutively when mutated. Together with the observation that a truncated receptor devoid of ectodomain displays significant increase in constitutive activity, this suggests that activation of the TSHR involves switching of specific extracellular portions from a tethered inverse agonist (maintenance of the basal state) to an intramolecular agonist (94). The resulting structural changes affecting the exoloops are expected to be directly conveyed to the transmembrane helices with the resulting breakage of silencing locks (arrows). From comparison of the inactive and active rhodopsin (59) or beta-2 adrenoreceptor structures (61), the largest spatial movement affects TMH6, involving a combination of horizontal and rotational (wound arrow) movements around a pivotal helix-kink at a proline (TSHR Pro639). These global changes result in the partial “opening” of the intra-helical crevice on the cytoplasmic side of the receptor (horizontal double-head arrows), allowing complete binding and activation of G-proteins.

 

In addition to the release of structural locks stabilizing the inactive conformation of GPCRs, activation of the GPHRs has been shown to involve a triggering mechanism exerted by an “intramolecular agonist” constituted by segments of the exoloops and the C-terminal portion of their ectodomain (94-96). According to this model, it is this module, activated by the binding of TSH, thyroid stimulating auto-antibodies, thyrostimulin (97,98) or mutations (see below) which would be the immediate agonist of the serpentine portion of the receptor (94,95,99,96,100,101). In all cases, however, mutations are expected to affect the local three dimensional structure of the receptor with a resulting global effect on its activation state. Amongst these are modification of “knob and hole” interactions (e.g. by repulsion) in tightly packed local microdomains and breakage, or creation of intramolecular interactions by changing the biophysical characteristics of side chains (e.g. (6)). As exclusive examples of these, mutations at Asp633 (57,102) or Asp619 (47) are expected to break interhelical locks between transmembrane helices 6 and 7 or 3, respectively. Interestingly, even mutations affecting an important residue of the trigger in the ectodomain (Ser281) seem also to be responsible for a “loss-of-local structure”. Indeed, substitution of the wild type residue (serine) by almost any amino acid results in constitutive activation (103,104). This implicates that predictions of phenotype-genotype relationships must always be considered with much caution if they are not backed by detailed structural and functional knowledge.

 

Familial gestational hyperthyroidism

Some degree of stimulation of the thyroid gland by human chorionic gonadotrophin (hCG) is commonly observed during early pregnancy. It is usually responsible for decrease in serum thyrotropin with an increase in free thyroxine concentrations that remains within the normal range (for references see (105)). When the concentrations of hCG are abnormally high, like in molar pregnancy, true hyperthyroidism may ensue. The pathophysiological mechanism is believed to be the promiscuous stimulation of the TSHR by excess hCG, as suggested by the rough direct or inverse relation between serum hCG and free T4 or TSH concentrations, respectively (106,105). A convincing rationale is provided by the close structural relationships of the glycoprotein hormones and their receptors, respectively (107).

A new syndrome has been described in 1998 in a family with dominant transmission of hyperthyroidism limited to pregnancy (Figure 4) (108). The proposita and her mother had severe thyrotoxicosis together with hyperemesis gravidarum during the course of each of their pregnancies. When non pregnant they were clinically and biologically euthyroid. Both patients were heterozygous for a K183R mutation in the extracellular amino-terminal domain (Figure 3) of the TSHR gene. When tested by transient transfection in COS cells, the mutant receptor displayed normal characteristics towards TSH. However, providing a convincing explanation to the phenotype, it showed higher sensitivity to stimulation by hCG, when compared with wild type TSHR (108).

The amino acid substitution responsible for the promiscuous stimulation of the TSHR by hCG is surprisingly conservative. Also surprising is the observation that residue 183 is a lysine in both the TSH and LH/CG receptors. When placed on the available three dimensional model of the hormone-binding domain of the TSHR (109), residue 183 belongs to one of the beta-sheets which constitute the putative surface of interaction with the hormones (Figure 3).

