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Hypothyroidism in Older Adults

ABSTRACT

 

Hypothyroidism is more common among elderly individuals due to the increasing incidence and prevalence of autoimmune thyroiditis that occurs with aging. Accurate diagnosis of this condition in the elderly may be challenging due to a number of factors including a relative paucity of referable symptoms, confounding findings that may be related to comorbid disorders, changes in thyroid hormone levels that may be related to nonthyroidal illness, and upward shifts in TSH levels that may occur with normal aging. Effective treatment of hypothyroidism in the elderly relies on consideration of potential drug interactions and changes in the metabolic clearance of thyroid hormone that occur with aging. Specific attention should be paid to minimizing the risks of atrial arrhythmias and progressive bone loss that may be associated with iatrogenic thyrotoxicosis caused by over-treatment with excessive doses of levothyroxine. Mild hypothyroidism identified in the elderly does not appear to be associated with any changes in cognitive function or functional status. Studies that have sought to determine the risk of cardiovascular disease associated with mild hypothyroidism and the potential benefits of treatment targeted to normalize thyroid hormone levels in elderly individuals with mild hypothyroidism have reported conflicting results. Elderly patients presenting with untreated or undertreated severe hypothyroidism may be particularly susceptible to decompensation that may progress to a state of myxedema coma.

INTRODUCTION

 

Hypothyroidism increases in prevalence and incidence among the elderly. It is important for clinicians to appreciate certain aspects of hypothyroidism in older individuals. Its clinical manifestations may be less obvious in the setting of somatic complaints and other conditions related to aging. Thyroid function test interpretation may be altered due to the presence of nonthyroidal illness. Special considerations may apply in planning treatment due to changes in the metabolic clearance of thyroid hormone, drug interactions, and potential adverse reactions.

Figure 1. Percentage of Population with High Serum TSH Level (>4.5mU/L). Adapted from Hollowel et al. (1). *Excluding persons with reported histories of thyroid disease, goiter, or treatment with thyroid medications. ** Excluding persons with reported histories of thyroid disease, goiter, treatment with thyroid medications, conditions predisposing to thyroid function test abnormalities, or positive antithyroid antibodies (2)


PREVALENCE

 

Hypothyroidism is more common in older persons than younger individuals, especially among women, principally due to the rising incidence and prevalence of autoimmune thyroiditis. Furthermore, the incidence of hypothyroidism steadily increases with advancing age (Figure 1).  Estimates of the prevalence of hypothyroidism among the elderly have varied depending on the populations studied and the criteria used to define the condition. An older survey employing the calculated free thyroxine index found that 2.3% of elderly inpatients met criteria for hypothyroidism (2).  More recent community surveys of populations of healthy adults have found that 7%-14% of elderly subjects have serum thyroid stimulating hormone (TSH) levels above the upper limit of reference ranges (3–7).  Comparable prevalence’s of hypothyroidism have been found in community dwelling and hospitalized older persons. A screening study that evaluated more than 25,000 individuals attending a health fair in Colorado revealed that 10% of men and 16% of women age 65-74 had TSH levels that were increased above the upper limit of the reference range, while 16% of men and 21% of women age 75 and older had increased TSH levels (8).  The Third National Health and Nutrition Examination Survey (NHANES III) reported that a significantly greater number of women aged 50-59 and 60-69 met criteria for subclinical and clinical hypothyroidism compared to men in the same age ranges. This survey also reported a higher prevalence of increased TSH levels and anti-thyroid antibody titers among whites and Mexican Americans compared to blacks (1).  A study evaluating geriatric patients under medical care demonstrated that 15% of the women and 17% of the men had previously undiagnosed hypothyroidism (9).  Similar studies evaluating skilled nursing facility and nursing home residents demonstrated that 7%-12% had evidence of previously undiagnosed hypothyroidism at the time of admission (10,11).  A treatment survey of an unselected population of older adults revealed that 10% of the women and 2% of the men studied were taking a prescribed form of thyroid hormone (12).  Among this population, 12% of the women and 29% of the men were reportedly taking thyroid hormone preparations for inappropriate reasons.

 

Future estimates of the prevalence of hypothyroidism among the elderly based on current definitions may need to factor in growing evidence that normal TSH distribution curves appear to be shifted towards higher value ranges in older individuals. Age-specific analysis of TSH levels and anti-thyroid antibody titers measured as part of the most recent NHANES study demonstrated that 12% of subjects aged 80 and older without any evidence of underlying autoimmune thyroiditis had TSH levels greater than 4.5 mIU/L (13). In this analysis, the upper 95% confidence limit for TSH in euthyroid individuals over age 80 was 7.5 mIU/L (13).

 

Dietary iodine content appears to have an impact on the prevalence of hypothyroidism in the elderly. A survey of Chinese adults living in a region of low iodine intake revealed that only 1.0% of elderly subjects studied met criteria for hypothyroidism, while a study of Eastern European nursing home residents revealed that subjects living in regions of abundant iodine intake had six-fold higher rates of hypothyroidism than subjects living in regions of low iodine intake (14,15).  These findings suggest that iodine deficiency may have a protective effect against the development of hypothyroidism in the elderly.

 

ETIOLOGY

 

Autoimmune thyroiditis is the most common cause of hypothyroidism among the elderly, as it is in younger persons (16–18).  A survey of endocrinology clinic patients revealed that 57% of patients aged 55 and older presenting with primary hypothyroidism carried a diagnosis of autoimmune thyroiditis, while 32% carried a diagnosis of postsurgical hypothyroidism and 12% had a diagnosis of post-radioiodine hypothyroidism (19).  Only 2% of the patients in this referral population presented with documented evidence of secondary hypothyroidism. The incidence of post-ablative hypothyroidism has been noted to be higher in patients aged 55 and older (20).  The annual incidence of post-ablative hypothyroidism in this population is estimated to be 8%, with 12% of patients presenting with evidence of thyroid failure in the first year after undergoing treatment with radioactive iodine (21,22).  The incidence of postsurgical hypothyroidism following subtotal thyroidectomy for treatment of hyperthyroidism has been estimated to be 16-27%, with 19% of patients presenting with evidence of thyroid failure in the first year after surgery (23).  External beam radiation therapy for treatment of head and neck malignancies has been associated with a high incidence of primary hypothyroidism. Up to 28% of patients treated with this modality eventually develop primary hypothyroidism at a median time of 15 months after completion of radiotherapy (24).  The risk of developing thyroid failure in this setting increases with advancing age.

 

CLINICAL FEATURES

 

Symptoms

 

Elderly patients developing hypothyroidism may present with classic symptoms, but complaints are generally even less specific than those reported by younger patients presenting with evidence of thyroid hormone deficiency (25–27).  In part this may be due to patients and physicians ascribing nonspecific complaints to other comorbid disorders common among the elderly, or to the effects of aging itself (28).  A study that compared the frequency of 24 symptoms of hypothyroidism reported by elderly and nonelderly patients found that complaints of fatigue and weakness were reported by more than 50% of elderly patients, but that significantly fewer complaints were reported by the elderly compared to a nonelderly group (29).  Elderly patients less often complained of cold intolerance, weight gain, paresthesias, and muscle cramps. Evaluation of a questionnaire administered to patients newly diagnosed with hypothyroidism ascribed to autoimmune thyroiditis showed that while all 13 referable symptoms were more prevalent in subjects younger than 60 years of age, the only referable symptoms that were more prevalent in older subjects were fatigue, dyspnea, and wheezing (30).  Other neurological symptoms that have been reported to occur more commonly in older patients include hypogeusia and dysgeusia, impaired hearing, and ataxia.

 

Signs

 

Physical findings evident in hypothyroid elderly individuals may include bradycardia, diastolic hypertension, pallor, dry skin, coarse hair, hoarseness, dysarthria, delayed relaxation of deep tendon reflexes, and mental status changes (31). The severity of specific findings may be exacerbated by comorbid cardiovascular, neuropsychiatric, dermatologic, or rheumatologic conditions that are more common among the elderly (32).  In some cases it may be necessary to evaluate responses to thyroid hormone replacement to determine the extent to which certain findings represent manifestations of thyroid hormone deficiency.

 

Morphologic changes in the size and appearance of the thyroid do not appear to increase with aging (33).  Elderly patients with autoimmune thyroiditis are more likely to present with the atrophic form of the disorder without goiter (34).  Neuropsychological testing of elderly patients with hypothyroidism has demonstrated that they score lower on Mini-Mental Status Tests and on 5 of 14 specific indices of visual-spatial function, memory, word fluency, attention, and psychomotor function (35).  Analysis of laboratory test results has demonstrated that 54% of patients diagnosed with hypothyroidism have increased serum creatinine levels that may be correlated with advancing age (36).  Pericardial effusion is one of the few radiographic findings associated with hypothyroidism, but the true incidence of this complication appears to be lower than previously estimated (37).

 

Morbidity

 

Severe medical complications of hypothyroidism are more common in affected elderly persons. The majority of patients presenting with myxedema coma are elderly. Elderly patients with unrecognized hypothyroidism may be at greater risk for the development of perioperative and intraoperative complications. One study that compared patients with unrecognized hypothyroidism with controls matched for age, sex, and operative procedure identified higher rates of intraoperative hypotension, heart failure, and postoperative gastrointestinal and neuropsychiatric complications in hypothyroid patients (38).  A prospective study that screened hospitalized patients aged 60 and older for thyroid dysfunction reported that unrecognized overt hypothyroidism in this population may be associated with significantly higher mortality (39).

 

 

A number of studies have sought to determine whether biochemical diagnosis of thyroid disorders in the elderly may be confounded by age-related changes in thyroid function (40).  An early study of thyroid function profiles in women aged 60 and older reported higher serum thyroxine (T4) and TSH levels, and decreased triiodothyronine (T3) and reverse triiodothyronine (rT3) levels in comparison to reference ranges (41).  Similar findings were confirmed in a contemporaneous study comparing thyroid function profiles in elderly men and women to those of younger persons, and in a more recent study comparing thyroid function profiles in women aged 70 and older to those in their middle-aged offspring (42,43).  In contrast, when other investigators stratified elderly patients by health status (i.e. healthy elderly adults, nursing home residents, or hospitalized elderly adults), they found that lower serum T3 levels and higher rT3 levels were only detected in the institutionalized elderly adults (44).  Consequently, previously observed patterns of age-related changes may have actually reflected effects of nonthyroidal illness. Two studies that evaluated thyroid hormone profiles in healthy adults have clarified this issue. One study that measured T3 and free T3 levels in healthy adults aged 65 and older determined that while levels of these hormones were lower than in younger adults, they fell well within the limits of reference ranges (45).  Another study of thyroid hormone profiles in a range of healthy adults who were not taking prescribed medications determined that there were no significant differences in T4, free T4, T3, free T3, or rT3 levels between groups stratified by age (46). These findings thus argue against the existence of a “low T3” syndrome associated with normal aging.

 

Studies of hypothalamic-pituitary function in the elderly have shown that blunted circadian fluctuations in TSH levels and diminished TSH responses to TRH stimulation may be detected in elderly males (47–49). The cause of this phenomenon is unclear. There are no histological or immunoreactive differences in the thyrotroph cells of elderly patients (50).  Measurement of serum deiodinase levels in a range of healthy adults has demonstrated a significant inverse correlation of 3’,3’-diiodothyronine, 3’.5’-diiodothyronine, and 3,5-diiodothyronine levels with increasing age (51). One study showed that the decline in deiodinase activity noted with increasing age was paralleled by a decline in selenium levels. Furthermore, selenium supplementation may effectively increase selenium levels, deiodinase activity, and T3/T4 ratios in elderly patients (52).

 

THYROID FUNCTION TESTS

 

Accurate diagnosis of primary hypothyroidism in the elderly relies primarily, as it does in all patients, on the measurement of a sensitive serum TSH level. Although data from the NHANES III study has established that median TSH levels appear to increase with advancing age, the normal upper limit of an established reference range may still be used as a cutoff to confirm the diagnosis of primary hypothyroidism in most elderly patients. While a blood spot TSH level has been shown to be an adequate screening test for the detection of overt primary hypothyroidism in the elderly, it may not be sensitive enough to detect cases of subclinical hypothyroidism characterized by elevated serum TSH levels with normal T4 or free T4 levels (53). One study has determined that there may be a negative correlation between age and the degree to which TSH levels are elevated in elderly patients presenting with primary hypothyroidism (54).

 

In cases of suspected secondary hypothyroidism that may result from disruption of the anatomy or function of the hypothalamic-pituitary axis, the TSH level may not be relied upon as an accurate index of thyroid function. In this setting the free T4 level may serve as more reliable measure of thyroid hormone production.

 

The interpretation of thyroid function test profiles in hospitalized or institutionalized patients must be tempered by an understanding of how nonthyroidal illnesses may produce changes in TSH and thyroid hormone levels (55). The direction and extent of changes observed may depend on the severity of an underlying illness and the point in the course of recovery at which thyroid function tests are measured (56). Longitudinal studies have demonstrated that early on in the course of severe illnesses or protracted procedures, TSH levels in euthyroid patients may decline to levels that fall below the lower limits of normal reference ranges (57). This change may be paralleled by a decline in T4 and T3 levels that may be particularly pronounced in elderly patients. One study demonstrated that 59% of elderly patients known to be euthyroid had documented low T3 levels measured during a course of hospitalization, whereas another demonstrated that changes in T3 levels detected in elderly hospitalized patients were more closely correlated with the severity of each underlying illness than with advanced age itself (58,59). Studies have demonstrated a correlation between declining T4 levels and increasing mortality rates in critical care patients (60). Free T4 levels measured by equilibrium dialysis or ultrafiltration methods, if they are within reference ranges, may help to distinguish hypothyroidism from the effects of altered thyroid hormone binding that may occur in critically ill patients (61).

 

Current data indicates that the normal or low TSH levels found in the presence of low T4 and T3 levels in the setting of nonthyroidal illness likely reflect the combined effects of central hypothyroidism and reduced peripheral generation of T3, effectively representing a deficiency of thyroid hormone. Whether this condition should be treated with administration of thyroid hormone preparations remains controversial. Some observers argue in favor of thyroid hormone replacement, while others weigh against it, without conclusive data to support either viewpoint (62,63).

 

If a patient survives to recover from nonthyroidal illness, TSH levels may transiently rise above the upper limits of reference ranges (64). If thyroid function tests are checked when a transiently increased TSH level precedes increases in low T4 and/or T3 levels, the profile that emerges may appear to be consistent with primary hypothyroidism (65). This could lead to unnecessary treatment with thyroid hormone, which would probably be inconsequential. In cases where changes in TSH and thyroid hormone levels may be plausibly ascribed to nonthyroidal illness, the patient’s thyroid function tests should be reassessed one to two weeks later to see if observed changes are resolving. One study that tracked thyroid function test profiles in hospitalized elderly female patients showed that while 14% of the subjects had increased TSH levels and decreased T4 and T3 levels on initial assessment, only 2% were proven to have evidence of underlying primary hypothyroidism during follow up (66).

 

Measurement of anti-thyroid antibody levels may help to confirm a suspected diagnosis of autoimmune thyroiditis as the underlying cause of primary hypothyroidism. However, the presence or absence of elevated anti-thyroid antibodies may not be an absolute indicator of the likelihood of eventual development of primary hypothyroidism in elderly individuals. One study that measured TSH and anti-microsomal antibody levels in healthy elderly adults showed that positive titers were detected in only 67% of subjects with TSH levels > 10.0 mIU/L and 18% of subjects with normal TSH levels (67). A similar study that measured anti-thyroid antibody levels in nursing home residents detected positive titers in only 64% of the women and 32% of the men presenting with increased TSH levels (68). Comparative measurements of anti-thyroglobulin, anti-microsomal, and anti-thyroid peroxidase antibodies have demonstrated that while there may be a similar prevalence of positive anti-microsomal and anti-thyroid peroxidase titers among elderly adults, mean values of anti-thyroid peroxidase antibody levels tend to be much more commonly elevated in this population (69). Nonetheless anti-thyroid antibody measurements in the elderly may help to predict the likelihood of progression from subclinical to overt hypothyroidism (70).

 

Abnormalities in other routine laboratory test parameters may suggest possible undetected hypothyroidism. Hyponatremia caused by decreased free water excretion may complicate moderate and severe cases of primary hypothyroidism (71). Hyperlipidemia characterized by hypercholesterolemia is commonly evident (72). Cases of primary hypothyroidism that are severe enough to precipitate myopathy may present with increased creatine phosphokinase levels (73). A hypochromic microcytic anemia that is not associated with any detectable hemoglobinopathy or iron deficiency state may be evident in up to 15% of cases of moderate primary hypothyroidism (74). Homocysteine and lipoprotein (a) levels may be increased in patients with primary hypothyroidism, potentially contributing to an increased risk of atherosclerotic disease (75).

 

TREATMENT

 

Initial treatment of hypothyroidism in elderly patients should typically start with sodium levothyroxine (thyroxine) administered in lower doses than those usually prescribed for healthy younger patients (e.g. 0.25 to 0.5 mcg/kg/day). Once cardiovascular tolerance of a starting dose has been assessed, most experts recommend gradually increasing daily doses by 12.5-25 mcg every four to six weeks until adequate replacement is confirmed by repeat TSH measurement. The degree to which this general strategy has been adopted in practice was confirmed by a recent survey of members of the American Thyroid Association (76). A recent trial demonstrated that older patients without any underlying cardiovascular disease could be safely started on full replacement doses of thyroxine (1.6 mcg/kg) without any adverse effects (77). While a great deal of interest has arisen regarding the potential benefits of adding doses of liothyronine (T3) to thyroxine to approximate physiologic thyroid hormone secretion, a number of randomized trials have shown that this mode of treatment does not have any significant impact on identified symptoms, mood, cognitive function, or quality of life (78–81).

 

Serial measurements of TSH levels four to six weeks after each change in thyroxine dosage should be used to monitor thyroid hormone replacement therapy. In a comparison trial based on a reference standard of measured TSH response to TRH administration, basal TSH levels proved to be more sensitive to fine alterations in thyroxine doses than basal free T4 or free T3 levels. Most experts recommend targeting a normal TSH range in elderly patients (82). While 39% of ATA members recommended targeting a TSH range of 0.5-2.0 mIU/L when treating younger patients, a comparable number reported that they were generally more liberal in their approach to elderly patients, targeting TSH ranges of 1.0-4.0 mIU/L. Treatment with thyroxine has been shown to increase cognitive testing performance and reduce oro-cecal transit time from an average of 135 minutes in a hypothyroid state to 75-95 minutes with adequate replacement (83,84).

 

While thyroid hormone supplementation to a level that completely corrects the hormonal deficiency may be an optimal goal, some patients with ischemic heart disease may not be able to tolerate full replacement doses of thyroxine (85,86). One study of patients with known coronary artery disease and primary hypothyroidism reported that precipitation of angina symptoms limited titration of thyroxine in two-thirds of cases, while precipitation of hypothyroid symptoms limited titration of antianginal agents in one-third of cases. Even with the addition of propranolol at maximally tolerated doses, 46% of the patients surveyed rated control of their angina and hypothyroid symptoms as fair to poor (87).

 

Thyroxine dose requirements in elderly patients may be related to several factors including declining metabolic clearance, progression of underlying thyroid failure, declining body mass, and interactions with other medications prescribed for the treatment of co-morbid conditions (88,89). On average, elderly patients with primary hypothyroidism receive initial daily doses that are 20 mcg lower and maintenance daily doses that are 40 mcg lower than those prescribed for younger and middle-aged patients (90–92). One study suggested that lean body mass may be a better predictor of daily replacement doses than age or weight alone (93). Another reported that most of the age-dependent differences in thyroxine requirements noted might be attributed to the effects of chronic disease, since substantially lower average daily replacement doses were reported by elderly patients treated for other chronic medical disorders (94). A study that tracked changes in elderly patients’ thyroxine requirements over time based on the etiology of their primary hypothyroidism reported that daily replacement doses increased in patients who initially presented with autoimmune thyroiditis or postsurgical hypothyroidism, decreased in patients who initially presented with post-ablative hypothyroidism, and did not change in patients who initially presented with subclinical hypothyroidism or drug-induced hypothyroidism (95).

 

In situations where cognitive or functional impairment may make it difficult for patients to comply with daily administration of thyroxine, alternative dosing schedules may be considered. A study that compared daily administration of thyroxine to twice weekly administration of comparable cumulative daily doses in elderly women showed that both regimens produced similar peak and trough free T4, T3, and TSH levels (96). Trials of regimens based on once weekly administration of cumulative daily doses of thyroxine have demonstrated similar results without any evidence of precipitation of thyrotoxicosis (97).

 

A number of medications used to treat other comorbid conditions in the elderly may interfere with absorption and metabolism of thyroxine (98). Ingestion of 2,000 mg of calcium carbonate has been shown to interfere with the peak and total incremental absorption of a concomitantly administered treatment dose of thyroxine (99). Ferrous sulfate, sucralfate, aluminum hydroxide, cholestyramine, colestipol, and raloxifene have also been reported to impair absorption of thyroxine (100,101). In postmenopausal women with primary hypothyroidism, treatment with estrogen replacement therapy may lead to increased thyroxine dose requirements as a consequence of increased production of thyroid binding globulin (TBG) (102). Women with hormonally-responsive breast cancer who receive fluoxymesterone may require substantially lower doses of thyroxine during courses of treatment, as exposure to this androgenic steroid may decrease effective TBG production (103). Long-term administration of phenytoin, carbamazepine, phenobarbital, or rifampin in the setting of treated primary hypothyroidism typically increases metabolism of thyroxine, increasing the dose of thyroxine required to provide optimal replacement (104–106).

 

Overtreatment with excessive doses of thyroxine may be associated with significant morbidity in the elderly. Palpitations, anxiety, tremulousness, irritability, insomnia, heat intolerance, hyperdefecation, and weight loss may be precipitated or exacerbated by iatrogenic thyrotoxicosis. In elderly patients, exposure to excessive amounts of thyroid hormone may be associated with increased risks of atrial fibrillation, other tachyarrhythmias, and progressive declines in bone mineral density (107). A prospective study of the incidence of atrial arrhythmias in patients aged 60 and older determined that over the course of a 10-year period, the relative risk of development of new-onset atrial fibrillation in subjects with initial TSH levels < 0.1 mIU/L was 3.1 when compared to subjects with normal TSH levels (108). Further analysis revealed that suppressed TSH levels identified in 77% of these subjects were attributable to iatrogenic thyrotoxicosis resulting from overtreatment. A study that tracked bone mineral density changes in women treated with thyroxine documented greater mean rates of decline in the lumbar spines of those with suppressed TSH levels (109). A recent cohort study that tracked TSH and free T4 and T3 levels in healthy aging adults in tandem with inventories of medication use reported that half of the cases of prevalent and incident thyrotoxicosis identified could be attributed to over-treatment with levothyroxine (110).

 

MILD HYPOTHYROIDISM (SUBCLINICAL HYPOTHYROIDISM)

 

Mild or subclinical hypothyroidism, which is characterized by an increased TSH level with concomitant free thyroid hormone levels that fall within normal limits, is very common among elderly men and women. The estimated prevalence of this condition has varied from 4-15%. A study evaluating a community of healthy elderly adults in the southwest of France reported that 4.2% of subjects presenting with increased TSH levels had normal free T4 levels (111). Within this group, mild hypothyroidism was linked with an increased prevalence of symptoms of depression. A study that evaluated thyroid function profiles in a bi-ethnic urban community reported that mild hypothyroidism was more commonly identified in females and non-Hispanic white subjects than Hispanic subjects (112). Stratified analysis of the impact of mild hypothyroidism in this population revealed no significant alterations in health status measures in subjects with TSH levels ranging between 4.7-10.0 mIU/L. A study that inventoried clinical findings of hypothyroidism in a population of geriatric clinic patients reported that while 15.4% of the men and 14.6% of the women screened met criteria for mild hypothyroidism, the incidence of symptoms and signs consistent with thyroid hormone deficiency detected in these subjects was similar to that reported for euthyroid subjects (113).  An array of studies that have tracked changes in thyroid function in cohorts of aging subjects in the United States, Australia, the Netherlands, Spain, the United Kingdom, and China have reported that the development of hypothyroidism in elderly patients does not appear to be associated with any change in cognitive function, increased levels of depression, or diminished ability to perform activities of daily living (114–120). A study that measured an array of anthropometric, biochemical, and neuropsychiatric parameters in Korean subjects aged 65 years and older showed that subclinical hypothyroidism did not appear to be associated with any discernible metabolic or neuropsychiatric derangements (121). A study that evaluated subgroups of subjects enrolled in the Health, Aging, and Body Composition study found that those determined to have mild subclinical hypothyroidism (defined by a TSH level of 4.5-7.0 mIU/L with normal thyroid hormone levels) demonstrated better mobility, cardiorespiratory fitness, and walking ease than subjects who were euthyroid or determined to have moderate subclinical hypothyroidism (defined by a TSH level of 7.0-20.0 with normal thyroid hormone levels) (122). An analysis of subgroups in this cohort study identified increased odds of prevalent metabolic syndrome among subjects with TSH levels > 10 (123). A study that evaluated postmenopausal women at risk for development of osteoporosis reported that subclinical hypothyroidism was not associated with decreased bone mineral density or an increased risk of vertebral or non-vertebral fracture (124).

 

Several longitudinal studies have tracked the natural history of untreated mild hypothyroidism in elderly persons. A study of nursing home residents confirmed that over time TSH levels declined to normal ranges in 51% of subjects with initial TSH levels that were lower than 6.8 mIU/L (125). Serial TSH levels were persistently elevated in the remainder of these subjects and in all subjects with initial TSH levels greater than 6.8 mIU/L. A similar study that stratified subjects on the basis of anti-thyroid antibody levels reported that 80% of elderly adults with mild hypothyroidism with initial measured anti-microsomal antibody titers greater than 1:1,600 eventually progressed to develop overt hypothyroidism requiring treatment with thyroxine replacement therapy (69). A study that tracked 505 subjects diagnosed with mild hypothyroidism over time showed that positive anti-thyroid peroxidase antibodies and higher total cholesterol levels measured at baseline were associated with increased odds of eventual progression to overt hypothyroidism (126). Two studies showed that when elderly patients diagnosed with subclinical hypothyroidism were tracked over a span of 4-4.2 years, 44-54% demonstrated normalization of TSH levels consistent with reversion to a euthyroid state (127,128). Findings that were associated with reversion included lower baseline TSH levels, homogenous echotexture of thyroid tissue on ultrasound imaging, and an absence of detectable anti-thyroid peroxidase antibodies.

 

Questions have been raised about the possible association of mild hypothyroidism with an increased risk of cardiovascular disease in the elderly. One study that confirmed the presence of mild hypothyroidism in 10.8% of subjects drawn from a cohort of postmenopausal women reported a greater age-adjusted prevalence of coronary and aortic atherosclerosis in mildly hypothyroid women (129). Even stronger associations between mild hypothyroidism and atherosclerotic disease were noted among postmenopausal women with elevated anti-thyroid antibody levels. Another study that evaluated the prevalence of peripheral vascular disease among nursing home residents reported that 78% of subjects with mild hypothyroidism presented with reproducible claudication, whereas symptomatic peripheral vascular disease was only identified in 17% of euthyroid subjects (130). A study that evaluated thyroid function in patients enrolled in a study of pre-existing heart failure reported that subclinical hypothyroidism presenting with TSH levels > 7 mIU/L was associated with an increased risk of a need for the use of ventricular assist devices, heart transplantation, and death (131).

 

Population-based studies that have tracked thyroid function in elderly subjects have reported differing results regarding risks of cardiovascular disease. A study that examined community-dwelling subjects aged 70-79 years enrolled in the Health, Aging, and Body Composition study found that subclinical hypothyroidism was associated with an increased incidence of congestive heart failure (132). A study that examined subjects aged 65 years and older enrolled in the Cardiovascular Health study found that subclinical hypothyroidism was not associated with an increased incidence of coronary artery disease, cerebrovascular disease, cardiovascular mortality, or all-cause mortality (133). Analysis of subgroup data tracked over the course of 12 years and echocardiographic parameters tracked over the course of 5 years demonstrated that subjects with TSH levels >10.0 mIU/L had a higher incidence of heart failure events, a greater increase in left ventricular mass, and appreciable changes in measurements reflecting changes in diastolic function compared to euthyroid subjects (134).  Two meta-analyses that analyzed data from a range of prospective cohort studies incorporating measurements of thyroid function identified a modest increase in the risk of coronary artery disease and associated mortality in subjects determined to have evidence of subclinical hypothyroidism (135,136). More recent analyses of subgroups tracked in cohort studies have reported that persistent subclinical hypothyroidism does not appear to be associated with an increased risk of all-cause mortality, cardiovascular mortality, coronary artery disease, myocardial infarction, or congestive heart failure (137–139). An analysis of NHANES III data has identified increased mortality in subjects diagnosed with concurrent subclinical hypothyroidism and congestive heart failure, and a retrospective cohort study from Israel involving 17,440 patients with subclinical thyroid disease showed that TSH levels > 6.35 mIU/L were associated with increased mortality (140,141).

 

Consideration of treatment of mild hypothyroidism in the elderly is often predicated on the notion that restoration of normal thyroid hormone levels might help to relieve symptoms that could be exacerbated by a deficiency of thyroid hormone. The Thyroid Hormone Replacement for Untreated Older Adult with Subclinical Hypothyroidism (TRUST) trial was specifically designed to address this question (142). It randomized 737 subjects > 65 years of age with persistent subclinical hypothyroidism to double-blinded placebo-controlled administration of doses of thyroxine adjusted to normalize TSH levels. Assessment based on a thyroid-related quality-of-life questionnaire after one year of treatment showed no difference in hypothyroid symptom scores or tiredness scores. An analysis that combined data from 146 TRUST trial subjects > 80 years of age with data from 145 subjects enrolled in the Institute for Evidence-Based Medicine in Old Age 80-plus trial who were evaluated with a similar protocol also showed no improvement in hypothyroid symptoms or fatigue; however, a majority of those with elevated TSH levels had values below 7 mIU/L (143). The attendant risks of iatrogenic thyrotoxicosis in elderly individuals must be taken into account when weighing the potential risks and benefits of thyroid hormone replacement (144).

 

Partial or complete reversibility of hypercholesterolemia has been shown to accompany thyroxine treatment of mild hypothyroidism in the majority of small interventional trials addressing this issue (145).  Lowering of lipoprotein (a) levels has been shown in some, but not all studies (146). Hyperhomocysteinemia in patients with mild hypothyroidism has not been shown to be reversed with thyroxine therapy. A nested trial incorporated in the TRUST trial showed that normalization of TSH levels with levothyroxine for a span of one year did not have any impact on carotid intima media thickness or carotid atherosclerosis (147).

 

MYXEDEMA COMA

 

Patients with severe hypothyroidism may present in a state of pronounced multisystem failure termed myxedema coma (148,149). Elderly patients with untreated or undertreated primary hypothyroidism and comorbid disorders may be particularly susceptible to decompensation that leads to onset and progression of this life-threatening condition (150,151). In addition to coma, there may be hypothermia, bradycardia, hypotension, congestive heart failure, ileus, and hypoventilation with hypercapnia and respiratory acidosis. In situations where historical information may be unobtainable, physical examination may reveal evidence of prior thyroid surgery, laryngeal surgery, or head and neck external beam radiation therapy. Radiographic studies may reveal pericardial effusions, which may also be reflected in low voltage waves on electrocardiograms. Although such pericardial fluid collections may be large, they are usually not hemodynamically significant. Laboratory evaluation confirming severe hypothyroidism may also reveal evidence of hyponatremia, hypoglycemia, and/or adrenal insufficiency.

 

Myxedema coma is an endocrine emergency with a mortality rate that may approach 40% (152). In addition to older age, factors that may be associated with an increased risk of mortality include comorbid cardiovascular disease and treatment with high-dose thyroxine replacement therapy (153). Generally recommended supportive measures include critical care-level monitoring of vital signs, careful external rewarming with heating blankets, correction of fluid and electrolyte imbalances, avoidance of hypnotics and sedatives, empiric treatment of suspected underlying infections, and mechanical ventilatory support as indicated. Given the theoretical risk of concomitant adrenal insufficiency due to polyglandular autoimmune syndromes or hypothalamic-pituitary compromise, many experts recommend empiric treatment with stress-dose glucocorticoids until definitive stimulatory testing can be performed.

 

Recommendations regarding the dose and composition of thyroid hormone preparations that should be administered to treat myxedema coma differ. Most experts concur that intravenous thyroxine should be used to circumvent impaired gastrointestinal absorption. Some have recommended initial thyroxine loading doses, while others have advocated co-administration of liothyronine (T3). Treatment of critically ill hypothyroid patients with high-dose thyroxine has been associated with a significant increase in cardiac index due to increased heart rate and stroke volume with decreased systemic vascular resistance (154). Although the onset of action of liothyronine is more rapid than thyroxine, supraphysiologic T3 levels measured after treatment have been correlated with increased mortality in older patients presenting with myxedema coma (155). A judicious approach may involve administration of a loading dose of 200-300 mcg of intravenous thyroxine followed by administration of 50 mcg daily. Depending on the estimated risk of underlying cardiovascular disease, a loading dose of 5-25 mcg of liothyronine may be administered concomitantly followed by doses of 2.5-5 mcg every eight hours until clinical improvement is evident. Intravenous hydrocortisone may be administered in stress doses of 50-100 mg every 8 hours while testing for underlying adrenal insufficiency is performed.

 

SCREENING AND CASE-FINDING RECOMMENDATIONS

 

Professional organizations and task forces have issued a range of recommendations concerning the advisability and timing of biochemical screening for hypothyroidism in adult populations (Table 1) (156–160).

 

Table 1. Screening Recommendations for Hypothyroidism in Adults

Guideline

Methods used to analyze evidence

Organization

Year of publication

American Thyroid Association guidelines for the detection of thyroid dysfunction

Narrative literature review Expert opinion

American Thyroid Association

2000

Consensus statement for good practice and audit measures in the management of hypothyroidism and hyperthyroidism

Narrative literature review Expert opinion

Royal College of Physicians of London Society for Endocrinology

1996

Laboratory medicine practice guideline for the diagnosis and monitoring of thyroid disease testing

Narrative literature review Expert opinion

American Association of Clinical Chemists American Association of Clinical Endocrinologists American Thyroid Association Endocrine Society National Academy Clinical Biochemistry

1990, in progress

Periodic health examinations: summary of AAFP policy recommendations & age charts

Based on systematic review performed by US Preventive Services Task Force Expert opinion

American Academy of Family Physicians

1996, 2001

Screening for thyroid disease

Systematic review Meta-analysis of observational trials

American College of Physicians - American Society of Internal Medicine

1997

Screening for thyroid disease

Systematic review

US Preventive Services Task Force

1996

AACE clinical practice guidelines for the evaluation and treatment of hyperthyroidism and hypothyroidism

Narrative literature review Expert opinion

American Association of Clinical Endocrinologists American College of Endocrinology

1996

Treatment guidelines for patients with hyperthyroidism and hypothyroidism

Narrative literature review Expert opinion

American Thyroid Association

1995, 1999

Screening for thyroid disorders and thyroid cancer in asymptomatic adults

Systematic review

Canadian Task Force on Preventive Health Care

1994, 1999

 

A panel of invited experts representing the American Thyroid Association, the American Association of Clinical Endocrinologists, and the Endocrine Society at a consensus development conference found a paucity of evidence regarding the morbidity and impact of subclinical thyroid disease, as well as the potential complications of instituting therapy. Consequently, this panel concluded that there was insufficient evidence to support routine population-based screening of asymptomatic adults. However, the panel did conclude that the weight of available evidence supported the adoption of aggressive case-finding strategies in patients at high risk for the development of hypothyroidism. Specific groups identified as being at increased risk for thyroid dysfunction include women aged 60 years and older and patients with histories of atrial fibrillation, thyroid surgery, radioactive iodine treatment, external beam radiation therapy, or family members with confirmed thyroid disease. A guideline issued by the American College of Physicians states that it is reasonable to check TSH levels in women aged 50 years and older presenting with symptoms that may be consistent with thyroid dysfunction, given the high prevalence of undiagnosed thyroid disorders among that population (161–163).  The Policy Recommendations for the Periodic Health Exam published by the American Academy of Family Physicians take a more neutral stance, recommending against routine screening in patients less than 60 years old without any specific provisions (164). The United States Preventive Services Task Force and the Canadian Task Force on the Periodic Health Examination have both concluded that there is not enough evidence regarding the impact of diagnosis and treatment of detectable thyroid disease to rule for or against routine screening of asymptomatic adults (163,165). Utility analysis based on decision modeling has demonstrated that routine periodic screening for mild hypothyroidism may become more cost-effective with increasing age (166).

 

Studies focusing on actual screening of identified populations of elderly adults have reported mixed results. One study reported that selection of candidates based on body mass index, symptoms consistent with thyroid dysfunction, or a family history of thyroid disease failed to identify the majority of elderly patients eventually confirmed to have elevated or suppressed TSH levels (167). Another study that evaluated elderly patients presenting with suspected dementia revealed that hypothyroidism was the second most common undiagnosed disorder contributing to cognitive impairment (168). A similar study reported that measurement of TSH levels identified hypothyroidism in 3.6% of elderly adults presenting for evaluation of mental status changes (169). Screening studies involving hospitalized patients reported that 2.3% of geriatric inpatients and 11.2% of patients admitted for elective cardiac surgery had thyroid function profiles consistent with hypothyroidism (170). These findings are not surprising in light of the substantial prevalence of hypothyroidism among elderly patients in general. An analysis of profiles of TSH and thyroid hormone levels tracked in subjects enrolled in the Birmingham Elderly Thyroid Study reported high stability of euthyroid and subclinical hypothyroid indices over a 5 year interval, indicating that repeat testing may not be warranted in this population (171).

 

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ACTH Action on the Adrenals

ABSTRACT

 

The adrenocorticotropin hormone (ACTH) is synthesized by the corticotroph cells of the anterior pituitary gland. ACTH, a post-translational product of the proopiomelanocortin protein (POMC), is a 39-amino acid peptide, its sequence being highly conserved in mammals. ACTH binds to the highly specific, for ACTH, melanocortin (MC) 2 receptors (MC2R) located on the surface of adrenal zona fasciculata cells producing cortisol. MC2R belongs to a superfamily of type 1 G protein-coupled receptors. The family of melanocortin receptors includes five members each having characteristic size, tissue distribution, and biological significance. Thus, the MC1R is the principal melanocortin receptor in the skin where it regulates its pigmentation. The MC3R and the MC4R in the central nervous system regulate food intake and energy homeostasis, and knockout animals for these receptors are obese. The MC5R exhibits a wide distribution although its levels in the central nervous system are low. In the adrenal cortex, it induces aldosterone production from the zona glomerulosa cells. ACTH-mediated cortisol synthesis from the zona fasciculata cells depends on a large number of factors of the adrenal microenvironment, such as chromaffin and immune cells, adipocytes, and adrenal innervation. Circadian rhythm of cortisol secretion is ensured by the central and peripheral local adrenal clock system. To activate ACTH the MC2R needs the presence of a small trans-membrane protein, the MC2 accessory protein (MRAP). Mutations of this protein result in the type 2 familial glucocorticoid deficiency (FGD) (FGD) syndrome. Type 1 FGD-syndrome is the result of mutations of the MC2R itself. ACTH stimulates cortisol synthesis and secretion by regulating multiple steps in the steroidogenetic pathway including an increase of the number of low-density lipoprotein (LDL) receptors and the cleavage of the side-chain of cholesterol converting it to pregnenolone, the first and rate-limiting step in cortisol production.

INTRODUCTION

It is now more than 80 years since Selye introduced the concept of “general adaptation syndrome”, later renamed “stress syndrome” as a state of threatened homeostasis in response to stressful stimuli, the stressors (1). Selye was also the first to describe “corticoids” and to propose that glucocorticoids and mineralocorticoids regulated not only carbohydrate and electrolyte metabolism, respectively, but also exerted anti- or pro-inflammatory effects. By stimulating adrenal corticosteroids synthesis, the adrenocorticotropin hormone (ACTH), which was first isolated in 1943 and synthesized in the 1970s, plays a central role in homeostasis and stress and is a key component of the hypothalamic-pituitary-adrenal axis (HPA) axis (2, 3, 4).

 

The corticotroph cells of the anterior pituitary synthesize and secrete the ACTH which via the circulation binds and activates its receptors in the adrenal fasciculate cells affecting most steps in the synthesis of cortisol. This widely accepted model has been extensively advanced and enriched during the last few years. More specifically, it has been found that for the ACTH receptor, the melanocortin receptor 2 (MC2R), needs the presence of a small trans-membrane protein, the MC2 accessory protein (MRAP) to respond to ACTH. Mutations of this protein result in the type 2 familial glucocorticoid deficiency (FGD) syndrome. Type 1 FGD is the result of mutations of the MC2R itself. Newer data reveal the role of the autocrine-paracrine micro-regulation of ACTH-mediated cortisol synthesis by a large number of intra-adrenally produced factors deriving from chromaffin cells, resident immune cells, intra- and peri-adrenal adipocytes, and adrenal innervation. Moreover, there is an increasing interest in the role of a central and peripheral (endogenous) adrenal clock system exerting a circadian regulation of ACTH secretion and action (5, 6). Great progress has been also made in our understanding of the pathophysiology of the triple A syndrome, which is caused by mutations in the gene encoding the regulatory protein ALADIN, a product of the ADRACALIN gene. The updated version of this chapter includes the classical data regarding ACTH-induced cortisol production by the adrenal gland, as well as a description of the new findings.

ACTH AND ITS PRECURSOR MOLECULE PRO-OPIOMELANOCORTIN (POMC)

The ACTH hormone is the primary regulator of cortisol production synthesized in the human adrenal fasciculate cells. ACTH is a post-translational product of the proopiomelanocortin protein (POMC), which is synthesized in the corticotroph cells of the anterior pituitary gland. ACTH is a 39-amino acid peptide. Its sequence is highly conserved in mammals since only amino acids 31 and 33 vary between higher mammals and primates. The biological activity of the ACTH molecule depends on its first 24 amino-terminal amino acids while fragments of less than 20 amino acids long are completely inactive. However, the residue 25-39 is important for the stability of the molecule, increasing its otherwise short half-life. Truncation of ACTH from the C-terminus gradually reduces its activity while removal of the four basic residues (Lys–Lys–Arg–Arg) in positions 15–18 inactivates it completely. Finally, it should be noted that its first 13 residues activate all melanocortin receptors in addition to the ACTH receptor. ACTH acts through the formation of cAMP which facilitates the transfer of cholesterol into the mitochondrial inner membrane for the synthesis of adrenal steroids (7,8) (Figure 1). 

Figure 1. POMC products after enzyme-mediated cleavage. 

The synthesis of POMC, its post-translational modification and the secretion of ACTH are under the control of corticotropin-releasing hormone (CRH or CRF) and to a lesser degree to arginine vasopressin (AVP). Both these hormones are synthesized in the parvocellular cells of the paraventricular (PVN) hypothalamic nucleus and are under the negative control of the circulating glucocorticoids. It should be noted that the AVP derived from PVN follows a distinct regulatory and secretory path, completely different from that of AVP synthesized in the magnocellular cells and transferred and secreted from the posterior pituitary as a regulator of water balance. Indeed, the magnocellular-derived AVP is transferred to posterior pituitary by axonal transport and its synthesis and secretion are under the influence of osmotic and oncotic stimuli and plays no part in stress response. On the other hand, the parvocellular-derived CRH and AVP travel, via axonal transport, to the median eminence (ME) at the lower part of hypothalamus from where they are both secreted into the vascular connection between hypothalamus and anterior pituitary, the portal circulation. Multiple neural signals regulate the synthesis of CRH and AVP as well as their secretion from ME.

In addition, a complex central clock synchronized by light information received via the retino-hypothalamic tract from the eye, is located in the suprachiasmatic nucleus (SCN) and sends circadian oscillatory stimuli to the PVN, influencing the secretion of CRH and AVP, and generating the circadian secretion of ACTH (9). This central clock entrains the peripheral oscillators in the adrenal gland via three pathways: (a) the neurohumoral pathway via the HPA axis, (b) the neural pathway via the autonomic nervous system, and (c), a local circadian intra-adrenal regulation of ACTH action (10). However, besides the central circadian regulation of ACTH secretion, local adrenal clocks are thought to regulate also the responsiveness of the adrenal cortex to ACTH in a circadian fashion. Moreover, it is known since the 1960s that adrenals tissues can exhibit an intrinsic rhythmicity of cortisol secretion independently of the upstream rhythm of the HPA axis (10). (Figure 2)

Figure 2. The pathway of stimulation of ACTH secretion from the pituitary and its action on the adrenal gland (6).

CRH reaching the anterior pituitary corticotrophs and binds to the CRH-R1 receptors. The corticotrophs represent approximately 10% of anterior pituitary cells. Their main product, POMC is a 260 AA protein, which is post-translationally cleaved into several bioactive peptides that are secreted from the corticotrophs along with ACTH, including β-lipotropin, the endogenous opioid peptide beta-endorphin, and melanocyte stimulating hormones (MSH) (11,12).

Glucocorticoids exert their negative feedback control on both the hypothalamus at the PVN and anterior pituitary corticotrophs suppressing POMC synthesis and ACTH secretion. Furthermore, chronic exposure to high levels of endogenous or exogenous glucocorticoids results in characteristic corticotropic cell degeneration. The immune system participates in the regulation of ACTH production via interleukins (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha and the interferons alpha and gamma which affect the axis at all its levels i.e. hypothalamus, pituitary, and adrenal cortex (13). Finally, the intra-adrenal production of cytokines appears to play an important modulator of the ACTH-mediated effect on adrenocortical cells (14).