 

Figure 4

Legend to figure 4: Familial gestational hyperthyroidism secondary to mutation of the TSHR gene. Upper panel displays the pedigree, with the two ladies affected, together with a “snake plot “of the TSHR, with the mutation indicated. Lower right panel illustrates the increased sensitivity of the K183R mutant TSHR vis-à-vis hCG. Binding of TSH, or hCG to the ectodomain of the TSHR, according to models based on crystallographic data from (110,82) is visualized in figure 3.

Detailed analysis of the effect of the K183R mutation by site-directed mutagenesis indicated that any amino acid substitution at this position confers a slight increase in stability to the illegitimate hCG/TSHR complex (111). This increase in stability would be enough to cause signal transduction by the hCG concentrations achieved in pregnancy, but not by the LH concentrations observed after menopause. Indeed, the mother of the proposita remained euthyroid after menopause. This finding is compatible with a relatively modest gain-of-function of the K183R mutant upon stimulation by hCG. A second family with the same phenotype has recently been identified. Interestingly, the mutation affects the same residue (K183N) (112).

 

Contrary to other mammals, human and primates rely on chorionic gonadotropin for maintenance of corpus luteum in early pregnancy (113). The frequent partial suppression of TSH observed at peak hCG levels during normal pregnancy indicates that evolution has selected physiological mechanisms operating very close to the border of thyrotoxicosis. This may provide a rationale to the observation that, in comparison to other species, the glycoprotein hormones of primates display a lower biological activity due to positive selection by evolution of specific amino acid substitutions in their alpha-subunits (114). Up to now no spontaneous mutation has been identified which would increase the bioactivity of hCG. An interesting parallel may be drawn between familial gestational hyperthyroidism and cases of spontaneous ovarian hyperstimulation syndrome (sOHSS) (86,115). In sOHSS, mutations of the FSH receptor gene render the receptor abnormally sensitive to hCG. The result is recurrent hyperstimulation of the ovary, on the occasion of each pregnancy.

 

LOSS OF FUNCTION MUTATIONS

Loss-of-function mutations in the TSHR gene are expected to cause a syndrome of “resistance to TSH.” The expected phenotype is likely to resemble that of patients with mutations in TSH itself. These mutations have been described early because of the prior availability of information on TSH alpha and beta genes (114). Mouse models of resistance to TSH are available as natural (hyt/hyt mouse) (116) or experimental TSHR mutant lines (117,118). Interestingly, and contrary to the situation in human (see below), the thyroid of newborn TSHR knockout mice is of normal size. As expected, the homozygote animals displayed profound hypothyroidism. Their thyroids do not express the sodium-iodide symporter, but showed significant (non-iodinated) thyroglobulin production. From this information one would expect patients with two TSHR mutated alleles to exhibit a degree of hypothyroidism in accordance with the extent of the loss-of-function, going from mild, compensated, hypothyroidism, to profound neonatal hypothyroidism with absent iodide trapping. Heterozygous carriers are expected to be normal or display minimal increase in plasma TSH.

 

Clinical cases with the mutations identified

A few patients with convincing resistance to TSH had been described before molecular genetics permitted identification of the mutations (119,120). The first cases described in molecular terms were euthyroid siblings with elevated TSH (121). Sequencing of the TSHR gene identified a different mutation in each allele of the affected individuals, which made them compound heterozygotes. The substitutions were in the extracellular amino-terminal portion of the receptor (maternal allele, P162A; paternal allele, I167N). The functional characteristics of the mutant receptors showed that the paternal allele was virtually completely non-functional, whereas the maternal allele displayed an increase in the median effective TSH concentration for stimulation of cAMP production. Current knowledge of the structure of part of the ectodomain of the receptor allows to establish structure-function relationships for mutations affecting this portion of the receptor (122,109,123-126,101).

A large number of familial cases with loss-of-function mutations of the TSHR have been identified in the course of screening programs for congenital hypothyroidism (127-140) [(Figure 5, For a complete list of naturally occurring and side-directed TSHR single amino acid substitutions with their functional characteristics see the GPHR information resource “SSFA” available under: http://www.ssfa-gphr.de (29,30)]. Some of the patients displayed the usual criteria for congenital hypothyroidism, including high TSH, low free T4, and undetectable trapping of 99Tc. In some cases, plasma thyroglobulin levels were normal or high. The patients can be compound heterozygotes for complete loss of function mutations (129), or homozygotes, born to consanguineous (128) or apparently unrelated parents (134).