 

EFFECTS OF ACTH ON ADRENAL CORTICAL CELLS

 

ACTH enters the systemic circulation and binds to the highly specific, for ACTH, MC2R located on the surface of adrenal cortical cells. The adult mammalian adrenal cortex is composed of three zones. The outermost or zona glomerulosa produces aldosterone, the middle or zona fasciculata is the largest producing cortisol, while the innermost or zona reticularis produces the weak adrenal androgens. ACTH is the main stimulus of the zona fasciculata and zona reticularis, stimulating glucocorticoid secretion, while angiotensin II and potassium are the main stimuli of aldosterone secretion by the zona glomerulosa. Most MC2R are localized in the zona fasciculata. In general, the steroids produced by the adrenal cortex are classified as 21-carbon steroids (glucocorticoids and mineralocorticoids), as 19-carbon steroids (adrenal androgens), and 18-carbon (adrenal estrogens). Cortisol, the main endogenous glucocorticoid, is synthesized in zona fasciculata under the exclusive regulation of ACTH. ACTH is of secondary importance in aldosterone production (where plasma angiotensin II and serum potassium represent the main regulators). The production of adrenal androgens is more complicated with ACTH playing a minor role. Under normal circumstances, ACTH acts with equivalent potency as a secretagogue for cortisol and aldosterone. Recently, novel evidence suggested that aldosterone secretion stimulated by ACTH via its receptor [called melanocortin receptor 2 (MC2R)] is observed in adrenal tissues of patients with primary aldosteronism (15).

 

The mechanism of ACTH action follows the classical peptide hormone rules. Indeed, ACTH binds to its receptor, MC2R, located on adrenocortical cell membranes activating a Gs-protein resulting in an increase of intracellular cyclic adenosine monophosphate (cAMP). cAMP relays ACTH-mediated functions via the activation of the serine–threonine kinase cAMP-dependent protein kinase A (PKA) or the exchange proteins directly activated by cAMP (EPAC1 and 2 also named cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) I and II). Following cAMP activation, PKA and EPAC transmit signals differently. PKA phosphorylates numerous substrates, while EPACs act as GEFs catalyzing the conversion of the small GTPases Rap1 and Rap2 from an inactive (GDP-bound) to an active form [guanine triphosphate (GTP)-bound] (16).

 ACTH stimulates cortisol synthesis and secretion by regulating multiple steps in the steroidogenetic pathway. Steroid hormones are produced from the same precursor, cholesterol, by a set of cytochrome P450 steroid hydroxylases (CYP11A1, CYP11B1 and CYP11B2, CYP17 and CYP21) and the steroid dehydrogenase 3βHSD (16). The enzymes are differentially expressed in the three zones of the adrenal cortex (zona glomerulosa, zona fasciculata, and zona reticularis) giving rise to zone-specific hormone production. In humans, the primary source of cholesterol for steroid hormone production is LDL-cholesterol, which is imported via the LDL receptor (LDLR) from the blood stream. About 80% of the cholesterol needed for the synthesis of adrenal steroids is supplied by LDL. ACTH increases the number of LDLR resulting in an increase in overall uptake of cholesterol ester. Once cholesterol ester enters the cell, hormone-sensitive lipase (HSL) converts it to free cholesterol (15). Free cholesterol is then delivered to the inner mitochondrial membrane by the actions of steroidogenic acute regulatory protein (StAR) and cholesterol-binding proteins (17).  The precursor cholesterol for steroidogenesis can be derived from a combination of sources: (1) de novo cellular cholesterol synthesis, (2) mobilization of cholesterol’s esters (CE) stored in lipid droplets (LD), and (3) lipoprotein-derived CE delivered through endocytic uptake, which is mediated by the LDL receptor or “selective” cellular uptake of HDL cholesterol via the scavenger receptor, class B type 1 (SR-B1) (17). Acute and chronic ACTH stimuli can modulate SR-B1 function, resulting in changes in the ability of SR-B1 to mediate cholesterol uptake and use for steroids production. ACTH also regulates the formation of microvillar channels in the plasma membranes which retain HDL particles and contain high numbers of HDL receptors.  Once PKA is activated, both an acute and a chronic response occur, which contribute to increased steroid hormone synthesis. During the acute response, PKA phosphorylates HSL, which converts cholesterol esters to free cholesterol. This rapid response also involves an increase in StAR, which facilitates the movement of cholesterol to the inner mitochondrial membrane, where the limiting enzyme CYP11A1 resides (18). The chronic response corresponds to the transcriptional activation of all the other steroidogenic enzymes.  ACTH affects the cleavage of the side-chain of cholesterol converting it to pregnenolone, the first and rate-limiting step in cortisol production. The CYP11A1 gene which encodes the cholesterol side-chain cleavage is regulated by ACTH and by the steroidogenic factor 1 (SF-1). Moreover, ACTH hydroxylates the pregnenolone in the 17-OH position which is subsequently converted into 11-deoxycortisol. 11-deoxycortisol moves back to mitochondria where a hydroxylation at position 21 results in cortisol which is then rapidly secreted into the systemic circulation (19).  

Activation of the MC2R by ACTH in the adrenals also induces the adrenal production of factors affecting adrenal growth and its blood flow. Thus, among other things, ACTH stimulates the intra-adrenal production of vascular endothelial growth factor (VEGF) and the vaso-relaxant epoxy-eicosa-trienoic acids (EETs) (20, 21).

 

Finally, chronic exposure of adrenocortical cells to high levels of ACTH (from eutopic or ectopic production) results in the development of adrenal hyperplasia, nodules, and finally neoplasia. Activation of ACTH receptor and PKA are considered vital for maintaining the highly differentiated cellular phenotype of adrenal cells and the subsequent activation of ERK is of low importance for cell proliferation. In addition, ACTH signals inactivate Akt, a kinase that promotes survival and proliferation. On the other hand, ACTH receptors are up-regulated in adrenocortical adenomas of patients with ACTH-dependent hyper-cortisolemia, intensifying the adrenal response to the already elevated ACTH, aggravating their disease. ACTH also up-regulates the human homolog of Diminuto/Dwarf1 gene, which is associated with benign adrenocortical adenomas. Low expression of this gene correlates with apoptosis, indicating that its intensified expression may contribute to cell survival.  

Wnt-signalling is the main pathway controlling cortisol secretion. Recent studies have shown that cortisol-producing neoplasms frequently display somatic or germline mutations that affect proteins of the cAMP/PKA pathway, leading to constitutive activation of PKA. These mutations include gain-of-function mutations of the MC2R, GNAS, the catalytic subunit α of protein kinase A (PRKACA) and PRKACB genes and inactivating mutations of the regulatory subunit R1α of PKA (PRKAR1A), and of two cAMP-binding phosphodiesterases (PDE11A and PDE8B) genes, mimicking the action of ACTH to stimulate glucocorticoid production, providing a molecular basis for the pathogenesis of primary adrenal Cushing syndrome (22).

 

It has also been shown that cortisol secretion by adrenocortical adenomas and hyperplasias could be stimulated by both locally produced ACTH (23) and aberrantly expressed membrane receptors, such as those of serotonin (24). Prolonged activation of the cAMP/PKA pathway by ACTH induces an aberrant serotonergic stimulatory loop in the adrenal cortex that likely participates in the pathogenesis of corticosteroid hypersecretion.

 

Mutations of the PRKAR1A are considered the main cause of familial and sporadic primary pigmented nodular adrenocortical disease (PPNAD). Moreover, inactivation of PDE11A and PDE8B are associated with isolated micronodular disease (iMAD) which can be also found in PPNAD and primary bilateral macronodular adrenal hyperplasia (PBMAH) cases. Recently, a germline mutation in Armadillo repeat containing protein 5 (ARMC5) gene was found in 25–50 % of PBMAH patients (25). PBMAH that results from ARMC5 mutations have been shown to contain clusters of ACTH-producing cells that stimulate cortisol secretion in an autocrine/paracrine fashion in adrenal tissues through the MC2R (23).

 

The role of ACTH in adrenocortical tumors remains to be elucidated. It may depend on the state of differentiation of the particular cell or the presence of additional events that may decide the direction of the ACTH signal towards cell survival or inhibition of proliferation (26,27). 

MELANOCORTIN 2 (MC2), THE ACTH RECEPTOR

ACTH exerts its effects on the adrenals via a highly selective receptor, a member of the MC2R superfamily of type 1 G protein-coupled receptors. As mentioned above, the MC2 receptor is highly specific for only one ligand, ACTH (28). The family of melanocortin receptors includes five members, each having characteristic size, tissue distribution, and biological significance (29). The MC system and its receptors regulate multiple physiological processes including skin pigmentation, glucocorticoid production, food intake and energy balance. The MC2R is a 297 amino acid transmembrane G-protein coupled receptor. In humans, it maps to 18p11.2. Activation of the MC2R initiates a cascade of events affecting multiple steps in adrenal cortisol production. The MC2R is dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP), which is essential for both trafficking of MC2R to the plasma membrane and for ACTH binding and activation of MC2R. MC2R–MRAP interactions may affect the trafficking of certain receptors to the cell membrane or allow activation of the receptor by its ligand. Specific mutations in the region of the N-terminal tail of MRAP1 and MRAP2 are essential for promoting only the trafficking of receptors to the plasma membrane (MRAP2) or essential for ACTH-MCR2 receptor ligand recognition and function (MRAP1). (see below).

 Mutations in the MC2 may result in familial glucocorticoid deficiency, a group of autosomal recessive disorders characterized by resistance to ACTH. It should be noted that although the MC2R is expressed predominantly in the adrenal cortex, it is also present in skin melanocytes where its ligand ACTH also binds to the MC1 thus affecting skin pigmentation. Indeed, chronically elevated ACTH in the circulation (chronic adrenal insufficiency or ectopic ACTH production or in Nelson’s syndrome following adrenalectomy) can induce skin and gum hyper-pigmentation. MC2R is also expressed in adipocytes and mediates stress-induced lipolysis via central ACTH release. The MC2R is localized in all three zones of the adrenal cortex. Results from binding studies indicate that in the adrenal cortex MC2R can be subdivided into a type with a KD of 1 nM, but with only 60 binding sites per cell and into a second type with a KD of 300 nM, but with several orders of magnitude more binding sites (about 600,000) per cell. The presence of high and low affinity receptors for ACTH means that the adrenal cortex is highly sensitive and specific to the usual concentrations of ACTH in the systemic circulation (30).  

Intra-Adrenal Regulation of Cortisol Production

The fasciculate cells of the adrenal cortex are affected by multiple factors produced within the adrenal gland. It should be noted that in addition to steroidogenic cells, the adrenals contain the chromaffin cells in the adrenal medulla arranged in columns crisscrossing the length of the gland, nerve fibers from intra- and extra-adrenal neurons, multiple cells of the immune system including monocytes / macrophages, mast cells, lymphocytes, vascular endothelial cells, and adipocytes within and around the gland. All these cells form complex intra-adrenal networks of interaction affecting, among other things, the response of fasciculata cells to ACTH, the expression of the MC2 receptors and their associated proteins, the growth and vascularization of the gland and many other functions. In addition, it has been also shown that the adrenal glands exhibit an intrinsic rhythmicity of corticosterone secretion in animal studies. Indeed, adrenal denervation leads to an abolishment of both the circadian corticosterone rhythm, as well as of the daily variation of the adrenal responsiveness to ACTH (9).

Role of Adrenal Chromaffin Cells

Chromaffin cells in the adrenal medulla originate from neural crest, their main products being the catecholamines epinephrine and norepinephrine. Chromaffin cells also produce neuropeptides and cytokines released together with catecholamines. Chromaffin cells are not clearly separated from the adrenal cortex as previously thought. Indeed, chromaffin cells can affect adrenal cortical cells in a paracrine mode of action since they can be found in all zones of the adult adrenal cortex up to the outer layer of the cortex i.e. zona glomerulosa and may form larger conglomerates of chromaffin in the adrenal subcapsular region. On the other hand, cortical cells are also located in the medulla, where they may form islets surrounded by chromaffin cells. This close association between cortical and chromaffin cells allows a paracrine regulation of adrenocortical steroidogenesis. Indeed, adrenal chromaffin cells synthesize a multitude of neuropeptides including beta-endorphin, the enkephalins, the dynorphins, CRH, substance P, adrenomedullin, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), atrial natriuretic peptide (ANP), somatostatin etc. These neuropeptides can affect either the response of cortical cells in zona fasciculate to ACTH, the vascularization of cortex, its growth, or they may exert direct modulatory effects on the cortical cells themselves. These effects on the adrenal cortex coordinate the two stress axes and streamline steroidogenesis as per the needs of the adaptation response to stressful stimuli (31-35). (Figure 3).

Figure 3. Autocrine and paracrine effects of intra-adrenally-produced substances on ACTH-induced cortisol synthesis.

Role of Peri- and Intra-Adrenal Adipocytes on the Adrenal Effects of ACTH

It is now suspected that the peri- and intra-adrenal adipocytes modulate the effects of ACTH on adreno-cortical cells. Thus, it has been shown that leptin exerts an inhibitory effect on ACTH-induced corticosteroid production by human adrenocortical cells without affecting their viability and proliferation. It should be noted that murine adipocyte cell lines and immortalized adipocytes express the MC2R suggesting that these adipocytes are also affected by ACTH (36-37).

The MC2 Signaling Pathway

As stated above, the MC2R is a G-protein coupled receptor. Among the G proteins Gs and Gi2 are implicated in ACTH signalling. ACTH also increases the transcription of G-alpha/q or G-q/11, a hetero-trimeric protein, which couples with the MC2R. G-q/11 activates the phospholipase C pathway. Mutations of the alpha subunits of Gs and Gi2 are associated with adrenocortical tumor formation. Signals that initiate from the MC2R and the G-proteins lead to cAMP formation and activation of PKA and PKC. As a result, several intermediate molecules are involved including kinases and transcription factors that orchestrate the ACTH actions on adrenal cells. The MC2R is a weak activator of MAP Kinases ERK1 and ERK2. ERK1 and ERK2 activation is important in ACTH-triggered mitogenic effects. In normal adrenal cortical cells, MC2 signals lead to activation of the Stress Activated Protein Kinase (SAPK) JNK. Activation of JNK depends on PKC activity and mobilization of intracellular Ca++ implying that both PKC activation and Ca++ influx result from the binding of ACTH to its receptor. In tissue culture experiments using the Y1 adrenocortical tumor cell line, ACTH exerts an antiproliferative effect, mediated by cAMP. ACTH signals result in dephosphorylation and inactivation of Akt/PKB kinase thus inhibiting the proliferation of adrenocortical tumor cells. Such anti-proliferative effect is most likely associated with increased steroidogenesis and suppression of the malignant phenotype of this particular cell line. The MC2R effects are mediated via activation of the cAMP pathway, which includes the cAMP-dependent transcription factors CREM (cAMP responsive element modulator) and CREB (cAMP responsive element binding protein) that result in transcriptional activation of steroidogenic enzymes, cell proliferation and differentiation. Activation of the MC2R leads to stimulation of Fos and Jun transcription, which by heterodimerizing form the AP1 complex. It should be noted here that the Fos gene family consists of four members, c-Fos, FosB, Fra1 and Fra2, while the Jun family consists of three members, c-Jun, JunB and JunD. These proteins form hetero- or homo- dimers inducing transcription through binding to AP1- binding sites. Activation of AP1-dependent transcriptions leads to the production of several pro-mitotic proteins while its inhibition results in a blockade of cell cycle to the G1 to S phase transition.

THE MELANOCORTINERGIC SYSTEM

 Conceptually, the fact that the ACTH receptor belongs to the melanocortin receptor family implies a close association between several physiological processes including stress, homeostasis, regulation of food intake and regulation of energy balance, immunity, and skin function. Indeed, ACTH can bind receptors in melanocytes, adipocytes, mononuclear/ macrophages and several areas within the central nervous system, with a much lower affinity compared to that of the MC2R. However, direct actions of ACTH through the MC2R have also been reported in several peripheral tissues. For instance, ACTH inhibits leptin secretion from adipocytes via the MC2R present in adipocytes, an affect indirectly contributing to the regulation of energy homeostasis during stressful periods (38). The melanocortinergic system in the central nervous system consists of the endogenous agonists alpha-, beta-, and gamma-MSH (post-translational products of POMC), the naturally occurring antagonists, the agouti-related protein (AGRP) produced by the arcuate nucleus neurons in hypothalamus and the agouti protein found in the skin. The AGRP antagonizes alpha-MSH in the hypothalamus at the level of MC3 and MC4R. The agouti protein and AGRP require the presence of a third protein, Mahogany, to antagonize MSH. Mahogany protein is widely expressed and it is a close relative of Attractin, an immunoregulatory protein made by human T lymphocytes.
Activation of the central melanocortin receptors (MC3 and MC4) by alpha MSH inhibits feeding and alters the rate of energy consumption leading to weight loss, whereas its blockade results in obesity. Development of MC3 and MC4 knockout mice revealed differential actions of each receptor. MC4 -/- mice were hyperphagic with partially increased metabolic efficiency while MC3 -/- animals developed obesity due to increased metabolic efficiency, thus underlying their significance in metabolism and obesity. The MCR is also involved in the regulation of autonomic nervous system tone and of arterial pressure at the level of the central nervous system. The MC receptor appears to be also involved in several higher learning processes. Outside the central nervous system, the MC4 receptor is expressed in osteoblasts where it may be involved in bone remodeling facilitating the communication between osteoblasts and osteoclasts (39-41) (Table). 

The MC1 receptor (MC1R) is a 315 amino acid transmembrane protein which in humans is mapped to 16q24. It is the principal melanocortin receptor in the skin where it regulates its pigmentation. It exhibits high affinity for most MSH isoforms and a much lower affinity for ACTH. Its highest affinity is towards alpha–MSH (Ki = 0.033 nmol/l). Stimulation of MC1R in the skin and the hair follicles by alpha-MSH results in induction of melanogenesis producing dark skin and hair in several species including the humans. The MC1R is also present in the adrenals, the leukocytes, lungs, lymph nodes, ovaries, testes, pituitary, placenta, spleen and the uterus. The agouti protein is an endogenous antagonist of alpha-MSH at the level of the MC1R in the skin. Over-expression of the agouti protein results in fair skin, reddish hair and disturbances of energy balance. Variants of the MC1R in humans are associated with red hair, pale skin, and increased risk for skin cancer. The MC1R in leukocytes and macrophages has been associated with the immune effects of alpha-MSH (42).

The MC3 receptor (MC3R) is expressed mainly in the brain. In humans it is a 360 amino acid protein and maps to 20q13.2. The MC3R and the MC4R in the Central Nervous System regulate food intake and energy homeostasis. Knockout (KO) animals for these receptors are obese. The MC4R KO mice are hyperphagic while the MC3R KO animals are not hyperphagic but still obese signifying the effect of this receptor on the overall energy homeostasis. The agouti and the agouti-related protein are endogenous natural antagonists of the MC1R, MC3R and MC4R. Finally, the MC3R may be involved in the mechanism turning off the inflammatory response mainly via suppression of macrophage migration. In the brain the MC3R is mainly expressed in the arcuate nucleus at the basis of hypothalamus where it regulates hunger and satiety.

The MC4 receptor (MC4R) is a 332 amino acid trans-membrane protein. It is expressed in the central nervous system (mainly in the hypothalamus), the gastrointestinal tract and the placenta. In humans, it maps to 18q22. The MC4R is a major regulator of food intake. Inactivating mutations of MC4R cause obesity both in mice and humans. Global homozygous deletion of MC4R in mice results in hyperphagia, increased fat and lean mass, increased body length, reduced activity, and a suppressed metabolic rate. Inactivating mutations in MC4R are the single most common form of monogenic obesity in humans. Common variants near the MC4R locus are associated with adiposity, body weight, risk of obesity, and insulin resistance. In addition to the homeostasis of energy and thermogenesis the MC4R receptor plays other roles including regulation of autonomic control of blood pressure. Finally, the MC4R plays an important role in the production of the neuropeptides YY and glucagon-like peptide 1 by the enteroendocrine cells (43-47).

 

The MC5 receptor (MC5R) is a 325 amino acid trans-membrane protein. It is expressed in the adrenals, skin, stomach, lung and spleen. Its levels in the central nervous system are very low. In the adrenal cortex, it is expressed in all three layers but predominantly in the aldosterone-producing zona glomerulosa cells. The presence of MC5R expression in zona glomerulosa may be involved in melanocortin-induced aldosterone production. In the skin, the MC5R affects exocrine function. It is expressed in peripheral lymphocytes and in splenocytes indicating that this may be the receptor utilized by ACTH in those cells. MC5R is expressed in articular chondrocytes mediating cytokine production in the inflamed joints in rheumatoid arthritis. The MC5 receptor also mediates the production of IL-6 from adipocytes contributing to metabolic inflammation and insulin resistance. Indeed, stimulation of the MC5R in 3T3-L1 adipocytes with αMSH induces lipolysis and suppresses re-esterification of fatty acids through the ERK1/2 pathway.

Table. The Melanocortin Receptor Family

Receptor

Ligand affinity

Main site of expression Primary Function

Primary Function

Disease phenotype with loss of function mutations

MC1R

αMSH =ACTH>βMSH> γMSH

 

Melanocytes

Pigmentation, inflammation

Increased risk for skin cancer

MC2R

ACTH only

Adrenal cortex

Adrenal steroidogenesis

FGD

MC3R

γMSH> αMSH =βMSH>ACTH

CNS, GI tract, Kidney

Energy homeostasis, inflammation, food intake

Obesity

MC4R

αMSH = βMSH = ACTH >> γMSH

CNS

Energy homeostasis, thermogenesis, appetite regulation, erectile

Obesity

MC5R

αMSH > ACTH>βMSH> γMSH

Lymphocytes, exocrine cells

Exocrine function, regulation of sebaceous glands

Decreased production of sebaceous lipids in mice

Abbreviations: MSH: Melanocyte-stimulating hormone, CNS: central nervous system, GI: gastrointestinal tract, FGD: familial glucocorticoid deficiency.

Regulation of MC2 Receptor Gene Expression

The MC2R gene has one untranslated exon (exon one), an 18kb intron, and the coding exon (exon two). The existence of different MC2 transcripts in human adrenal cortical cells suggests the presence of multiple transcription initiation sites. An alternate exon 1 (exon1f) is transcribed in adipose tissue but not in the adrenals. This exon appears to be transcribed by a different promoter region from that reported in the adrenal, thus conferring tissue specificity. Studies on the MC2 promoter polymorphism reveal a single nucleotide polymorphism close to the transcriptional initiation site (-2C/T) resulting in inhibition of transcription causing reduced MC2 levels even in the heterozygous state. This allele is present in 10% of the population.

The MC2R promoter contains binding sites for several transcription factors. Transcription factors are nuclear proteins modifying the expression of genes by binding to specific DNA sequences usually located upstream of gene promoters. Phosphorylation of a transcription factor results in its activation and modulation of the transcriptional activity of a promoter containing response elements for the specific factor (48).

Factors Affecting the Expression of MC2 Receptor Gene

EFFECTS OF ACTH ON THE EXPRESSION OF MC2R

Several studies have shown that the MC2R gene is up regulated by its own ligand, ACTH. Indeed, ligand-induced up-regulation of MC2R expression may be a crucial adaptive process directed towards optimizing adrenal responsiveness to ACTH. The effect of ACTH on MC2R expression is dependent on cAMP and probably mediated through AP-1(49).

EFFECTS OF GLUCOCORTICOID REGULATORY ELEMENTS (GRE) ON THE MC2R GENE

Glucocorticoids are major regulators of MC2 expression. Glucocorticoids exert an enhancing effect on basal, ACTH- and cAMP-induced MC2 expression.

Steroidogenic Factor-1 (SF-1) is an orphan nuclear receptor. The MC2R gene contains three SF-1 binding sites in the proximity of the transcription initiation site. In addition to its effect on the transcription of the MC2R gene, SF-1 also affects the transcription of genes involved in steroidogenesis in the adrenals and the gonads as well as the organogenesis of both glands. SF-1 knockout mice lack adrenal glands and gonads. SF-1 is also essential for the compensatory adrenal growth following unilateral adrenalectomy. In steroidogenesis, SF-1 affects the transcription of CYP11A1 gene which encodes the P450scc cholesterol side-chain cleavage enzyme, the first step in steroidogenesis. Several SF-1-binding sites on the promoter of CYP11A1 modulate its transcription rate (50).

DAX-1 (Dosage-sensitive sex reversal, Adrenal hypoplasia congenital critical region on the X chromosome, gene 1) is a transcription factor expressed in the adrenal gland and gonads. DAX-1 encodes an orphan member of the nuclear hormone receptor super family. DAX-1 inhibits SF-1-mediated steroidogenesis while its absence augments the adrenal responsiveness to ACTH most probably through an up-regulation of the MC2R transcription via SF-1. A cAMP-dependent PKA augments the SF-1-mediated induction of steroidogenesis. Generally speaking, DAX-1 is a suppressor of the transcription of several genes involved in the steroidogenic pathway. Indeed, inactivating mutations of DAX-1 results in the X-linked form of adrenal hypoplasia congenital (AHC) with associated hypogonadotropic hypogonadism. AHC presents as adrenal failure in early infancy, although a wide range of phenotypic expressions have been reported. Interestingly, the MC2 promoter contains several DAX-1 sites. As expected, DAX-1 suppresses the expression of the MC2 gene when transfected in adrenocortical Y-1 cells. In adrenocortical tumors there is a distinct negative correlation between DAX-1 and MC2 (51-52).  Steroidogenic acute regulatory protein (StAR) does not appear to affect the MC2 promoter but regulates steroidogenesis, an effect augmented by ACTH via the MC2R. StAR promotes intra-mitochondrial cholesterol transfer in the adrenal cortical cells. StAR is thus the only major adrenal transcription factor which has not been associated with the expression of the MC2R gene (53).

The activator protein-1 regulatory element (AP-1) is the product of the hetero-dimerization of the proto-oncogenes Fos and Jun following activation of several signalling pathways including that of PKA and PKC. Two AP-1 binding sites have been identified upstream of the MC2R. Deletion of the AP-1 binding sites on MC2 gene abolishes the stimulatory effect of cAMP. The effect of glucocorticoids and Angiotensin II on the expression of MC2R gene is carried out via a glucocorticoid-mediated inhibition of AP-1 binding sites on the ACTH receptor promoter. The angiotensin II protein stimulates the expression of MC2R gene in the adrenal cortex. Promoter deletion studies revealed that the two AP1 binding elements on MC2 promoter mediate the Angiotensin II stimulatory signals. Indeed, Angiotensin II rapidly activates Fos and Jun to promote MC2 transcription.

The MC2 Accessory Proteins MRAP and MRAP2

For many years’ researchers, in the field of adrenal physiology, suspected that an unidentified adrenal factor was needed in order for the effect of ACTH to take place. Indeed, ACTH was effective only in transfected cells with the MC2R of the adrenal lineage. In other transfected cell with the MC2R, ACTH was ineffective i.e. a crucial factor present only in cells of adrenal lineage was necessary for the effect of ACTH to take place. It was ssubsequently found that MC2R depended, for its trafficking to cell surface, on a small single trans-membrane domain protein the malfunction of which caused a clinical syndrome indistinguishable from that caused by the absence or malfunction of the MC2R. This was shown to be the MC2 accessory protein (MRAP).

MRAP is peculiar in that it naturally exists as an antiparallel homodimer formation (MRAPalpha and MRAPbeta) each pair associated with the MC2R. Later it was also shown that the MRAP protein is necessary not only for the trafficking of the receptor to cell surface, but also for conformational changes necessary for the binding of the ACTH ligand either by influencing ACTH ligand binding or by facilitating the interaction of the Gas protein with the receptor, or both (54). The MPAR gene is mapped in human chromosome 21 (C21orf61) corresponding to a murine adipocyte transmembrane protein. Two isoforms have been identified each conferring a different affinity of the MC2 receptor towards ACTH, thus explaining the observed two subpopulations of MC2 receptor as far as its affinity towards the ACTH is concerned (see above). MRAP has no effect on the trafficking of either MC1R or MC3R, while it may suppress the trafficking of MC4R and MC5R to cell surface (55-60) (Figure 4).

 

Figure 4. ACTH receptor protein expression. MC2R mRNA is translated at the endoplasmic reticulum and is unable to traffic beyond this point to the plasma membrane. MRAP mRNA is translated and adopts an anti-parallel homodimeric conformation at the endoplasmic reticulum. Only the MRAP-MC2R membrane complex is competent to bind ACTH at physiological concentrations and to generate a steroidogenic signal.

THE FAMILIAL GLUCOCORTICOID DEFICIENCY (FGD) SYNDROMES

Hereditary ACTH resistance syndromes encompass the genetically heterogeneous isolated or Familial Glucocorticoid Deficiency (FGD) and the distinct clinical entity known as Triple A syndrome. The molecular basis of adrenal resistance to ACTH includes defects in ligand binding, MC2R/MRAP receptor trafficking, cellular redox balance, cholesterol synthesis, and sphingolipid metabolism. Biochemically, this is manifested by ACTH excess in the setting of hypocortisolemia.

FGD is an autosomal recessive condition characterized by the presence of isolated glucocorticoid deficiency, classically in the setting of preserved mineralocorticoid secretion. Primarily there are three established subtypes of the disease: FGD 1, FGD2 and FGD3 corresponding to mutations in the MRC2R (25%), Melanocortin 2 receptor accessory protein MRAP (20%), and Steroidogenic acute regulatory protein STAR (5–10%), respectively. Mutations in these 3 genes account for approximately half of cases. Whole exome sequencing in patients negative for MC2R, MRAP and STAR mutations, identified mutations in mini-chromosome maintenance (MCM), nicotinamide nucleotide transhydrogenase (NNT), thioredoxin reductase 2 (TXNRD2), cytochrome p450scc (CYP11A1), and sphingosine 1-phosphate lyase (SGPL1), accounting for a further 10% of FGD. These novel genes have linked replicative and oxidative stress and altered redox potential as a mechanism of adrenocortical damage. However, a genetic diagnosis is still unclear in about 40% of cases (61).

The FGD syndromes are autosomal recessive diseases characterized by atrophic zona fasciculata and zona reticularis, accompanied by low plasma cortisol levels and elevated ACTH. FGD syndromes exhibit an isolated defect in the endogenous production of cortisol without a parallel defect in the production of aldosterone. The cortisol insufficiency is usually accompanied by hyperpigmentation of the skin and of the mucous membranes due to the high levels of circulating ACTH activating the cutaneous MCR. Recurrent episodes of hypoglycemia are also present due to the lack of the counter-regulatory effect of cortisol on the hypoglycemic effects of insulin. The affected neonates present with failure to thrive, repeated episodes of hypoglycaemia, and seizures.

Several types of FGD are recognized as per the pathophysiological defect on the ACTH receptor pathway. The type 1 FGD and type 2 FGD cause ‘pure’ isolated glucocorticoid deficiency, however over the last 25 years it has become clear that glucocorticoid deficiency itself may occur as part of a syndrome with a much more complex clinical picture.

The defect in type 1 FGD is localized in the MC2R gene usually consisting of single point mutations. These inactivating mutations of the MC2R may result from the introduction of a stop codons within the coding region of the ACTH receptor, frameshift mutations, and mutations that cause single amino acid substitutions and structural disruption of the ACTH receptor affecting the ligand-binding domain resulting in loss of ligand-binding capability. Type 1 FGD represents approximately 25-40% of all patients with FGD.

The defect in type 2 FGD appears to be due to mutations in the MC2R accessory protein, the MRAP mentioned above. It represents around 15-20% of all cases of. At least 8 different mutations in MRAP have been identified in type 2 FGD patients. Most mutations of MRAP cluster around the first coding exon (exon 3) especially at the splice donor site. The same mutation has been found in genetically unrelated individuals suggesting that this is a true ‘hot spot’ area for mutation. The other common site for mis-sense mutations is in the initiator methionine. This mutation prevents translation of the full-length protein. The next in-frame methionine is at position 60 which, if translated, would result in a severely truncated protein. The adrenal histology of FGD type 2 is typical of all other cases of FGD. They are characterized by a relatively preserved glomerulosa cell layer with highly atrophic and disorganized fasciculata and reticularis cell layers.

The defect in type 3 FGD concerns the regulatory alacrima-achalasia-adrenal insufficiency neurologic defect (ALADIN) protein causing the Allgrove syndrome.

The defects in the remaining cases of FGD are attributed in problems within the MC2R signaling transduction. Mutations in the intracellular portion of the MC2R may result in the loss of its signal transduction properties. Absence of a biological response to ACTH may thus be due to impaired binding of ACTH to its receptors or inability of the bound ACTH to initiate its post-receptor effects (62-65).

THE ACHALASIA-ADDISONIANISM-ALACRIMA (TRIPLE A) OR ALLGROVE SYNDROME

The triple A syndrome is caused by mutations in the gene encoding the regulatory protein ALADIN, a product of the ADRACALIN gene. ALADIN is a WD-repeat regulatory protein, part of the nuclear pore complex. It is crucial for the development of the peripheral and central nervous system. Mutations of ALADIN lead to a syndrome characterized by achalasia, alacrima, and addisonism (66-72). The underlining pathology of this syndrome appears to be a systemic and progressive loss of cholinergic function.

Alacrima is often manifested at birth, the patients exhibiting conjunctival irritation which if not treated leads to severe keratopathy and corneal dehydration-induced ulcerations. Alacrimia is diagnosed by Schirmer's test.

Achalasia is a neuromuscular disorder of the esophagus resulting in elevated lower esophageal sphincter pressure and lack of peristaltic waves of the esophagus, and recurrent lung infections resulting in respiratory failure.

The neurologic manifestations of the disease include motor neuron disease-like presentations, motor-sensory or autonomic neuropathy, optic atrophy, cerebellar ataxia, Parkinsonism, and mild dementia. The autonomic nervous system dysfunction may be manifested as papillary abnormalities, an abnormal reaction to histamine test, abnormal sweating, orthostatic hypotension, and disturbances of the heart rate. Cognitive deficits, pyramidal syndrome, cerebellar dysfunction, dysautonomia, neuro-ophthalmological signs and bulbar and facial symptoms also occur. The neurological features may appear at a later age.

Only half of the patients develop adrenal insufficiency accompanied by episodes of hypoglycemia which intensify the problems of cognition.

Using genetic linkage analysis, a causative locus has been identified on chromosome 12q13 coding the alacrima-achalasia-adrenal insufficiency neurologic defect (ALADIN) regulatory protein, a product of the ADRACALIN gene which is encoding the ALADIN protein of the nuclear pore complex. This protein is crucial in the development of the nervous system, especially its peripheral parts. Several mutations have been described including homozygous mutations of c.771delG (p.Arg258GlyfsX33) in exon 8 and c.1366C>T (p.Q456X) in exon 15 and a missense mutation in p.R155H.

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Sexual Differentiation

ABSTRACT

 

The chromosomal sex of the embryo is established at fertilization. However, 6 weeks elapse in humans before the first signs of sex differentiation are noticed. Sex differentiation involves a series of events whereby the sexually indifferent gonads and genitalia progressively acquire male or female characteristics. Believed initially to be governed entirely by the presence or absence of the SRY gene on the Y chromosome, gonadal determination has proven to rely on a complex network of genes, whose balanced expression levels either activate the testis pathway and simultaneously repress the ovarian pathway or vice versa. The presence or absence of primordial germ cells, of extragonadal origin, also has a sexually dimorphic relevance. Subsequently, internal and external genitalia will follow the male pathway in the presence of androgens and anti-Müllerian hormone (AMH), or the female pathway in their absence. Here we review the sexually undifferentiated stage of embryonic development, and the anatomic, histologic, physiologic and molecular aspects of the fetal sexual differentiation of the gonads, the internal reproductive tract and the external genitalia.

 

INTRODUCTION

 

Genital sex differentiation involves a series of events whereby the sexually indifferent embryo progressively acquires male or female characteristics in the gonads, genital tract and external genitalia. Sex development consists of several sequential stages. Genetic sex, as determined by the chromosome constitution, drives the primitive gonad to differentiate into a testis or an ovary. Subsequently, internal and external genitalia will follow the male pathway in the presence of specific testicular hormones, or the female pathway in their absence. Since the presence of the fetal testis plays a determining role in the differentiation of the reproductive tract, the term "sex determination" has been coined to designate the differentiation of the gonad during early fetal development.

 

THE BIPOTENTIAL GONAD

 

No sexual difference can be observed in the gonads until the 6th week of embryonic life in humans and 11.5 days post-coitum (dpc) in mice. Undifferentiated gonads of XX or XY individuals are apparently identical and can form either ovaries or testes. This period is therefore called indifferent or bipotential stage of gonadal development.

 

The Gonadal Ridge

 

The urogenital ridges are the common precursors of the urinary and genital systems and of the adrenal cortex (1). In the human, they develop during the 4th week post-fertilization at the ventral surface of the cranial mesonephroi, and are formed by intermediate mesoderm covered by coelomic epithelium. Each urogenital ridge divides into a urinary and an adreno-gonadal ridge in the 5th week (Table 1). The adreno-gonadal ridge is the common precursor of the gonads and adrenal cortex. The gonadal ridge is bipotential and can develop into an ovary or a testis. Gonads are subsequently colonized by the primordial germ cells, of extra-gonadal origin. The mesonephroi also give rise to components of the internal reproductive tract and of the urinary system.

 

The molecular mechanisms underlying the specific location of the gonads on the surface of the mesonephroi begin to be unveiled in chicken embryos, where Sonic hedgehog (SHH) signaling mediated by the bone morphogenetic protein 4 (BMP4) establishes the dorsoventral patterning of the mesoderm and induces coelomic epithelium cell ingression, thus probably initiating gonadal development (2). However, since there are significant differences in gonadal development between birds and mammals, these mechanisms need to be explored to establish whether they are conserved amongst vertebrates.

 

TABLE 1. Chronology of Human Sex Differentiation*

Age from conception

CR length (mm)

Event

22 days

2-3

Intermediate mesoderm becomes visible

Primordial germ cells in the yolk sac

24 days

2.5-4.5

Formation of solid Wolffian ducts

Primordial germ cells migrate to the hindgut

26 days

3-5

Wolffian ducts develop a lumen

Primordial germ cells in the hindgut

28 days

4-6

Primordial germ cells migrate to the urogenital ridges

32 days

5-7

Gonadal primordia develop

Growth of Wolffian ducts

33-37 days

7-11

Primordial germ cells reach gonadal ridge

Urogenital sinus is distinguishable

Differentiation of Müllerian ducts

Genital tubercle and urethral folds are visible

41-44 days

11-17

Seminiferous cord differentiation

Differentiation between pelvic and phallic parts of the urogenital sinus

44-50 days

15-20

Seminiferous cords with germ cells

50-60 days

30

Beginning of secretion of AMH

Leydig cell differentiation

Cranial part of Müllerian ducts begins to regress

9 weeks

40

Leydig cells produce testosterone

Beginning of masculinization of urogenital sinus and external genitalia

10 weeks

45-50

Meiotic entry of oocytes in the medulla

Beginning of degeneration of female Wolffian ducts

Male Müllerian ducts have disappeared

Prostatic buds appear

12 weeks

55-60

The vaginal cord is formed

Primordial follicles appear

Seminal vesicles develop

Testis at internal inguinal ring

14 weeks

70

Completion of male urethral organogenesis

16 weeks

100

Primary follicles appear

20 weeks

150

Testosterone serum level is low

Formation of prostatic utricle

22 weeks

180

Vagina reaches perineum

24 weeks

200

Graafian follicles appear

Beginning of penile growth

27-30 weeks

230-265

Inguino-scrotal descent of the testis

36 weeks

300

Secondary and tertiary follicles produce AMH

* According to O’Rahilly (3).

 

Several general transcription factors belonging to the large homeobox gene family play an important role in the stabilization of the intermediate mesoderm and the formation of the urogenital ridges (Table 2). Mice in which Lhx1 (4), Emx2 (5, 6) or Pax2 (7) have been inactivated fail to develop urogenital derivatives. Most of these ubiquitous factors are essential for the development of other vital embryonic structures. However, another LIM homeobox gene, Lhx9, seems to be essential only for the proliferation of somatic cells of the gonadal ridge (8) by interacting with Wt1 to regulate Sf1 (9). LHX9 expression increases in both XX and XY undifferentiated gonads, and then decreases as Sertoli and granulosa cells differentiate (10, 11). Several other factors are involved in cell proliferation in the gonadal primordium both in XX and XY embryos. For instance, impairment of the signaling pathway of the insulin/insulin-like growth factor family in mouse knockout models with disrupted Insr, Igf1r and Insrr leads to a significant reduction of the size of adreno-gonadal ridges in both XX and XY embryos (12). Also in mice with a knockout of Tcf21, gonads are severely hypoplastic in both XX and XY fetuses (13). GATA4 (14) and the homeoproteins SIX1 and SIX4 are also essential for early proliferation of gonadal precursor cells and for FOG2- and SF1-regulated SRY expression (15). The Notch signaling pathway is also involved in somatic cell lineage commitment during early gonadogenesis in mice. Conditional knockout of Numb and Numbl (antagonists of Notch signaling) in the undifferentiated gonad results in disruption of the coelomic epithelium and reduction of somatic cell numbers in the gonads (16). Finally, NRG1 is also required in a dose-dependent manner in order to induce somatic cell proliferation in the gonads (17). Since cell proliferation is more important in the male than in the female early developing gonad (18, 19), sex-reversal is often observed in XY embryos with an alteration of gonadal cell proliferation (12). It has been suggested that this is due to a reduction in the number of SRY-expressing pre-Sertoli cells, resulting in very low levels of SRY expression that are insufficient to trigger testicular differentiation (discussed in ref. (20).

 

TABLE 2. Factors Involved in Early Gonadal Ridge Development

Gene

Chromosomal localization

Expression

Function

ATRX (Alpha-thalassemia/mental retardation syndrome, Helicase 2, X-Linked)

Xq21.1

Widespread

Nucleotide excision repair and initiation of transcription

CITED2 (CBP/p300-interacting transactivator, with glu/asp-rich c-terminal domain, 2)

6q24.1

Widespread

WT1 cofactor, regulating SF1expression in the adrenogonadal primordium

EMX2 (homolog of empty spiracles homeobox gene 2)

10q26.11

Telencephalon and epithelial components of the urogenital system

Arealization of the neocortex and induction of the mesenchyme

GATA4 (GATA-binding protein 4)

8p23.1

Widespread

Regulation of coelomic epithelium thickening

INSR (Insulin receptor)

IGF1R (Insulin growth factor 1 receptor)

INSRR (Insulin receptor-related receptor)

19p13.2

15q26.3

 

1q23.1

Widespread

Metabolic, cell proliferation

JMJD1A, or KDM3A(Lysine-Specific Demethylase 3A)

2p11.2

Testis, ovary, kidney, lung, heart, brain, liver, skeletal muscle, pancreas, and spleen

Demethylases histone H3 (epigenetic regulation by modification of chromatin conformation)

LHX1 (LIM homeobox gene 1)

17q12

Primitive streak, prechordal and intermediate mesoderm, brain, thymus, tonsil

Differentiation and development of the head, neural and lymphoid tissues and urogenital structures

LHX9 (LIM homeobox gene 9)

1q31.3

Central nervous system, forelimb and hind limb mesenchyme and urogenital system

Activation of SF1 in gonadal primordia

NR5A1 (Nuclear receptor subfamily 5, group A, member 1, also SF1: Steroidogenic factor 1, or AD4BP: Adrenal 4 binding protein, or FTZF1: Fushi tarazu factor homolog 1)

9q33.3

Gonadal ridges, adrenal gland primordia, hypothalamus and pituitary

Stabilization of intermediate mesoderm, and transcriptional regulation of several genes (StAR, steroid hydroxylases, aromatase, AMH, DAX1 and many other)

NRG1 (Neuregulin 1)

8p12

Widespread, including progenitors of somatic gonadal cells

Progenitor cell proliferation in the gonads

NUMB

and

NUMBL

14q24.2-q24.3

and

19q13.2

Widespread, including coelomic epithelium

Antagonize NOTCH signaling, involved in mediating asymmetric division of cells in the coelomic epithelium

PAX2 (Paired box gene 2)

10q24.31

Mesonephros, metanephros, adrenals, spinal cord, hindbrain and optic and otic vesicles

Regulation of WT1 expression and of mesenchyme- to- epithelium transition

SIX1 / SIX 4 (Sine oculis homeobox 1 and 4)

14q23.1

Urogenital ridge derivatives

Regulation of gonadal precursor cell proliferation, and of Fog2 and Sf1

TCF21 (Transcription factor 21, also POD1: Podocyte-expressed 1)

6q23.2

Epithelium of the developing gastrointestinal, genitourinary, and respiratory systems

Basic helix-loop-helix transcription factor

WT1 (Wilms tumor associated gene 1)

11p13

Urogenital ridge derivatives

DNA- and RNA-binding protein with transcriptional and post-transcriptional regulating capacity

 

The differentiation of the gonadal ridge from the intermediate mesoderm requires the expression of sufficient levels of WT1 and SF1. WT1 was initially isolated from patients with Wilms' tumor, an embryonic kidney tumor arising from the metanephric blastema. By alternative splicing and alternative translation initiation, WT1 encodes more than 20 isoforms of a zinc-finger protein acting as transcriptional and/or post-transcriptional regulator (20). The -KTS splicing variant of WT1, lacking the three amino acids lysine (K), threonine (T) and serine (S) at the end of the third zinc finger, is required for cell survival and proliferation in the indifferent gonad, whereas the +KTS variant is involved in the regulation of SRY expression (21). The first indication of a role for WT1 in gonadal and renal development was its expression pattern in the urogenital ridges (22). During gonadal differentiation, WT1 is expressed in the coelomic epithelium and later in Sertoli and granulosa cells (23). In mice with a knockout of WT1, neither the kidneys nor the gonads develop (24). In humans, mutations in the WT1 gene do not completely prevent urogenital ridge development but may result in gonadal dysgenesis associated with nephroblastoma (Wilms' tumor) and/or nephrotic syndrome owing to glomerular diffuse mesangial sclerosis (25-27).