 

Figure 5

Legend to figure 5: Structural model of the TSHR with indication of loss-of-function mutations. The location and substitutions responsible for known loss-of-function mutations (side-chains as blue sticks) are indicated on a three-dimensional receptor model. Single letter abbreviations of amino-acids are used. In contrast to activating mutations, many inactivating mutations are located also in the LRRD. As observable in this complex model inactivating mutations can have different molecular effects on TSHR functions dependent on their localization, like diminishing hormone binding (location in the LRRD, e.g. R109Q), G-protein binding (intracellular localization, e.g. M527T, R531W), leading to a decreased receptor cell surface expression by modification of the three-dimensional structure (e.g. mutations at extracellular cysteines which interrupts stabilizing disulfide bridges, e.g. C390W), or interrupting the signal transport in the serpentine domain (located at the helices, e.g. A593V).

 

In agreement with the phenotype of knock-out mice with homozygous invalidation of the TSHR, patients with complete loss-of-function of the receptor display an in-place, thyroid with completely absent iodide or 99Tc trapping. However, in contrast with the situation in mice, the gland is hypoplastic. Activation of the cAMP pathway, while dispensable for the anatomical development of the gland and thyroglobulin production, is thus absolutely required for expression of the NIS gene and, at least in human, for normal growth of the tissue during fetal life. This explains that in the absence of thyroglobulin measurements or expert echography, loss-of-function mutations of the TSHR may easily be misdiagnosed as thyroid agenesis. In the heterozygous state, complete loss of function mutations of the TSHR is a cause of moderate hyperthyrotropinemia (subclinical hypothyroidism), segregating as an autosomal dominant trait (141).

 

Resistance to thyrotropin not linked to the TSHR gene

Finally, it must be stressed that an autosomal dominant form of partial resistance to TSH has been demonstrated in families in which linkage to the TSHR gene has been excluded (142). A locus has been identified on chromosome 15q25.3-26.1 but the gene responsible for the phenotype has not been identified yet (143).

 

Polymorphisms

A series of single nucleotide polymorphisms affecting the coding sequence have been identified in the TSHR gene. After the initial suggestion that some of these (D36H, P52T, D727E) would be associated with susceptibility to autoimmune thyroid diseases (144-146) the current consensus is that they represent neutral alleles with no pathophysiological significance (147-150). However, a genome-wide study involving a large cohort of patients has recently demonstrated association between non-coding SNPs at the TSHR gene locus and Graves’disease (151,152). The genetic substratum responsible for this association is still under study (152). However, a large meta-analysis of genome wide association studies failed to identify the TSHR as a locus affecting plasma TSH values (153).

One polymorphic residue deserves special mention: position 601 was found to be a tyrosine or a histidine in the two initial reports of TSHR cloning (154,155). Characterization of the two alleles by transfection in COS cells indicated interesting functional differences: the Tyr601 allele displayed readily detectable constitutive activity, whereas the His601 was completely silent; the Tyr601 allele responded to stimulation by TSH by activating both the adenylylcyclase and phospholipase C dependent regulatory cascades, when the His601 allele was only active on the cAMP pathway (156,157). The Tyr601 allele is by far the most frequent in all populations tested. A Tyr601Asn mutation was found in a toxic adenoma. Characterization of the mutant demonstrated increase in constitutive activation of the cAMP regulatory cascade (157), making the 601 residue an interesting target for structure-function studies.

Acknowledgments

Research in the laboratory of the author was supported by the Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy (6/14), the Fonds de la Recherche Scientifique Médicale of Belgium, the Walloon Region (program “Cibles”) and the non-for-profit Association Recherche Biomédicale et Diagnostic. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), projects KL2334/2-2 and Cluster of Excellence ‘Unifying Concepts in Catalysis’ (Research Field D3/E3-1) to G.Kl..

 

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