 

SF1, also known as Ad4BP or FTZF1 (HGNC approved gene symbol: NR5A1), initially described as a regulator of steroid hydroxylases, is an orphan nuclear receptor expressed in the hypothalamus, the pituitary, the gonads and the adrenal glands (reviewed in refs. (28-30). In mice with a knockout of the SF1 gene, the intermediate mesoderm is not stabilized and the gonadal and adrenal primordia soon degenerate (31). SF1 also plays an important role in spermatogenesis, Leydig cell function, ovarian follicle development and ovulation, as demonstrated by a gonad-specific disruption of SF1 (32). A recurrent heterozygous p.Arg92Trp variant of the gene is associated with testicular development in XX subjects (33, 34). WT1, through interaction with CITED2 (35, 36), and LHX9 (8) regulate the expression of SF1 upstream of the gonadal development cascade. GATA4 and SOX-family factors also regulate SF1 expression in the gonad (28). In humans, the phenotype resulting from SF1 mutations does not exactly match that of Sf1 knockout mice: the clinical spectrum includes severe and partial forms of testicular dysgenesis, anorchidism, and even male infertility in normally virilized individuals; adrenal insufficiency is not always present. In 46,XX females, SF1 mutations have been described in patients with primary ovarian insufficiency (29, 30). SF1 is one of the increasing number of examples of dosage-sensitive mechanisms in human sex differentiation, since mutations at the heterozygous state are sufficient to induce sex reversal in XY individuals (reviewed in refs. (29, 30).

 

Recent studies using single-cell RNA sequencing (scRNA-seq) has shed light on the initial steps of lineage trajectories and cell fate in the developing gonads (1, 37). A subset of cells of the coelomic epithelium expressing GATA4, SF1 and WT1 are likely to be the precursors of the somatic lineages of the undifferentiated gonads: both the supporting (Sertoli and granulosa) and the steroidogenic (Leydig and theca) cell populations of the differentiating gonads seem to derive from SF1 and WT1-expressing cells present in the genital ridge (1, 37, 38).

 

The Germ Cells

 

Initially formed exclusively by somatic cells, the gonads are subsequently colonized by the primordial germ cells (PGCs). PGCs derive from pluripotent cells of the posterior proximal epiblast, which move, at a very early stage of embryonic life, through the primitive streak into the extra-embryonic region at the base of the allantois (39). Not all of these cells are committed to a germ cell lineage since they also give rise to extra-embryonic mesoderm cells (40).

The mechanisms responsible for specification of epiblast cells to become PGCs vary between species (41-43). In mice, PCG specification involves several extraembryonic ectoderm-derived factors, including bone morphogenetic protein 2 (BMP2) (44), BMP4 (45-47), BMP8B (46) and WNT3 (48). Cells of the adjacent epiblast become determined to develop through the germline as they start expressing BLIMP1 (44), encoded by Prdm1. BLIMP1 represses somatic fate in the epiblast cells, and together with PRDM14 and AP2G (encoded by Tfap2c), constitute a tripartite genetic network necessary and sufficient for mouse PGC specification (49). PRDM14 regulates the restoration of pluripotency and epigenetic reprogramming in PGCs, reestablishing the expression of the pluripotency factors OCT3/4 (encoded by Pou5f1), SOX2 and NANOG (41).

 

Instead, embryos of other mammals do not form a structure equivalent to the extraembryonic ectoderm, and the origin of the signals that initiate PGC specification remain largely unknown. Notably, in the human embryo, PGC-like cells express very low or no PRDM14, maintain NANOG expression, and do not express SOX2. Furthermore, the expression of SOX17 is detected before that of BLIMP1 and could be involved in the regulation of PGC specification and maintenance of their pluripotency in humans (49, 50).

 

Widespread chromatin modifications are observed: PGCs undergo genome-wide demethylation including erasure of genomic imprints (44), thus reaching a ‘ground state’ in terms of epigenetic marks. Re-methylation of germ cell genome occurs later during fetal life: in XY germ cells when they have committed to the spermatogenic fate, and in XX germ cells just before ovulation (45).

In the 4thweek, PGCs have migrated and are present in the yolk sac near the base of the allantois. They can be identified by their expression of alkaline phosphatase, OCT3/4 and the tyrosine kinase receptor C-KIT (Fig. 1A) (40). Subsequently, PGCs become embedded in the wall of the hind gut, gain motility and migrate through the dorsal mesentery to reach the gonadal ridges in the 5thweek (Fig. 1B). Early migration of PGCs is dependent on the expression of interferon-induced transmembrane proteins 1 and 3 (IFITM1 and IFITM3) in the surrounding mesoderm (51). During migration, PGCs proliferate actively but do not differentiate (40). Germ cell migration through the dorsal mesentery to the gonadal ridges and survival/proliferation in both XX and XY embryos is driven by signaling between kit ligand (KITL, also known as Stem cell factor [SCF], Steel factor or mast cell growth factor [MGF]), which is expressed in somatic cells of the gonadal ridge and the hind gut along the pathway of PGC migration, and its receptor present in germ cells, C-KIT (Fig. 1) (52). PGC migration and genital ridge colonization is also dependent on stromal cell-derived factor 1 (SDF1, also known as CXCL12) and its receptor CXCR4 (53) and on interactions with extracellular matrix proteins, like fibronectin and laminin, while proliferation and/or survival involve many other factors (39, 40, 52, 54).

 

PGCs are in a bipotential state when they colonize the gonadal ridges, i.e. they still have the capacity to enter either spermatogenesis or oogenesis. Shortly afterwards, induced by the gonadal environment, PGCs begin to express DAZL, DDX4 (also known as MVH) and low levels of SYCP3 (43), probably owing to promoter demethylation (55). DAZL seems to induce PGCs capacity to respond to specific male or female gonadal signals (56, 57).

FIGURE 1. Regulation of Germ Cell Migration. A: 4-week embryo. Differentiation of primordial germ cells (PGC) occurs from epiblast-derived cells present in the yolk sac near the base of the allantois. PGCs express PMRD1, the receptors C-KIT and CXCR4, OCT3/4 and alkaline phosphatase. Fibronectin and laminin, together with KITL, SDF1 and IFITM 1 and 3 are expressed in the mesoderm along the PGC pathway. B: 5-week embryo. PGCs migrate along the dorsal mesentery of the hind gut to the gonadal ridges.

SEX DETERMINATION

 

The Determining Role of Testicular Differentiation

 

The pioneering experiments of fetal sexual differentiation carried out by Alfred Jost in the 1940’s clearly established that the existence of the testes determines the sexually dimorphic fate of the internal and external genitalia (Fig. 2)(58, 59). Irrespective of their chromosomal constitution, when the gonadal primordia differentiate into testes, all internal and external genitalia develop following the male pathway. When no testes are present, the genitalia develop along the female pathway. The existence of ovaries has no effect on fetal differentiation of the genitalia. The paramount importance of testicular differentiation for fetal sex development has prompted the use of the expression “sex determination” to refer to the differentiation of the bipotential or primitive gonads into testes.

 

In the next section, we describe the morphological aspects of fetal testicular and ovarian differentiation and the underlying molecular mechanisms, involving genes mapping to sex-chromosomes (Fig. 3) and autosomes (Table 3).

FIGURE 2. Determining role of the testes in fetal sex differentiation. In normal females, Müllerian ducts are maintained, Wolffian ducts regress. In males, the opposite occurs. In castrated fetuses, irrespective of genetic or gonadal sex, the reproductive tract differentiates according to the female pattern.

The Fate of the Undifferentiated Gonadal Ridge

 

As already mentioned, the gonadal ridges are bipotential until the 6th week after conception in humans, i.e. they have the capacity to follow the testicular and the ovarian pathways. The discovery of the testis-determining factor SRY in 1990 was followed by the progressive unveiling of robust networks of genes, whose balanced expression levels either activate the testis pathway and simultaneously repress the ovarian pathway or vice versa (Fig. 4). During the formation of the undifferentiated gonadal ridges, a common genetic program is established in the supporting-cell lineage deriving from the multipotent somatic progenitor cells in both XX and XY embryos, characterized by a balanced expression of pro-Sertoli (SOX9, FGF9, PGD2) and pregranulosa (WNT4, RSPO1, FST and CTNNB1) genes (37, 60). Under physiological conditions in the XY gonad, the upregulation of SRY induces a destabilization of that balance, initiating the testis cascade.

 

THE MALE DETERMINING PATHWAY

 

Sex-Determining Region on the Y Chromosome (SRY)

 

Compelling evidence for the importance of the Y chromosome for the development of the testes, irrespective of the number of X chromosomes present, has existed since 1959 (61, 62). However, the identification of the testis-determining factor (TDF) on the Y chromosome did not prove easy and several candidates (e.g. HY antigen, ZFY) were successively proposed and rejected until the SRY (Sex-determining region on the Y) gene was cloned in 1990 in man (63) and mouse (64). Experimental (65, 66) and clinical (67, 68) evidence clearly established that SRY was the testis-determining factor. Considerable progress has been made since SRY was identified, and it has become clear that sex determination is a far more complex process, regulated by competing molecular pathways in the supporting cell lineage of the bipotential gonad. SRY has lost much of its prestige because it has a very weak transactivation potential, is expressed very transiently in the mouse, weakly at best in other mammals and not at all in sub-mammalian species (reviewed in ref. (20). Instead, its target gene encoding the transcription factor SOX9 has emerged as the master regulator of testis determination, the main role of SRY consisting in upregulating the expression of SOX9 during a very narrow critical time window (69). Once time is up, either SOX9 is able to maintain its own expression with the help of feed-forward enhancing mechanisms succeeding in triggering Sertoli cell differentiation or it is silenced by an opposing set of genes which impose ovarian differentiation. Timing and expression level determine which team wins (20, 70, 71) but the battle is never over, even after birth, at least in mice (72).

 

SRY is a member of a family of DNA-binding proteins bearing a high mobility group (HMG) box; its gene maps to the short arm of the Y chromosome (Table 3), very close to the pseudoautosomal region 1 (PAR1) (Fig. 3). PAR1 on Yp and PAR2 on Yq are the only regions of the Y chromosome that undergo meiotic recombination with homologous sequences of the X chromosome during male spermatogenesis. The proximity of SRY to PAR1 makes it susceptible to translocation to the X chromosome following aberrant recombination and provides an explanation for 80% of XX males (73) and for a low proportion of XY females. Indeed, mutations and deletions of the SRY locus only account for 15% of XY females (74, 75).

 

While SRY gene exists in almost all mammals as a single copy gene, the rat carries 6 copies and the mouse Sry gene has a distinct structure from other mammalian SRY genes because of the presence of a long-inverted repeat. Also, SRY expression varies between species: in mice a functional transcript is present only in pre-Sertoli cells for a very short period during early gonadogenesis, in goats SRY is expressed in all somatic and germ cells of the gonad during fetal life and restricted to Sertoli cells and spermatogonia in the adult testis. Human SRY is expressed in both Sertoli cells and germ cells at fetal and adult stages (reviewed in ref. (20). Proteins that interact with SRY and could have a relevant function in gonadal differentiation include SIP-1/NHERF2 (76) and KRAB-O (77).

FIGURE 3. X and Y chromosome genes involved in sex determination and differentiation.
SRY: Sex-determining region Y chromosome; DAX1: DSS-AHC critical region X chromosome gene 1; AR: Androgen receptor; and ATRX: Alpha-thalassemia/mental retardation syndrome X-linked are involved in in sex determination and differentiation. Other genes present in the X and Y chromosomes are: AZF: azoospermia factor; CSF2RA: Colony-stimulating factor 2 receptor alpha; DAZ: Deleted in azoospermia; FRA-X: Fragile X syndrome; DMD: Duchenne muscular dystrophy; GK: Glycerol kinase; HY: Histocompatibility antigen Y; IL3RA: Interleukin 3 receptor alpha; IL9R: Interleukin 9 receptor; Kal1: Kallmann syndrome 1; PAR: Pseudo-autosomal regions; POLA: DNA polymerase alpha; RBMY: RNA-binding motif protein Y chromosome; SHOX: Short stature homeo box; USP9Y: Ubiquitin-specific protease 9 Y chromosome; XIST: X inactivation-specific transcript; ZFX: Zinc finger protein X-linked; ZFY: Zinc finger protein Y-linked.

Owing to its Y-chromosome localization, SRY can only be expressed in the XY gonadal ridge, thus playing a paramount role in tilting the balance between testicular and ovarian promoting genes towards the male pathway.

 

A tight regulation of SRY expression is essential for fetal gonadogenesis: both timing and level of expression are determinant, as revealed by experiments in mouse showing that SRY levels must reach a certain threshold at a certain stage of fetal development to induce testis differentiation (69). SRY expression commences between days 41 and 44 post-fertilization in humans (78). The mechanisms underlying the initiation of SRY expression begin to be unraveled (Fig. 4). The +KTS splice variant of WT1 (21, 79, 80), SF1 (20) and SP1 (81, 82) are able to activate SRYtranscription. The transcriptional co-factor CITED2 acts in the gonad with WT1 and SF1 to increase SRY levels to attain a critical threshold to efficiently initiate testis development (35). The +KTS isoform of WT1 might also act as a posttranscriptional stabilizer of SRY mRNA (70).

 

The implication of GATA4 on SRY expression is less straightforward. The interaction between GATA4 and its cofactor FOG2 in the gonadal primordium is required for normal Sry expression and testicular differentiation in mice (83). However, whether the effect is specific on Sry transcription or more general on gonadal somatic cell development was not evaluated. Functional GATA-binding sites are present in the mouse and pig Sry promoter but not in the human SRY (84, 85). One possibility is that GATA4 interacts with WT1 (Fig. 4), mainly the +KTS isoform, which binds to the SRY promoter and increases its transcriptional activity (84). Alternatively, it has been proposed that GATA4 directly acts on the SRY promoter, based on the experimental observation that GADD45G binds and activates the mitogen-activated protein kinase kinase MAP3K4 (also known as MEKK4) to promote phosphorylation and activation of the p38 kinase (Table 3), which in turn phosphorylates GATA4 thus enhancing its binding to the Sry promoter (85, 86)(Fig. 4). These results are in line with those indicating that MAP3K4 is essential for testicular differentiation in mice (87).

 

SRY expression is also epigenetically regulated: the demethylase KDM3A, also known as JMJD1A, positively regulates the expression of Sry in mice, as shown by the absence of testicular development and consequent sex reversal in Jmjd1a-deficient XY mice (88). Histone methylation is an important mechanism of epigenetic regulation: methylation of lysine 9 of histone H3 (H3K9) is a hallmark of transcriptionally suppressed chromatin. JMJD1A demethylates H3K9, thus allowing transcriptional activation of Y chromosome genes, amongst which is SRY. ATRX,also known as XH2, is an X-encoded DNA-helicase whose mutation results in mental retardation, α-thalassemia and gonadal dysgenesis in XY individuals (89-91). ATRX has a more general effect on chromatin remodeling, which seems to play an important role in the epigenetic regulation of sex determination (92).

 

Several other experimental models impairing the expression of signaling molecules, which are expressed SRY in the early gonadal ridge in normal conditions, show reduced or absent SRY expression, develop gonadal agenesis and a female phenotype of the internal and external genitalia. LHX9 (8) is a potential regulator of SRY expression. A direct effect of LHX9 on the SRY gene has not been demonstrated but an indirect effect through SF1 upregulation has been postulated (20). Loss-of-function mutations of the mouse genes encoding the insulin receptor (Insr), the IGF1 receptor (Igf1r) and the insulin related receptor (Insrr) also result in decreased or absent Sry expression (12). However, these factors and signaling pathways affect cell proliferation, and decreased SRY expression might only reflect the reduced number of cells in the gonadal primordium. Indeed, many of these potential regulators have not yet been proven to affect SRY expression directly.

 

TABLE 3. Factors Involved in Gonadal Differentiation

Gene

Chromosomal localization

Expression

Function

ATRX (Alpha-thalassemia/mental retardation syndrome, Helicase 2, X-Linked)

Xq21.1

Widespread

Nucleotide excision repair and initiation of transcription

CBX2 (Chromobox homolog gene 2; or M33 mouse homolog of)

17q25.3

Widespread

Regulation of homeotic genes. Represses WNT4 signaling

CITED2 (CBP/p300-interacting transactivator, with glu/asp-rich c-terminal domain, 2)

6q24.1

Widespread

WT1 and SF1 cofactor, regulating SRYexpression in the gonad

COUP-TF2 (Chicken ovalbumin upstream promoter transcription factor 2), or NR2F2(Nuclear receptor subfamily 2, group F, member 2)

15q26.2

Widespread

Transcription factor (orphan nuclear receptor) likely involved in mesenchymal-epithelial interactions

CTNNB1 (β-catenin)

3p22.1

Widespread

Upregulates WNT4, FST and FOXL2

DAX1: DSS-AHC critical region on the X chromosome 1); orNR0B1 (Nuclear receptor subfamily 0, group B, member 1).

Xp21.2

Gonads, pituitary, adrenals

Antagonizes SRY, SOX9. Essential for normal testicular and ovarian development

DHH (Desert hedgehog)

12q13.12

Sertoli cells (testis), Schwann cells (peripheral nerves)

Morphogenesis

DKK1 (Dickkopf, xenopus, homolog of, 1)

10q21.1

Widespread

Represses WNT4 binding to the LRP5/6 co-receptor

DMRT1 (Doublesex- and mab3-related transcription factor 1)

9p24.3

Gonads and several other tissues

Antagonizes FOXL2

FGF9 (Fibroblast growth factor 9)

13q12.11

Gonads and several other tissues

Upregulation of SOX9 and downregulation of WNT4

FGFR2 (FGF receptor 2)

10q26.13

Gonads and several other tissues

Upregulation of SOX9 and downregulation of WNT4

FOG2 (Friend of GATA, gene 2, or ZFPM2: zinc finger protein multitype 2)

8q23.1

Widespread

Repression of DKK1

FOXL2 (Forkhead transcription factor 2)

3q22.3

Gonads and eyelids

Antagonizes SOX9. Survival of meiotic germ cells

FST (Follistatin)

5q11.2

Widespread

Antagonizes Activins. Survival of meiotic germ cells

GADD45G (Growth arrest- and DNA damage-inducible gene, gamma)

9q22.2

Widespread

Phosphorylation of GATA4

GATA4 (GATA-binding protein 4)

8p23.1

Widespread

Regulation of SRY expression

HHAT (Hedgehog acyltransferase)

1q32.2

Gonads

Two INHBB subunits form Activin B dimer, which induces vascular endothelial cell migration to the gonad

INHBB (Inhibin βB, Activin βB)

2q14.2

Gonads

Two INHBB subunits form Activin B dimer, which induces vascular endothelial cell migration to the gonad

JMJD1A; or KDM3A(Lysin-specific demethylase 3A)

2p11.2

Testis, ovary, kidney, lung, heart, brain, liver, skeletal muscle, pancreas, and spleen

Demethylases histone H3 (epigenetic regulation by modification of chromatin conformation)

MAP3K1 (MAP/ERK Kinase Kinase 1; MEKK1; MAPKKK1; MEK Kinase)

5q11.2

Widespread

Phosphorylation of GATA4

MAPK14 (Mitogen-activated protein kinase 14; or p38-MAPK)

6p21.31

Widespread

Phosphorylation of GATA4

NR5A1 (Nuclear receptor subfamily 5, group A, member 1, also SF1: Steroidogenic factor 1, or AD4BP: Adrenal 4 binding protein, or FTZF1: Fushi tarazu factor homolog 1)

9q33.3

Gonadal ridges, adrenal gland primordia, hypothalamus and pituitary

Transcriptional regulation of several genes (SRY, SOX9, STAR, steroid hydroxylases, aromatase, AMH, DAX1 and many other)

PDGFB (Platelet-derived growth factor, beta polypeptide)

22q13.1

Endothelial cells

Increase in cell proliferation in the gonadal interstitial tissue

PDGFRA (PDGF receptor α)

4q12

Gonadal interstitial cells and several other tissues

Increase in cell proliferation in the gonadal interstitial tissue

PTGDS (or PGDS2: Prostaglandin D2 synthase)

9q34.3

Gonads and several other tissues

Synthesis of prostaglandin D2 (PGD2), upregulation of SOX9 and its nuclear translocation

RSPO1 (R-spondin family, member 1)

1p34.3

Gonads and skin

Upregulates WNT4 by sequestering the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43.
Cooperates with WNT4 signaling, by antagonizing DKK1, to stabilize β-catenin and FST

SOX8 (SRY box 8)

16p13.3

Gonads and several other tissues

Transcriptional regulation of SOX9, in cooperation with SF1

SOX9 (SRY box 9)

17q24.3

Testis, cartilage

Triggers testis differentiation, and regulates several testis-specific genes

SOX10 (SRY box 10)

22q13.1

Gonads and several other tissues

Transcriptional regulation of SOX9, in cooperation with SF1

SP1 (Specificity protein 1)

12q.13.13

Widespread

Regulation of SRY expression

SRY (Sex-determining region on the Y chromosome)

Yp11.31

Male gonadal ridge

Regulates SOX9 and triggers testis differentiation

VEGFA (Vascular endothelial growth factor A)

6p21.1

Mesenchymal cells of the gonadal ridge and other organs

Induces vascular endothelial cell migration to the gonad

WNT4 (Wingless-type MMTV integration site family, member 4)

1p36.12

Gonads and several other tissues

Induces β-catenin and silences FGF9 and SOX9 by binding to Frizzled receptor

WT1 (Wilms tumor associated gene 1)

11p13

Urogenital ridge derivatives

Transcriptional regulation and post-transcriptional stabilization of SRY

ZNRF3 (Zinc finger and ring finger protein 3)

22q12.1

Widespread

Inhibition of WNT signaling by targeting Frizzled receptor for degradation by ubiquitination and increased membrane turnover

 

SOX9: A Target of SRY

 

SOX9, an autosomal member of the HMG-box protein superfamily mapped to chromosome 17 q24 (93), is the master regulator of Sertoli cell differentiation (94). In the mouse, SOX9 is expressed at low levels in the bipotential gonads of both sexes under SF1 regulation (95), but persists only in testicular Sertoli cells after SRY expression has peaked (96-98). SRY and SF1 directly bind to several sites within a 3.2-kb testis-specific enhancer (TES) or 1.4-kb of its core element (TESCO), present approximately 14 kb upstream of the Sox9 promoter and responsible for this expression pattern (95, 99). Together with SF1, SOX9 also binds and activates TES, thus maintaining its own expression by autoregulation after transient SRY expression has ceased in the mouse.

 

SOX9 mimics SRY effects independently of SRY expression. In fact, overexpression of SOX9 during early embryogenesis induces testicular differentiation in two different models of transgenic XX mice (100, 101). Functional analysis of SOX9 during sex determination, by conditional gene targeting in mice, has shown that homozygous deletion of Sox9 in XY gonads interferes with sex cord development and with activation of testis specific markers (102). Further evidence for the role of SOX9 in testicular development comes from observations in humans, in whom a double dose of SOX9 expression is required. Heterozygous mutations result in haploinsufficiency resulting in campomelic dysplasia, a polymalformative syndrome that includes sex-reversal due to gonadal dysgenesis in XY individuals (93, 103), whereas gain-of-function of SOX9 in XX individuals leads to sex reversal (104).

 

In humans more distant regulatory regions of SOX9 have been identified (105), and confirmed by observations in patients with XY gonadal dysgenesis. No mutation has been found in the TES sequence (106), instead a 1.9 kb SRY-responsive subfragment of a 32.5 kb interval lying 607.1–639.6 kb upstream of SOX9 —termed XY SR for XY Sex Reversal— seems to be the core of the Sertoli-cell enhancer of human SOX9. Heterozygous deletions encompassing these sequences were identified in four families with SRY-positive 46,XY gonadal dysgenesis without campomelic dysplasia (107) and a deletion of a 557–base pair element named enhancer 13 (Enh13), reproduced in mice, led to XY sex reversal (108). This region is included in a 1.2-Mb deletion previously described in a case of 46,XY DSD with gonadal dysgenesis and no skeletal phenotype (109). Finally, in line with these observations, overexpression of SOX9 is supposed to underlie testicular development in familial 46,XX SRY-negative males with a 178-kb duplication or a 96-kb triplication in sequences lying 500–600 kb upstream of SOX9 (110, 111).

SOX9 also affects the differentiation of the reproductive tract by upregulating the expression of anti-Müllerian hormone (AMH) (112, 113), a Sertoli cell factor involved in male differentiation of the internal genitalia (see below).

 

SOX8 and SOX10 are two other members of the SOX family expressed in the gonads and in several other tissues. During mouse embryo development, the expression of SOX8 and SOX10 is triggered shortly after that of SOX9, but at lower level (114-117). SOX8 is regulated by SOX9 (102). Like SOX9 itself, SOX8 and SOX10 can synergize with SF1 and upregulate SOX9 expression (Fig. 4) upon binding to TESCO (20). SOX8 can bind the canonical target DNA sequences and activate AMH transcription acting synergistically with SF1, but with less efficiency than SOX9 (114, 118). Later during fetal development, an interaction between SOX9 and SOX8 is required for basal lamina integrity of testis cords and for suppression of FOXL2, two events essential to the normal development of testis cords (117).

 

An X-linked member of the SOX family, SOX3, although not involved in the normal pathway of fetal gonadal differentiation, is capable of inducing SOX9 expression and testis differentiation when ectopically expressed in the XX gonad (119). It is also possible that indirect mechanisms mediate Sox9 activation, in line with the hypothesis indicating that SRY might act as a repressor of a negative regulator of the male cascade (120). For instance, targeted disruption of Foxl2 leads to SOX9 upregulation in the XX gonad (121), and prostaglandin D2 (PGD2) has been shown to upregulate SOX9 in the absence of SRY (122).

 

SOX9 expression is maintained at high levels in the male gonad despite down-regulation of SRY soon after testicular determination, at least in the mouse (97, 98). As mentioned, SOX9 is capable of autoregulating its expression (95), and other members of the SOX family like SOX3, SOX8 and SOX10 are also able to interact with SF1 to maintain SOX9 expression in the male gonad (20, 117).

 

Observations made in XY intersex patients with normal SRY together with the discovery of proteins showing a sexually dimorphic pattern of expression in the gonads following SRY peak have helped to identify other loci, likely to be involved in testicular differentiation, which are discussed below.

 

FGF9 and PGD2: Maintaining SOX9 Expression Levels

 

SOX9 upregulates the expression of FGF9 and the synthesis of prostaglandin D2 (PGD2) catalyzed by PGD synthase. FGF9 interacts with its receptor FGFR2, initiating a feed-forward loop that maintains SOX9 expression and also results in downregulation of WNT4 expression (123-126) (Fig. 4). Independently of FGF9, PGD2 interacts with its receptor DP to induce SOX9 expression (122, 127) and its nuclear translocation (127, 128), thus increasing its availability to target genes (80).

FIGURE 4. Schematic representation of molecular mechanisms involved in determining the fate of the undifferentiated gonadal ridge. Black arrows indicate a positive regulation; double arrows indicate a positive feedback loop; red lines indicate a negative regulation; double red lines indicate a mutual antagonism. In the 6th week of embryonic life, the gonadal ridge is sexually undifferentiated, and various factors are expressed at the same levels in the XX and the XY gonads. During the 7th week, in the XY gonad, SRY expression is triggered, and the male pathway prevails driving to the formation of the coelomic vessel. In the XX gonad, the female pathway prevails, and there is no formation of the coelomic vessel. Reprinted with permission from ref. (129) Freire AV, Ropelato MG, Rey R. Ovaries and Testes. In: Kovacs C, Deal C, Eds. Maternal-Fetal and Neonatal Endocrinology. 1st Edition. Boston: Academic Press-Elsevier, 2020, pp. 625-641. ISBN 9780128148235. Copyright © 2020 Elsevier Inc.

As already discussed, somatic cell proliferation is critical for early testicular differentiation (18). FGF9 and WNT4 act as antagonistic signals in the first steps of differentiation of the gonadal ridge (130). FGF9 controls cell proliferation in a sexually dimorphic fashion: the disruption of FGF9 expression by targeted deletion in transgenic mice does not affect XX gonads but prevents testicular differentiation and results in sex reversal in XY mice (131). In the mouse, FGF9 and WNT4 are expressed in the undifferentiated XX and XY gonads at the same levels: FGF9 near the coelomic surface and WNT4 near the mesonephric border (130). When SRY expression is initiated and upregulates SOX9 in the XY gonadal ridge, the balance between FGF9 and WNT4 is disrupted: SOX9 enhances FGF9 expression which in turn maintains high SOX9 levels thus resulting in a feed-forward loop that accelerates commitment to the male pathway. WNT4 expression is downregulated when a threshold level of FGF9 is reached (130). FGF9 controls the proliferation of a cell population that gives rise to Sertoli progenitors (19). In Fgf9 knockout mice, initial Sertoli cell differentiation is not hindered: SRY and SOX9 expression is observed but soon weakens resulting in an aborted differentiation of Sertoli cell precursors (130). Although in experimental conditions, FGF9 is capable of inducing proliferation of coelomic epithelium cells in XX gonadal ridges, this does not result in Sertoli cell differentiation, clearly indicating that increasing cell proliferation is not sufficient to induce testicular differentiation, and that other pro-testicular signals are also required (131). FGF9 and SOX9 also upregulate AXIN1 and GSK3β, which promote the destabilization of β-catenin and, thus, serve to block ovarian development (132).

 

DMRT1, DAX1 and Other Factors Modulating Testis Versus Ovary Antagonism

 

DMRT1 is a member of the DM domain transcription factor family which appears to play a conserved role in vertebrate male gonad development. In mice, DMRT1 –but not DMRT2 or DMRT3– is expressed and required in both germ cells and Sertoli cells of the testis (133). Overexpression of DMRT1 in XX mice inhibits WNT4 and FOXL2 expression and results in partial testicular differentiation and male genital development (134), while loss of DMRT1 expression activates FOXL2 and reprograms Sertoli cells into granulosa cells, even in postnatal life, suggesting that DMRT1 is essential to maintain mammalian testis differentiation life-long in mice (135, 136).

 

In humans, deletions of chromosome 9p involving DMRT1, DMRT2 and DMRT3 genes are associated with XY male-to-female sex reversal due to gonadal dysgenesis. Patients also present with mental retardation and typical craniofacial dysmorphia, including trigonocephaly, upward-slanting palpebral fissures, and less frequently hypertelorism, epicanthus, flat nasal bridge, low-set ears, microstomia, micrognathia, short neck, widely spaced nipples, square hyperconvex nails, dolichomesophalangy and hypotonia (137, 138).

 

DAX1 (HGNC approved gene symbol: NR0B1), encoding for an orphan nuclear receptor and mapping to the DSS(Dosage Sensitive Sex-reversal) region on Xp21, was the first putative testis repressor and/or ovarian determining gene. A duplication of DSS results in sex-reversal in 46,XY patients (139), and DAX1 overexpression in transgenic XY mice impairs testis differentiation by antagonizing the ability of SF1 to synergize with SRY action on SOX9 (140, 141)(Fig. 4). However, the disruption of Dax1 gene in XX mice does not prevent ovarian differentiation (142). Furthermore, DAX1 is essential for normal testicular cord formation (143, 144). These observations in rodent models, together with DAX1 expression pattern in the human fetus showing persistently low levels in both XX and XY gonads from 33 days post-fertilization (i.e. the bipotential stage) through 15 fetal weeks (78), strongly suggest that low DAX1 levels are necessary for gonadal development in both sexes. Abnormally low or high DAX1 expression result in abnormal gonadal differentiation (145).

 

CBX2, the human homolog of murine M33 (146), does not seem to activate SRY expression as initially proposed (147), but may act as a stabilizer of SRY action and the testis pathway by repressing WNT4 downstream target LEF1, involved in ovarian differentiation (148). Interestingly, biallelic mutations in CBX2 were found in a 46,XY girl with ovarian tissue (149), and XY mice with inactivated Cbx2 developed as female (146).

 

MAP3K1, unlike MAP3K4 (87), is not essential for testicular differentiation and development in mice (150), but it modulates the balance between testicular and ovarian male pathways by sequestration of AXIN1 (see “Genetic pathways of ovarian differentiation”). In humans, mutations in the MAP3K1 gene have been associated with testicular dysgenesis (151, 152).

 

Similarly, inactivating variants that disrupt ZNRF3 function result in 46,XY DSD in humans and to sex reversal in mice, likely due to gonadal dysgenesis (153).ZNRF3 is an E3 ubiquitin ligase that promotes the degradation by ubiquitination and the turnover of Frizzled, a WNT receptor (Fig. 5) (154, 155).

FIGURE 5. WNT and RSPO actions. Under steady state conditions (red dotted arrows), ZNRF3 provokes the ubiquitination and degradation of Frizzled, receptor of WNT family factors. GSK3 phosphorylates β-catenin, which is then degraded. R-spondin (RSPO) family members binding to their receptors LGR4/5/6 results in complex formation with ZNRF3. Consequently, more Frizzled molecules become available for WNT signaling. Under these conditions (blue full arrows), the complex formed by GSK3, Axin, CKIα and APC is recruited to the WNT–receptor complex and inactivated, allowing β‑catenin to translocate to the nucleus and regulate target genes.

COUP-TF2, encoded by NR2F2, is a transcription factor likely involved in mesenchymal-epithelial interactions required for organogenesis. In the fetal gonads, COUP-TF2 expression increases as the ovaries develop, and loss-of-function mutations in NR2F2 have been described in 46,XX ovotesticular SRY-negative DSD (156), indicating that COUP-TF2 may be involved in driving the balance towards ovarian differentiation.

 

MAMLD1 is expressed in fetal Sertoli and Leydig cells, under the control of SF1 (157, 158), and gene variants have been associated with a broad phenotypic spectrum of DSD (159). However, Mamld1 knockout mice depict a very mild reproductive phenotype (160). The precise role of MAMLD1 still needs to be established.

 

Stabilization of Testis Differentiation: Vascular, Cellular and Molecular Pathways

 

In the XY fetus, the initially amorphous cluster of gonadal cells becomes segregated in two compartments, testicular cords and interstitial tissue, during the 7th week of gestation (3). These architectural changes are heralded by gonadal ridge vascularization, a highly dynamic and sexually dimorphic process. At variance with the differentiating ovary that recruits vasculature by typical angiogenesis, the XY gonad recruits and patterns vasculature by a remodeling mechanism: pre-existing mesonephric vessels disassemble and generate a population of endothelial cells that migrate to the gonad, below the coelomic epithelium, where they reaggregate to form the coelomic vessel, an arterial vessel that runs the length of the testis at its antimesonephric margin (161, 162). The formation of this vessel is one of the earliest hallmarks of testis development that distinguishes it morphologically from the developing ovary (161, 163). Evidence now exists for a close spatial relationship between testis vascularization and cord formation (162, 164). Furthermore, all of the cells migrating from the mesonephros to the coelomic zone of the differentiating testis express endothelial markers such as VE-cadherin, an indication that incoming endothelial, rather than peritubular myoid cells, are required for testicular cord formation (164). Subsequently, Sertoli cells aggregate and enclose germ cells. The interaction between differentiating peritubular myoid cells and Sertoli cells results in the formation of basement membrane of the testicular cords. Mesenchymal cells and matrix and blood vessels fill the interstitial space, in which Leydig cells will soon appear. Beyond vascularization, which is necessary to allow efficient export of testosterone, cell migration from the mesonephros largely contributes to testicular organogenesis (165, 166) and is antagonized by the initiation of meiosis in germ cells (167).

 

The molecular mechanisms underlying sex-specific gonadal vascularization are being progressively unraveled. A vascular-mesenchymal cross-talk between VEGFs and PDGFs drives gonadal patterning during early fetal life (Fig. 4). VEGF-A, expressed in interstitial mesenchymal cells of the undifferentiated gonadal ridge, induces vascular endothelial cell migration to the gonad. In turn, PDGF-B expressed by the endothelial cells is responsible for an increase in cell proliferation in the gonadal interstitium, upon binding to its receptor PDGFRα. Disruption of vascular development blocks formation of testis cords (168, 169) while not affecting Sertoli and Leydig cell specification(169). In the XX gonadal ridge, WNT4 and its downstream target follistatin (FST) repress endothelial cell migration, probably by antagonizing Activin B (Fig. 4). In the XY gonad, the SRY/SOX9 pathway downregulates WNT4/FST thus allowing Activin B, VEGF and other potential as yet unidentified factors to induce male-specific gonadal vascularization (170). Genes involved in male sex determination are shown in Fig. 6.

FIGURE 6. Sex determination and differentiation. Reprinted with permission from ref. (171): Grinspon RP, Rey RA Molecular Characterization of XX Maleness. International Journal of Molecular Sciences (2019) 20:6089, © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Differentiation of Sertoli and Leydig Cells

 

As already mentioned, both the supporting and the steroidogenic cell lineages derive from WT1-positive somatic progenitors present in the undifferentiated gonadal ridges. Wt1-positive cells can express HES1, a Notch effector, or not (38). In the subset of WT1-positive and HES1-negative cells, having delaminated from the coelomic epithelium in the central part of the indifferent gonad, SRY expression is induced giving rise to the supporting cell lineage (pre-Sertoli cells) (38, 172-174). SRY-expressing pre-Sertoli cells lying beneath the coelomic epithelium play a central role in the migration of cells from the mesonephric mesenchyme into the differentiating gonad (175). Experimental work using XX-XY chimeras has shown that not 100% of Sertoli cell precursors need to express SRY to differentiate along the male pathway: in fact, up to 10% of Sertoli cells were XX. However, a threshold number of SRY expressing –i.e. XY– cells seems to be essential in order for Sertoli cell differentiation, and thus testicular development, to be guaranteed (176).

 

Along with SRY, FGF9 might have a role in inducing mesonephric cell migration into the developing fetal testis and Sertoli cell differentiation. FGF9 is expressed in Sertoli cells of the fetal testis and Fgf9-null mice have dysgenetic gonads (131, 177) (see below).

 

Vanin-1, a cell-surface molecule involved in the regulation of cell migration, might also be responsible for differentiating Sertoli cell association with, and adhesion to, migrating peritubular cells (178). Nexin-1, expressed by early Sertoli cells, could act to maintain the integrity of the basal lamina (178).

 

Desert hedgehog (DHH) and its receptor PATCHED2 might also play a role in Sertoli-peritubular cell interaction and basal lamina deposition (179, 180). DHH is a protein secreted by fetal Sertoli cells, but not by somatic components of the fetal ovary, immediately after testicular determination (181). Patched2 is expressed in germ, peritubular and interstitial cells of the testis (182). Testes develop abnormally during fetal life in Dhh null mice, resulting in XY sex-reversal. Seminiferous cords are disorganized owing to defects in the basal lamina and peritubular cells, with germ cells occasionally lying in the interstitial tissue, and Leydig cells are hypoplastic (179, 180). Homozygous mutations of DHH in 46,XY patients are associated with gonadal dysgenesis (183, 184).

 

DHH, like other members of the hedgehog family, undergoes post-translational modifications including N-terminal palmitoylation by HHAT (hedgehog acyl-transferase), which is essential for efficient signaling. A mutation leading to defective HHAT function was found to cause complete gonadal dysgenesis and female phenotype in two 46,XY patients (185).

 

Testicular cord formation can be detected in human fetuses 13-20 mm crown-rump length (43-50 days) beginning in the central part of the gonad (186). Cord formation is heralded by the development of a new type of cell, the primitive Sertoli cell, characterized by a polarized, large and clear cytoplasm with abundant rough endoplasmic reticulum and complex membrane interdigitations (187), a downregulation of desmin and an upregulation of cytokeratins (188), and the expression of SOX9 (97), AMH (189, 190) and DHH (184, 191, 192). Differentiating Sertoli cells also express growth factors, like nerve growth factors (NGFs), which can induce cell migration from the mesonephros acting through their receptors TRKA (NTRK1) and TRKC (NTRK3) (193, 194). Sertoli cells aggregate around large, spherical germ cells, with a large nucleus and pale cytoplasm, called gonocytes at this stage, which can be observed in the center of testicular cord cross-sections (186). The structural basis of cord formation seems to be dependent on basal lamina deposition between Sertoli and peritubular cells with myofibroblastic characteristics (166). In the interstitial compartment, connective tissue, blood vessels and Leydig cells can be observed. As described above, one particular feature of testicular vasculature is the formation of the coelomic vessel, a large vessel that appears below the coelomic epithelium very early in testicular differentiation (161, 195). Surrounding the gonad, the basement membrane layer underlying the coelomic epithelium thickens to form the tunica albuginea.

 

Sertoli and germ cell numbers increase exponentially in the human fetal testis throughout the second trimester (196)in response to FSH acting through its receptor in Sertoli cells (197-199), and androgens acting indirectly through the peritubular myoid cells (200). This probably explains why newborns with congenital hypogonadotropic hypogonadism have small testes and low serum levels of Sertoli cell markers, such as AMH and inhibin B (201, 202). Sertoli cells do not reach a mature state, and meiosis is not initiated in the human testis until pubertal age, when all Sertoli cells reach a high expression level of the androgen receptor (203-206). In mice, NRG1 and its receptors ERBB2/3 are also essential for Sertoli cell proliferation, and Nrg1 gene invalidation leads to Sertoli cell hypoplasia and micro-orchidism (17).

 

Morphologically and functionally distinct from testicular cords, the interstitial compartment contains developing Leydig cells (Fig. 7), the main androgen producing cells in the male. The origin of Leydig cells has not been clearly established: the precursors of fetal Leydig cells have been proposed to be either migrating cells from the coelomic epithelium, the mesonephros or the neural crest or resident cells present in the adreno-gonadal primordium (reviewed in refs (20, 207, 208). According to the latter hypothesis, a subset of SF1-expressing cells gives rise to all steroidogenic lineages of the gonads and adrenal cortex. This is supported by the finding of adrenal markers (209)and adrenal-like cells in the fetal testis (210, 211) and of adrenal rests in the testes of male patients with congenital adrenal hyperplasia (212). Mesonephric cells expressing nestin, a cytoskeletal filament initially characterized in neural stem cells, are a multipotent progenitor population that gives rise to Leydig cells, pericytes and smooth muscle cells. However, the first cohort of Leydig cells derive from nestin-negative cells, confirming the multiple origins of fetal Leydig cells (213).

 

Another particular feature of the mouse testis is that Leydig cell populations can be divided into fetal and adult Leydig cells according to the time they arise. Fetal Leydig cells disappear after birth and are replaced by adult Leydig cells at puberty (214). Despite their similar functions, fetal and adult Leydig cells show morphologic and gene expression differences: some progenitor cells that lose Wt1 expression and are HES1-negative/GLI1-negative become located to the interstitial tissue, do not express SOX9 and differentiate into fetal Leydig cells, under the effect of the Notch signaling pathway. Another subset of cells that expresses HES1 and GLI1, under the Hedgehog signaling pathway, are not initially steroidogenic, but give rise to adult Leydig cells in postnatal life (38).

 

In the human fetus, Leydig cells can be identified in the interstitial tissue by the beginning of the 8th week (215) —after testicular cords have completely formed— and soon begin to produce testosterone, which plays an essential role in the stabilization of Wolffian ducts and the masculinization of external genitalia. Leydig cells also produce insulin-like growth factor 3 (INSL3), a growth factor responsible for the transabdominal phase of testicular descent (216-218). Although the initial differentiation of fetal Leydig cells depends, at least partially, on Sertoli cell-secreted PDGFs binding to PDGFRα (219) independently of gonadotropin action (220), further Leydig cell differentiation and proliferation depends on placental hCG in the first and second trimesters of fetal life and on fetal pituitary LH thereafter acting on the LH/CG receptor (221). At mid-gestation, interstitial tissue is literally packed with Leydig cells; afterwards their number decreases (196, 215).

 

SF1 action, is suppressed by WNT4-activated DAX1 expression (222). By counteracting WNT4, and thus downregulating DAX1 in interstitial cells of XY gonads, SRY might indirectly enhance SF1 action (223, 224). Finally, ARX is an X-chromosome gene identified in patients with X-linked lissencephaly and genital abnormalities probably associated with a block in Leydig cell differentiation (225). FGF9 (131, 177) and DHH (180) are Sertoli cell-secreted signals involved in Leydig cell differentiation.

FIGURE 7. Leydig cells accumulate in the testicular interstitial tissue of a 90-mm male human fetus (11th week). Large eosinophilic Leydig cells with a prominent nucleus are interspersed with mesenchymal cells.

Timing of Testicular Differentiation

 

In order for the fetal testis to adequately differentiate and secrete masculinizing hormones, not only do all these factors need to be present at sufficient levels in the right cell lineage, but their expression must also be initiated within a narrow time window. In mice, the ability of SRY to induce testis development is limited to a time window of only 6 hours after the normal onset of expression in XY gonads. If SRY is expressed later, Sox9 gene activation is not maintained due to failure of FGF9/WNT4 signaling to switch to a male pattern (69).

 

Germ Cell Interaction with Somatic Cells in the Developing Testis: Repression of Meiosis

 

Upon arriving in the undifferentiated genital ridge, by the end of the 5th week, germ cells continue to proliferate by mitosis and maintain bipotentiality for approximately one week. Then germ cells in the male gonad become enclosed in the seminiferous cords and differentiate into the spermatogonial lineage, which does not enter meiosis until the onset of puberty. Gonocyte proliferation in the fetal testis is inhibited by androgens (226). Prevention of entry into meiosis was first thought to be a specific effect of male somatic cells since germ cells entering a prospective ovary or those which have failed to enter gonads of either sex enter meiosis at approximately the same time and develop into oocytes, irrespective of their chromosomal pattern (227). Subsequent studies shed light on the sexually dimorphic evolution of gametogenesis in the fetal gonads. The mesonephros from the indifferent gonad, as well as the lung and adrenal gland, synthesize retinoic acid that acts as a meiosis inducer (228, 229). Germ cells embedded in the seminiferous cords do not enter meiosis because they are protected from retinoic acid action: mouse Sertoli cells express two factors that prevent meiosis onset: FGF9 (230) and CYP26B1, an enzyme that catabolizes retinoic acid (231, 232). NANOS2, expressed in germ cells, is also a meiosis-preventing protein, since in the fetal testis it represses the expression of STRA8 (233) (for details on STRA8, see “Genetic control of oogenesis and folliculogenesis“. In human fetal testis, CYP26B1 does not seem to be expressed, and the mechanism underlying the inhibition of germ cell entry into meiosis needs to be elucidated (234, 235).

 

Chromosomal constitution does not influence sex differentiation of germ cells: XX germ cells surrounded by Sertoli cells differentiate into spermatogonia, whereas XY germ cells in an ovarian context differentiate into oogonia and then enter meiosis (236). However, germ cells whose karyotype is discordant with the somatic lineages fail to progress through gametogenesis and enter apoptosis later in life.

 

The influence of germ cells on the developing gonad is sexually dimorphic: Germ cell progression through meiosis is essential for the maintenance of the fetal ovary, otherwise prospective follicular cells degenerate and streak gonads result. In contrast, the development of the testes is not hindered by the lack of germ cells (195).

 

STABILIZATION OF OVARIAN DIFFERENTIATION: CELLULAR AND MOLECULAR PATHWAYS

 

Genetic Pathways of Ovarian Differentiation

 

The pathway leading to ovarian differentiation and stabilization is far more complex than what was originally hypothesized. In humans, the absence of an active SRY gene –e.g. SRY mutations or deletions of the Y chromosome involving the SRY locus– results in gonadal dysgenesis of variable degrees, but is not sufficient to allow ovarian differentiation: no oocyte meiotic progression or follicle development has been described, even during fetal life. Recent findings suggest that most probably the coordinated action of several factors is needed for the differentiation and stabilization of the ovaries (237-239) (Table 3, Figs. 4, 6 and 8).

 

WNT4 is a secreted protein that functions as a paracrine factor to regulate several developmental mechanisms. WNT proteins bind to the frizzled (FZ) family of membrane receptors and LRP5/6 co-receptors, leading to the activation of the phosphoprotein disheveled (DVL) and a subsequent increase in cytoplasmic β-catenin levels owing to an inhibition of its degradation rate (240). In turn, WNT4 is upregulated by the action of β-catenin, which establishes a positive feedback loop, and also indirectly by the GATA4/FOG2 complex, which represses DKK1 (241). DKK1 is capable of binding to the LRP5/6 co-receptor, thus preventing the formation of the WNT-FZ-LRP5/6 signaling complex. WNT4 is expressed at similar levels in the XY and XX bipotential gonads. When SRY upregulates SOX9 in XY gonads, and the feed-forward loops with FGF9 and PGD2 are established, WNT4 is silenced (130) (Fig. 4). In XX gonads, the absence of SRY releases WNT4 expression, which stabilizes β-catenin and silences FGF9 and SOX9 (130). WNT4 also up-regulates DAX1 (222), which antagonizes SF1 and thereby inhibits steroidogenic enzymes. WNT4-deficient XX mice express the steroidogenic enzymes 3b-hydroxysteroid dehydrogenase and 17a-hydroxylase, which are required for the production of testosterone and are normally suppressed in the developing female ovary (242). In humans, a duplication of chromosome 1 containing 1p36.12, where human WNT4 maps, causes ambiguous genitalia of XY patients, probably due to low testosterone production (222), whereas inactivation of both copies of WNT4 in XX human fetuses results in alterations in gonadal morphology, ranging from ovotestes to testes, associated with renal agenesis, adrenal hypoplasia, and pulmonary and cardiac abnormalities (SERKAL syndrome: Sex reversal with kidney, adrenal and lung abnormalities) (243). WNT4 is also involved in the development of the internal genital tract (see below).

 

Like WNT4, RSPO1 is expressed in the undifferentiated gonadal ridge of XY and XX embryos and increases in the XX gonads in the absence of SRY. RSPO1 binds to G protein–coupled receptors LGR4 and LGR5 (244), stimulates the expression of WNT4 and cooperates with it to increase cytoplasmic β-catenin (Fig. 5) and FST levels (245-248). RSPO1 is thought to facilitate WNT-FZ-LRP complex formation through fending off DKK1 and by sequestering ZNRF3, which promotes FZ degradation by ubiquitination and increased turnover (154, 155, 249). The increase in WNT4/β-catenin counteracts SOX9, thus leading to the ovarian pathway (170). Loss of function mutations in the human RSPO1 gene and Rspo1 gene ablation in mice result in the formation of ovotestes in the XX fetus probably owing to SOX9 upregulation (75, 170, 250).

 

β-catenin also activates FOXL2 winged helix/forkhead transcription factor, expressed in germ and somatic cells, more strongly in the female than the male fetal gonad from the 8th fetal week (251) and involved in granulosa cell differentiation (252, 253). The high levels of WNT4/β-catenin and FOXL2 counteract FGF9 and SOX9, thus leading to the stabilization of the ovarian differentiation pathway (238, 239). FOXL2 also represses SF1 expression by antagonizing WT1 in the XX mouse fetus (254). FOXL2 and FST are needed for the survival of meiotic germ cells (72, 255, 256). In the XY fetus, SOX9 represses FOXL2 expression in the gonad (257). Conversely, inducible deletion of Foxl2 in adult mouse ovarian follicles leads to upregulation of Sox9 and reprogramming of adult ovaries to testes (72). In goats, XX males develop in the event of a deletion in the autosomal PIS locus (258), where FOXL2 has been identified. In humans, FOXL2 mutations result in a variety of phenotypes, from streak gonads to adult ovarian failure associated with eyelid abnormalities characterized by blepharophimosis, ptosis and epicantus inversus (BPES) (259).

 

Germ cell entry into meiosis is a specific feature of initial ovarian differentiation (Table 3, Figs. 4 and 9). Once stabilized by the cooperative action of WNT4 and RSPO1, cytoplasmic β-catenin migrates to the nucleus and induces the expression of FST. The latter antagonizes Activin B, thus repressing endothelial cell migration and the coelomic vessel formation, one of the earliest testis-specific events (170). Wnt4 has a similar effect (256).

 

MAP3K1 modulates the balance between female and male pathways. As explained above (see “FGF9 and PGD2: maintaining SOX9 expression levels”), SOX9 and FGF9 upregulate AXIN1 and GSK3β, which promote the destabilization of β-catenin, thus blocking ovarian development. MAP3K1 sequestrates AXIN1; consequently, there is a stabilization of β-catenin, which favors the ovarian pathway (132). In XY patients with mutations of MAP3K1 that result in increased binding to AXIN1, there is an increase of β-catenin leading to defective testicular differentiation and finally resulting in gonadal dysgenesis (151).

FIGURE 8. Female sex determination. As in the male, general transcription factors, as LHX1, EMX2 and PAX2, are necessary for intermediate mesoderm development. The gonadal ridge differentiates from the intermediate mesoderm following the action of SF1, LHX9 and WT1. WNT4, FST, RSPO1 and β-Catenin should be expressed to antagonize testis differentiation and promote early ovarian differentiation. Germ cell development (dependent on BMP family members, KIT ligand and its receptor C-KIT, WNT4, FST, retinoic acid and its receptors, the existence of two X chromosomes as well as several factors like DAZLA, MSH5, STRA8 and DMC1) are essential for fetal ovary stabilization. A number of other factors are involved in early folliculogenesis (FOXL2, neurotrophins and neurotrophin tyrosine kinase receptors, FIGα, NOBOX, SOHLH and members of the TGFβ family like GDF9, AMH and BMP15).

Ovarian Morphogenesis

 

In the XX fetus, the gonad remains histologically undifferentiated after the 7th week from a histological standpoint, but a functional differentiation is already detectable: XX gonads become capable of estradiol production at the same time as XY gonads begin to synthesize testosterone (260). PGCs proliferate by mitosis and differentiate to oogonia. Ovarian maturation proceeds from the center to the periphery. At week 10, oogonia in the deepest layers of the ovary enter meiotic prophase, the first unequivocal sign of morphological ovarian differentiation. Subsequently, oogonia become surrounded by a single layer of follicular (granulosa) cells, they enter meiosis, become oocytes and form primordial follicles (Fig. 9). Initiation of meiosis in the fetal ovary is heralded by the increase in retinoic acid levels synthesized by retinaldehyde dehydrogenase isoform 1 (encoded by ALDH1A1), expressed in the developing female gonad (261).

 

The earliest primary follicles appear at 15-16 weeks and the first Graafian follicles at 23-24 weeks (262, 263). By the end of the 7th month of gestation, mitotic activity has ceased and almost all germ cells have entered meiotic prophase. Oocytes proceed to the diplotene stage, where they remain until meiosis is completed at the time of ovulation in adult life. However, not all oocytes undergo meiosis: from 6-7 million ovarian follicles at 25 weeks, only 2 million persist at term (264). Most oocytes undergo apoptosis and follicles become atretic. AMH is produced, albeit in low amounts, after the 23th week of development (265) by granulosa cells from primary to antral follicles, but not by primordial follicles (266-268). The dynamics of follicle development and entry of germ cells into meiosis is notably different in rodents, in whom meiosis and folliculogenesis only progress after birth (170).

 

The involvement of germ cells in the stabilization of the gonadal structure is one major difference between the ovary and the testis, with germ cells being critical only in the ovaries in terms of maintenance of the somatic component of the gonad. In fact, while fetal testis development progresses normally in the absence of germ cells (269), ovarian follicles do not develop when germ cells are absent (263, 270). Furthermore, if germ cells are lost after formation of follicles, these rapidly degenerate (263, 271, 272).

 

In XX gonads, very few endothelial cells migrate from the mesonephros to the gonad, which suggests that cortical and medullary domains of the ovary are already established in early gonadogenesis, although no morphological boundaries are evident, consistently with molecular evidence of discrete gene expression domains specified by 12.5 dpc in the mouse ovary (255). The coelomic vessel formation, characteristic of the differentiating testis, does not occur in the normal XX gonadal ridge.

 

Granulosa cells, the equivalent of the Sertoli cells of the testes, originate from 3 possible sources: the ovarian surface epithelium, mesonephric cells from the adjacent rete ovarii, and the existing mesenchymal cells of the genital ridge (170, 273). Recent evidence in mice shows that many coelomic epithelial cells ingress to ovarian cortex and give rise to FOXL2-positive granulosa cells (274), confirming that other potential granulosa cell precursors are present in the gonadal ridge prior to the start of coelomic cell migration (173, 274). Theca cells, the counterpart of testicular Leydig cells, are thought to derive from fibroblast-like precursors in the ovarian stroma under the control of granulosa cells (275).

FIGURE 9. Developing human fetal ovaries. At 45 days, the ovary is recognizable only because it has not yet undergone testicular differentiation. In the cortex of the 14-week-old gonad, germ cells are aligned in rows, some of them have entered the meiotic prophase (arrows). In the medulla, primordial (small arrow head) and primary (large arrowhead) follicles are visible.

Genetic Control of Oogenesis and Folliculogenesis

 

Two major steps mark ovarian development: germ cell migration, proliferation and meiosis onset, followed by folliculogenesis. For a long time, it has been known that two intact X chromosomes are required in the human for ovarian differentiation and development –in contrast to the mouse, in which XY oocytes can occur in experimental conditions (65)– for ovarian differentiation and development. The lack of two X chromosomes, e.g. in Turner syndrome, results in germ cell loss and, subsequently, gonadal dysgenesis (263, 271). Therefore, all the factors involved in the proliferation and migration of PGCs in early embryogenesis (see “The Germ Cells” section) are essential for ovarian formation.

In the female gonad, germ cells continue to proliferate by mitosis. Meiotic entry is delayed until the 10th week in the human fetus and the 13th day in the mouse fetus (Table 1), due to the suppressive effect of the Polycomb repressive complex 1 (PRC1), which represses STRA8 and other factors involved in the differentiation of primordial germ cells and in early meiosis programs until retinoic acid reaches a threshold (276). Retinoic acid, synthesized by retinaldehyde dehydrogenases present in the mesonephros and the developing ovary (261, 277, 278), binds to the retinoic acid receptor (RAR) present in the germ cells and induces the expression of STRA8 (229, 234), a transcription factor that upregulates DAZL and SYCP3, two proteins involved in the formation of the synaptonemal complex essential for the onset of meiosis (39). Stabilization of oocytes requires the expression of MSH5, a protein involved in DNA mismatch repair (279). In Msh5 null mice, oocytes are lost before the diplotene stage resulting in ovarian dysgenesis. The expression of STRA8 takes place in an anterior-to-posterior wave and is followed by the upregulation of another meiotic gene Dmc1 (280). For a detailed description of other factors involved in oocyte development, see refs. (281) and (282).

A number of genes are upregulated in the human ovary before and during primordial follicle formation; their functional implications still need to be elucidated (283). In mice, neurotrophins (NTs) and their NTRK tyrosine kinase receptors facilitate follicle assembly and early follicular development (284). Factors involved in germ cell meiosis are also important. Although not essential to ovarian differentiation, several factors are involved in the development of ovarian follicles. FIGα is crucial for the formation of primordial follicles (285). AMH regulates the recruitment of primordial follicles into subsequent steps of folliculogenesis (286, 287), NOBOX, SOHLH1 and SOHLH2 are critical transcription factors during the transition from primordial to primary follicles (reviewed in ref. (39). GDF9 (288, 289) and BMP15 (290, 291) are important for follicle growth beyond the primary stage. An increasing number of factors are involved in later steps of folliculogenesis (for review, see ref. (39).

 

THE INTERNAL REPRODUCTIVE TRACT

 

The Indifferent Stage

 

Up to 8 weeks in the human embryo, the internal reproductive tract is similar in both sexes and consists of a set of two unipotential ducts, the Wolffian and Müllerian ducts (Fig. 10).

FIGURE 10. Undifferentiated reproductive tract. Both Wolffian and Müllerian ducts are present. Müllerian ducts open in the urogenital sinus at the level of the Müllerian tubercle between the orifices of the Wolffian duct.

Wolffian Ducts

 

In both the XX and the XY human embryo, Wolffian (mesonephric) ducts originate in the intermediate mesoderm, laterally to somites 8-13 in embryos 24 to 32 days old (Table 1) (3). Wolffian ducts elongate caudally and induce the formation of nephric tubules through a mesenchymal‑epithelial transition process. These tubules give rise, in a cephalic-to- caudal direction, to the three kidney primordia: pronephros, mesonephros and metanephros. While the pronephros and mesonephros are transient structures that soon degenerate, the metanephros is one of the main sources of the definitive kidney. Because Wolffian ducts are crucial for kidney development, abnormal formation of the Wolffian ducts is usually associated with other malformations in the urinary or genital systems.

 

Several factors have been identified in the induction and development of the Wolffian ducts (292, 293): PAX2 and PAX8, acting through GATA3, induce the initial formation, and LIM1 is required for the extension of the Wolffian ducts (293). EMX2 is necessary for their maintenance, whereas FGF8 and its receptors FGFR1 and FGFR2 seem to be important in the development and maintenance of different segments (cranial or caudal) of the Wolffian ducts (293).

 

A single ureteric bud evaginates from the Wolffian duct and grows dorsally, in response to inductive signals from metanephric mesenchyme involving GREMLIN1, BMP4 and BMP7 (294). RET signaling is involved in multiple aspects of early Wolffian duct development (295). Growing caudally, Wolffian ducts undergo extensive elongation and coiling while progressively acquiring a lumen. Factors involved in Wolffian duct stabilization, elongation and coiling include the SFRP1 and SFRP2, VANGL2, WNT5A and PKD1 (293).

 

As the Wolffian ducts elongate towards the cloaca, they induce the formation of the mesonephric tubules, most of which finally undergo regression, except close to the testes. There is a number of factors involved in mesonephric tubule development, including PAX2, PAX3, PAX8, GATA3, OSR1, WNT9B, WT1, SIX1, FGFR1, FGFR2, FGFR8 and SHH (292, 293, 296, 297). The mesonephric tubules give rise to the efferent ducts connecting the rete testis with the epididymis. WNT9B knockout male mice fail to develop the efferent ducts and the epididymis (298). Epididymal disjunction from the rete testis reflects a defect in these processes and can be found in approximately 40 % of patients with cryptorchidism (299).

 

The Wolffian ducts finally reach the caudal part of the hindgut, the cloaca. A spatiotemporally process of regulated apoptosis in both the Wolffian ducts and the cloaca is necessary for Wolffian duct insertion into the cloaca (300). The Wolffian ducts become incorporated into the male genital system when renal function is taken over by the definitive kidney, the metanephros (301).

 

Müllerian Ducts

 

Müllerian (paramesonephric) ducts, which give rise to most of the female reproductive tract, develop after Wolffian ducts in the urogenital ridges of both XX and XY embryos. They arise in 10-mm human embryo (5–6 weeks of gestation) as a cleft lined by the coelomic epithelium, between the gonadal and mesonephric parts of the urogenital ridge (3). This coelomic opening will later constitute the abdominal ostium of the Fallopian tube. The cleft is closed caudally by a solid bud of epithelial cells, which burrows in the mesenchyme lateral to the Wolffian ducts and then travels caudally inside their basal lamina. Initially, these cells are mesoepithelial, i.e. they exhibit characteristics of both the epithelium and the mesenchyme; they will become completely epithelial only in the female, at the time male ducts begin to regress (302, 303). At 8 weeks of development, the growing solid tip of the Müllerian duct, now in the pelvis, lies medial to the Wolffian duct, having crossed it ventrally in its downward course. For a while, the two Müllerian ducts are in intimate contact, then they fuse, giving rise to the uterovaginal canal (Fig. 11), which makes contact with the posterior wall of the urogenital sinus, causing an elevation, the Müllerian tubercle, flanked on both sides by the opening of the Wolffian ducts (Fig. 10).

FIGURE 11. Fused Müllerian ducts flanked by Wolffian ducts in the lower reproductive tract of a 50-mm female human fetus (10th week).

Development of the Müllerian duct occurs in three phases (Fig. 12) (302, 303). First, cells of the coelomic epithelium are specified to a Müllerian duct fate. These can be identified by a placode-like thickening of the coelomic epithelium and by the expression of LHX1 (302, 304) and anti-Müllerian hormone receptor type II (AMHR2) (305, 306). Transcriptional co-factors DACH1 and DACH2 are required for the formation of Müllerian ducts, possibly by regulating the expression of LHX1 and WNT7A or other factors important for Müllerian duct formation (307, 308).

During the second phase, these primordial Müllerian cells invaginate from the coelomic epithelium to reach the Wolffian duct. WNT4 expression in the mesonephric mesenchyme is essential for the Müllerian duct progenitor cells to begin invagination (304, 309).

 

The third or elongation phase begins when the invaginating tip of the Müllerian duct contacts the Wolffian duct. This phase consists in the proliferation and caudal migration of a group of cells at the most caudal tip. Müllerian duct elongation continues in close proximity to the Wolffian duct, then Müllerian ducts cross Wolffian ducts ventrally and fuse centrally close to the urogenital sinus.

 

As could be expected, integrity of protein kinase pathways is required for cell proliferation (310). Close contact with the Wolffian duct is also necessary to Müllerian growth; indeed, the lack of transcription factors required for Wolffian development, such as LIM1 or PAX2, leads to Müllerian truncation (see Table 4). Wolffian ducts do not contribute cells to the elongating Müllerian tip (302, 311), but act by supplying WNT9B, secreted by Wolffian epithelium (298).

FIGURE 12. Müllerian duct (MD) development can be subdivided into three phases. A. Phase I (initiation): MD progenitor cells in the mesonephric epithelium (ME) (yellow) are specified and begin to express LHX1. Phase II (invagination): in response to WNT4 signaling from the mesenchyme, LHX1+ MD progenitor cells invaginate caudally into the mesonephros towards the WD (blue). Phase III (elongation): the tip of the MD contacts the WD and elongates caudally in close proximity to the WD requiring structure and WNT9B signaling from the WD. B. Beginning at ∼ E11.5 in mice, the MD invaginates and extends posteriorly guided by the WD. During elongation, mesenchymal cells separate the WD and MD anterior to the growing tip (inset I). However, at the MD tip, the MD and WD are in contact (inset II). At ∼ E12.5, the MD crosses over the WD to be located medially. Elongation is complete by ∼ E13.5 with the MD reaching the urogenital sinus (UGS). A = anterior (rostral); D = dorsal; P = posterior (caudal); V = ventral. Reprinted with permission from ref. (303): Mullen RD, Behringer RR. Molecular Genetics of Müllerian Duct Formation, Regression and Differentiation. Sexual Development 8:281-296 (2014), Copyright 2014, Karger.

Caudally each Müllerian duct contacts the urogenital sinus at the Müllerian tubercle. This is a critical step and its failure can lead to lower vaginal agenesis, as it has been observed in Lhfpl2 mutant mice (312). In weeks 7 and 8, the caudal portions of the Müllerian ducts lie between the two Wolffian ducts near the urogenital sinus. Then during the 8thweek, Müllerian ducts fuse in the midline, leaving temporarily an epithelial septum that disappears one week later giving rise to the midline uterovaginal canal. The degree of midline fusion of Müllerian ducts is extensive in humans, but it is almost inexistent in mice, exhibiting paired oviducts and large bilateral uterine horns. Defects in Müllerian duct fusion and retention of the midline septum can lead to various congenital malformations in humans, including separate hemiuteri, uterus didelphys or unicornis, double vagina or cervix, vagina with septum etc. (313).

 

MALE DIFFERENTIATION OF INTERNAL GENITALIA

 

Male differentiation of the internal genital tract is characterized by regression of Müllerian ducts and differentiation of the Wolffian duct into male accessory organs.

 

Müllerian Duct Regression

 

Müllerian regression, the first sign of male differentiation of the genital tract, occurs in 55 to 60 day-old human embryos (Fig. 13), triggered by anti-Müllerian hormone (AMH) at the center of a complex gene regulatory network (reviewed in ref (314)). Once initiated, the regression of the Müllerian duct extends caudally as well as cranially, sparing the cranial tip which becomes the Morgagni hydatid, and the caudal end, which participates in the organogenesis of the prostatic utricle. Müllerian regression of the cranial part of the Müllerian duct begins while the duct is still growing caudally towards the urogenital sinus (315) and is characterized by a wave of apoptosis spreading along the Müllerian duct (316, 317). Extra-cellular matrix is deposited in the peri-Müllerian mesenchyme (318), which progressively strangles the Müllerian duct epithelium and finally remains the only witness of its former existence. Mesenchymal changes are preceded by the dissolution of the basement membrane, which precipitates apoptosis and allows extrusion of epithelial cells and their transformation into mesenchymal cells (317, 319). Epithelial-mesenchymal transformation is an important factor of epithelial cell loss during Müllerian regression.

 

Integrity of the WNT/β-catenin pathway is required for complete Müllerian duct regression in the male, perhaps through amplification of the AMH signal (320). β-catenin accumulates in the nucleus (317) upregulating Osterix (Osx), also called Sp7, an AMH-induced gene that regulates the expression of matrix metallopeptidase 2 (MMP2) (321). Osxis expressed in male, but not female, Müllerian ducts before and during regression. Overexpression of human AMH in female fetuses induces Osx, and Amhr2 knockout males lose Osx expression. Additionally, conditionally invalidation of β-catenin in the Müllerian ducts leads to a reduction in Osx expression, indicating that OSX is downstream of β-catenin in the regression pathway.

 

Wif1 (WNT inhibitory factor 1) encodes a secreted frizzled-related protein that inhibits WNT signaling. WIF1 shows many similarities to OSX: it is expressed in the male, but not the female, Müllerian duct and is not detected in Amhr2knockout mice. However, Müllerian ducts are absent in Wif1 knockout male mice, which implies that WIF1 is not indispensable for Müllerian duct regression (322).

FIGURE 13. Regressing Müllerian duct in a 35-mm male human fetus (9th week). Note the fibroblastic ring surrounding the epithelium of the Müllerian duct (right), the Wolffian duct is visible on the left.

 

Stabilization and Differentiation of Wolffian Ducts

 

The second aspect of male differentiation of the internal genital tract is the stabilization and differentiation of the Wolffian ducts (323). After the loss of mesonephric functional activity, the mesonephric nephrons and caudal tubules degenerate but the cranial tubules persist to form the male efferent ducts. The connections between the mesonephric tubules and the gonadal primordium are permanently established in the sixth week; in the male, they give rise to the rete testis, while in the female, they form the rete ovarii. Between weeks 9 and 13 in the human embryo, the upper part of the Wolffian duct differentiates into the epididymis. Below, it is surrounded by a layer of smooth muscle and becomes the vas deferens, which opens into the urogenital sinus at the level of Müllerian tubercle. In sexually ambiguous individuals, in whom Wolffian and Müllerian ducts coexist, the vas deferens is embedded in the uterine and vaginal walls (reviewed in ref. (324). The seminal vesicle originates from a dilatation of the terminal portion of the vas deferens in 12-week-old fetuses.

 

Testicular Descent

 

During human fetal development, the testis migrates from its initial pararenal position to its terminal location in the scrotum (Fig. 14). Testicular descent has been subdivided into several phases (325). Initially, the upper pole of the testis is connected to the posterior abdominal wall by the cranial suspensory ligament while a primitive gubernaculum extends from the caudal pole to the inner inguinal ring. At 12 weeks, the cranial suspensory ligament dissolves and the gubernaculum testis swells and pulls the testis down to the inguinal ring. After 25 weeks, the gubernaculum bulges beyond the external inguinal ring and is hollowed out by a peritoneal diverticulum called the processus vaginalis. The second –inguinoscrotal– phase of testicular descent occurs between 27 and 35 weeks after conception. « Physiological » cryptorchidism is frequent in premature infants. In the female, the cranial ligament holds the ovary in a high position and the gubernaculum, now the round ligament, remains long and thin.

FIGURE 14. Testicular descent. Left, Initial phase: the primitive gonad is located near the kidney, held by the cranial suspensory ligament (CSL) and the gubernaculum testis. Center, Transabdominal descent: androgen-mediated dissolution of the CSL and insulin like factor 3 (INSL3) mediated swelling of the gubernaculum bring the testis to the internal orifice of the inguinal canal. Right, Inguino-scrotal migration: the testis passes through the inguinal canal into the scrotum, this phase is androgen-dependent. Reprinted from ref. (325): Klonisch T, Fowler PA, Hombach-Klonisch S. Molecular and genetic regulation of testis descent and external genitalia development. Developmental Biology, 270:1-18 (2004), Copyright 2004, with permission from Elsevier. http://www.sciencedirect.com/science/article/pii/S001216060400137X

FEMALE DIFFERENTIATION OF INTERNAL GENITALIA

 

Female differentiation of the internal genital tract is characterized by the disappearance of the Wolffian ducts, which is complete at 90 days of human fetal development, except for vestiges such as the Rosenmüller organs or Gartner canals. Traditionally, the regression of Wolffian ducts in the female fetus has been ascribed to a passive process deriving from the lack of androgen action. Recent work using a Nr2f2 (encoding COUP-TF2) deletion, conditionally targeted to the Wolffian mesenchyme, has shown that the regression of Wolffian ducts in female embryos is an active process induced by COUP-TF2 through inhibition of the expression of FGFs, which otherwise activate the p-ERK pathway in the Wolffian duct epithelium for its maintenance (326). How androgens interact with this mechanism in males needs to be elucidated.

 

Müllerian ducts persist, establish apico-basal characteristics and develop into an epithelial tube that will give rise to the endometrium (302), while the surrounding mesenchyme differentiates into the myometrium of the uterus and Fallopian tubes (306). The acquisition of true epithelial characteristics signals the end of the AMH-sensitive window of Müllerian ducts (302). Tubal differentiation involves formation of fimbriae and folds in the ampullary region (Fig. 15) and acquisition of cilia and secretory activity by the high columnar epithelium. The uterotubal junction is demarcated by an abrupt increase in the diameter of the uterine segment and by the development of epithelial crypts. The early endometrium is lined by a closely packed columnar epithelium in which gland formation and vacuolated cells can be recognized as gestation advances. The cervix occupies the distal two-thirds of the fetal uterus.

FIGURE 15. Müllerian ducts develop into the uterus and fallopian tubes

THE UROGENITAL SINUS AND EXTERNAL GENITALIA

 

The Indifferent Stage

 

Up to approximately 9 weeks, the urogenital sinus and external genitalia remain undifferentiated (Fig. 16). The urogenital sinus is individualized in 7-9 mm (~5 week) human embryos, when a transverse urorectal septum divides the cloaca into the rectum dorsally and the primitive urogenital sinus ventrally. The Müllerian tubercle demarcates the cranial vesicourethral canal from the caudal urogenital sinus.

FIGURE 16. Sex differentiation of urogenital sinus (left) and external genitalia (right).

The cloaca is closed by the cloacal membrane, formed by ectoderm and endoderm, with no mesoderm in between. In the 5th week, mesodermal cells spread along the cloacal membrane and give rise to pair of swellings –the cloacal folds–, which form urogenital folds flanking the urogenital sinus and anal folds posteriorly. The urogenital folds fuse anteriorly to the cloacal membrane in the midline to form the genital tubercle. The cloacal membrane is divided by the urorectal septum into the genital membrane anteriorly and the anal membrane posteriorly. The genital membrane disappears in 20-22 mm (~8 week) embryos (327).

 

In embryos 8-15 mm long (~6 weeks), the opening of the urogenital sinus, the ostium, is surrounded by the labioscrotal swellings, which develop on each side of the urogenital folds. These are connected to the caudal poles of the genital ridges by fibrous bands which later develop into the gubernaculum testis in males and the round ligament in females.

 

The genital tubercle, consisting of lateral plate mesoderm and surface ectoderm, emerges as a ventral medial outgrowth just cranial to the opening of the ostium (328). Endodermal epithelial cells from the urogenital sinus are thought to invade the genital tubercle to form the midline epithelial urethral plate, which lies in the roof of the primary urethral groove and extends to the tip of the phallus (329, 330). After the corpora cavernosa and glans have differentiated, the ventral surface of the genital tubercle is depressed by a deep furrow, the urethral groove. The external genitalia remain undifferentiated up to approximately 9 weeks (327) (Fig. 16).

 

At 12 weeks in males and females alike, the vaginal primordium is formed by the caudal tips of the Müllerian ducts, and medial and lateral outgrowths of the urogenital sinus, the sinovaginal bulbs, which fuse to form the vaginal cord or plate. When the cells of the vaginal plate desquamate, the vaginal lumen is formed.

 

MALE DIFFERENTIATION  

 

Urogenital Sinus and Prostate

 

Male orientation of the urogenital sinus is characterized by prostatic development and by the repression of vaginal development. Prostatic buds appear at approximately 10 weeks at the site of the Müllerian tubercle and grow into solid branching cords. Maturation of the prostatic gland is accompanied by development of the prostatic utricle. Two buds of epithelial cells, called the sino-utricular bulbs in the male, develop from the urogenital sinus close to the opening of the Wolffian ducts and grow inwards, fusing with the medial Müllerian tubercle, to form the sino-utricular cord, enclosed within the prostate gland, which canalizes at 18 weeks to form the prostatic utricle, the male equivalent of the vagina (331).

 

External Genitalia

 

Masculinization of the external genitalia begins in human male fetuses 35-40 mm long (~9 weeks) by lengthening of the anogenital distance (327) (Fig. 16). Fusion of the labioscrotal folds, in a dorsal to ventral fashion, forms the epithelial seam (332), which closes the primary urethral groove. The literature concerning penile development is controversial. Most textbooks describe it as a two-step process, with the proximal urethra forming by fusion of the urethral folds around the urethral plate and the distal urethra arising from an invagination of the apical ectoderm. However, according to Cunha and colleagues (333), the entire human male urethra is of endodermal origin, formed by the urethral plate dorsally and the fused urethral folds ventrally. The seam is remodeled into the tubularized urethra without connection to the epidermis. The ventrally discarded excess epithelial cells migrate into the ventral skin of the penis. Abnormalities of seam formation or remodeling could explain the vast majority of cases of hypospadias in which defects of androgen synthesis or metabolism cannot be demonstrated (334).

 

Urethral organogenesis is complete at 14 weeks, apart from a physiological ventral curvature, which can persist up to 6 months of gestation. However, surprisingly, no size difference exists between penile or clitoral size until 14 weeks (335) despite the fact that serum testosterone levels peak between 11 to 14 weeks in males (336). The insensitivity of the male genital tubercle to high levels of androgens during the second trimester does not correspond to a low expression of the androgen receptor or of 5α-reductase type 2 in the corpora cavernosa (337). Maximal phallic growth occurs during the third trimester of fetal life, at a time when male testosterone levels are declining. The action of the growth hormone-insulin-like growth factor system (GH-IGFs) is partly responsible for penile growth, independently of androgens (338-340).

 

FEMALE DIFFERENTIATION  

 

Female orientation of the urogenital sinus is characterized by lack of prostatic differentiation and the acquisition of a separate vaginal opening on the surface of the perineum (Fig. 16). At the end of the ambisexual stage, the vaginal anlage is located just underneath the bladder neck. In females, the lower end of the vagina slides down along the urethra until the vaginal rudiment opens directly on the surface of the perineum at 22 weeks. The hymen marks the separation between the vagina and the diminutive urogenital sinus, which becomes the vestibule.

 

The embryological origin of the vagina is still hotly debated. In the generally accepted view, the upper part of the vagina derives from the Müllerian ducts and the lower part from the sinovaginal bulbs, which by fusion form the vaginal plate, derived from the urogenital sinus (341). It is now thought that the Wolffian ducts do not contribute cells to the sinovaginal bulbs but they may have a helper function during downward movement of the vaginal bud in the female (342). Atresia of the vagina in the Mayer-Rokitansky-Küster-Hauser syndrome could be explained by the failure of Wolffian and Müllerian ducts to descend caudally.

 

Development of female external genitalia is essentially static. The anogenital distance does not increase, the rims of the urethral groove do not fuse, the urethral plate persists as an epithelial cord, and the labioscrotal swellings give rise to the labia majora. The dorsal commissure forms at their junction. The genital folds remain separate and become the labia minora. When the vagina acquires a separate perineal opening, the diminutive pars pelvina and the pars phallica of the urogenital sinus become the vestibule.

 

CONTROL OF SEX DIFFERENTIATION

 

Growth Factors

 

GENITAL DUCT FORMATION

 

Molecular genetic studies in the mouse have contributed to the identification of growth factors essential for the formation of the sexual ducts (Table 4) [see refs. (323) and (343) for review]. Since Wolffian ducts are required for the elongation of Müllerian ducts, absence of growth factors necessary to Wolffian development will per se induce Müllerian truncation. Many growth factors, such as LIM1, EMX2, HOXA13, PAX2 and 8 and VANGL2 are essential also for the development of other organs. In contrast the role of WNT4A and WNT7A, a subset of the Wnt family homologous to the Drosophila wingless gene, is restricted to reproductive organs. WNT4 is required in both sexes for the initial formation of Müllerian ducts (309), mutations of WNT4 have been reported in three cases of Müllerian aplasia associated with hyperandrogenism in girls (reviewed in refs. (344-347), but have not been detected in classical forms of the Rokitansky-Küster-Mayer syndrome (240, 348). WNT7 is required for the expression of AMHR2; in its absence the Müllerian ducts do not regress in male fetuses (349). Members of the dachsung gene family, DACH 1 and 2 also play a role by regulating the expression of LIM1 and WNT7 (307).

 

Congenital bilateral absence of the vas deferens affects 97-98%% of patients suffering from cystic fibrosis, a bronchial and pancreatic disease due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) (350). Whether efferent duct maldevelopment is a primary defect of cystic fibrosis or a secondary degenerative change resulting from obstruction by mucus is not known at the present time.

 

TABLE 4. Consequences of Null Mutations of Growth Factors on Morphogenesis of Genital Ducts

Growthfactors

Wolffian ducts

Müllerian ducts

Gonads

References

β-catenin

Normal

Lack of oviduct coiling.

Lack of regression

Loss of germ cells in the ovary.

Testes normal

(320, 351, 352)

DACH1/DACH2

Normal

Hypoplasia of female reproductive tract

Normal

(307)

DICER1

Normal

Hypoplasia of female reproductive tract

Reduced ovulation rate

(353)

EMX2

Early degeneration

Do not form

Absent

(5)

HOXA13

Rostral ureteral junction

Agenesis of caudal portion

Normal

(354)

IGF1

Agenesis of caudal portion

Infantile uterus

No ovulation.

Abnormal Leydig cells.

(355)

LIM1 (LHX1)

Do not form

Do not form

Normal

(304)

PI3K/AKT

Increased apoptosis

Increased apoptosis

 

(310)

PAX2

Early degeneration

Early degeneration

Normal

(356)

PAX8

Normal

Endometrium does not form

Normal

(356, 357)

Retinoic acid receptors

Agenesis of vas deferens and seminal vesicles

Agenesis of uterus and cranial vagina

Normal

(358)

WNT4

Persist in females

No regression in males

Do not form

Ovary produces testosterone

(320)

WNT7A

Normal

Persist in males

 

(349, 359, 360)

 

VAGINA, PROSTATE, URETHRA, AND EXTERNAL GENITALIA

 

Correct vaginal development requires Wnt, Pax and Vangl2 genes (Table 5). Vaginal abnormalities similar to those elicited by diethylstilbestrol (DES) administration, i.e. vaginal clear-cell adenocarcinoma, vaginal adenosis, transverse vaginal ridges and structural malformations of the cervix and uterus, occur in transgenic mice deficient in WNT7A, a signaling molecule expressed by the Müllerian epithelium, suggesting that DES exposure acts by deregulating WNT7A during uterine morphogenesis (361). WNT7A deficiency could act by interfering with normal mesenchymal-epithelial signaling, which is required for correct morphogenesis of the reproductive tract. Vaginal opening is regulated by PAX8 (357) and VANGL2 as shown in the mutated the loop-tail mouse (362).

 

SOX9 (363) and FGF10 (364) both play a role in early prostate bud differentiation.

The secreted frizzled-related proteins (SFRP1 and 2) are required for correct gubernaculum development and testicular descent (365).

 

Early patterning of external genitalia is regulated by a cascade of signaling molecules which orchestrate interaction between tissue layers and mesenchymal/epithelial tissues (Table 5). External genitalia are appendages emerging from the caudal body trunk, hence many genes which pattern distal limb development also play a predominant role during genital tubercle formation, for example BMPs (328, 366), Fgf-8 and 10, Hox gene families (for reviews, see refs. (325, 367). β-catenin activates Fgf8 expression in the urethra, required for normal genital tubercle outgrowth (368). Sonic hedgehog (SHH) signaling regulates many of the mesenchymal genes involved (325, 328, 369-371) (Fig. 17). The homeotic genes Hoxa13 and Hoxd13 act in a partially redundant manner since double null mutants show more severe urogenital abnormalities than those with at least one functional allele (372).

 

Ephrin family factor EFNB2 and receptors EPHB2 and EPHB3 mediate cell adhesion and patterning events occurring at the midline, including urethral closure and scrotal fusion, as well as palate fusion (328, 373). Diacylglycerol kinase K (DGKK), an enzyme that phosphorylates diacylglycerol, is expressed in the epithelial cells of the urethral plate (374). In humans, DGKK is strongly associated with hypospadias risk (375, 376). Regulation of urethral tube closure during the androgen-dependent phase of penile development is mediated by FGF10, signaling through the IIIb isoform of fibroblast growth receptor 2 (FGFR2-3b), suggesting that these genes are downstream targets of the androgen receptor (377).

FIGURE 17. Growth factors regulating the outgrowth and ambisexual differentiation of the external genitalia. Role of sonic hedgehog (Shh) in the outgrowth and ambisexual differentiation of the genital tubercle (see table 5 for references). Most factors, with the exception of Hoxa13, are regulated by sonic hedgehog (Shh), expressed in the urethral epithelium (light green), and are identical to those regulating limb morphogenesis. Apoptosis is also affected by Shh. Data obtained from ref. (325): Klonisch T, Fowler PA, Hombach-Klonisch S. Molecular and genetic regulation of testis descent and external genitalia development. Developmental Biology, 270:1-18 (2004). http://www.sciencedirect.com/science/article/pii/S001216060400137X.

 

TABLE 5. Growth Factors in Urogenital Development

Growth factors

Role in urogenital development

References

BMP4

Restricts prostate ductal budding

(378)

BMP7

Closure of the distal urethra

(367)

FGF8

Initiation of genital swellings;

(379)

Ephrins

Urethral closure and scrotal fusion

(373)

FGF10

Development of the glans penis and clitoridis, and prostate

(364, 369, 379)

FGFR2-IIIB

Null mice exhibit severe hypospadias

(377)

HOXA10

Atrophic seminal vesicles in null mice

(380)

HOXA13

In mice, semi-dominant mutations lead to limb defects, vaginal hypoplasia and deficiency of the os penis (Hypodactyly syndrome)

In humans, an autosomal dominant mutation produces limb and uterine abnormalities and urinary tract malformations (Hand-Foot-Genital syndrome)

(354, 381)

HOXD13

Hoxd-13 null mice display decreased ductal branching in the prostate and seminal vesicle and agenesis of bulbourethral gland

(382)

HOXA13/HOXD13 null mutants

No genital tubercle, no partition of the cloaca in double mutants

(372)

LTAP

Vaginal opening

(362)

MSX2

Disruption of vaginal epithelium and lack of caudal Wolffian regression

(383)

PAX8

Vaginal opening

(357)

SHH/GLI2

Outgrowth and patterning of external genitalia and urogenital sinus

Development of prostatic ducts

Inhibition of apoptosis in penile smooth muscle

Masculinization of external genitalia

(369, 371, 384, 385)

SOX9

Lack of ventral prostate development

(363)

SFRP1 and 2

Testicular descent

(365)

VANGL2 (looptail mouse)

Imperforate vagina

(362, 386)

WNT/β-catenin

Masculinization of external genitalia

(387)

 

HORMONAL CONTROL OF MALE SEX DIFFERENTIATION

 

The classical experiments of Jost (58, 59) (Fig. 2) have taught us that the reproductive tract, whatever its genetic sex, will develop along female lines provided it is not exposed to testicular hormones, the main forces driving male sex differentiation (Fig. 18).

FIGURE 18. Hormonal control of male sex differentiation.

Anti-Müllerian Hormone (AMH)

 

Anti-Müllerian hormone (AMH), a member of the TGFβ family, triggers Müllerian regression, the first step of male sex somatic differentiation. AMH is expressed at high levels by Sertoli cells from the time of testicular differentiation (Fig. 19) until puberty and at lower levels thereafter (for reviews, see refs. (324, 388)). In the female, AMH begins to be produced in the second half of fetal life by granulosa cells of growing follicles (265, 266).

FIGURE 19. AMH protein expression by seminiferous tubules of an 11-week-old male human fetus, using an AMH-specific polyclonal antibody. Note the strong staining of seminiferous tubules.

Low expression of AMH and/or its type II receptor AMHR2 has also been identified in spermatocytes of maturing rat testis (389), the endometrium (390), the brain (391), hypothalamus (392), motor neurons (393) and female pituitary (394).

 

TGFβ family ligands are translated as dimeric precursor proteins comprising two polypeptide chains, each containing a large N-terminal pro-region and a much smaller C-terminal mature domain. Processing involves cleavage at sites between the two domains and dissociation of the pro-region domain. The AMH molecule is initially synthesized as a biologically inactive precursor. The precursor is cleaved by proteolytic enzymes into C and N terminal fragments which remain associated by non-covalent bonds (395, 396). Whether cleavage occurs at the time of secretion or within the target tissue is not clear at the present time. This step is required for binding of AMH to its primary receptor, at which time the AMH complex dissociates, releasing the mature ligand, the C-terminal homodimer and the N-terminal proregion (396). The homology of AMH to other members of the transforming growth factor-β (TGF-β) family is restricted to the C-terminus, for which a molecular model has been built, by analogy with crystallized members of the family (397) (Fig. 20). Cleavage and presumably bioactivity are enhanced if the endogenous cleavage site RAQR is replaced by a furin/kex2 RARR consensus site (398).

 

FIGURE 20. Molecular model of C-terminal AMH. A three-dimensional model of the C-terminal dimer was generated by comparative modeling using human BMP9 (399) as a template. The wrist epitope, the putative binding site for the type I receptor, is composed of the prehelix loop and alpha-helix of one monomer together with the concave side of the fingers of the second monomer (400). A mutation in the prehelix loop of AMH, Q496H, causes persistent Müllerian duct syndrome (397). Residues in the knuckle epitope of AMH, the putative binding site for AMHR2, are similar to those present in BMP7 and activin at the interface with ACTR2B (401, 402). Disulfide bonds (yellow) and Q496 residues (blue) are shown as sticks; residues in the knuckle epitopes are shown as spheres. Reprinted from ref. (397): Belville C, Van Vlijmen H, Ehrenfels C, Pepinsky RB, Rezaie AR, Picard J, Josso N, di Clemente N, Cate RL. Mutations of the anti-Müllerian hormone gene in patients with persistent Müllerian duct syndrome: biosynthesis, secretion and processing of the abnormal proteins and analysis using a three-dimensional model. Molecular Endocrinology 18:708-721 (2004). Copyright 2004 The Endocrine Society with permission. http://mend.endojournals.org/content/18/3/708.abstract?sid=22a37d21-69b5-499e-9996-8b1d4df81215

The human 2.8-kb gene has been cloned (403) and mapped to chromosome 19p13.3 (404). It consists of five exons, the last one coding for the C-terminal fragment. The AMH gene has been cloned in many other mammals (405-409), in the marsupial tammar wallaby (410), in the chick (411, 412) and American alligator (413), all of which carry Müllerian ducts which regress in the male. The gene is also present in the caudate amphibian, Pleurodeles waltl,whose Müllerian ducts persist in males (414). Even more surprisingly, AMH orthologs (415, 416) and the AMH type II receptor (417) have been cloned from the gonads of modern teleost fish, which do not possess Müllerian ducts at all. In fish, AMH appears to be involved essentially in germ cell proliferation and gonadal development (reviewed in ref. (418)), which suggests that AMH was initially a regulator of gonadal differentiation which acquired its anti-Müllerian activity during the course of evolution without completely relinquishing its former role. Indeed, in higher vertebrates, AMH inhibits Leydig cell differentiation (419) and follicle maturation (420).

 

The ontogeny of AMH expression differs widely between males and females. In the human fetal testis, AMH mRNA and protein can be detected from the 8th week, when Sertoli cells begin to form cord-like structures, the future seminiferous tubules (189) (Fig. 19). In the ovary, AMH production is detectable at 24 week gestation in granulosa cells of preantral follicles (265). The timing of the expression of AMH is crucial. In the male, high amounts of AMH must be expressed before Müllerian ducts lose their responsiveness, i.e. before the end of the 8th week in the human fetus. In the female, to avoid destroying the reproductive tract, it must be expressed after the window of sensitivity of the Müllerian ducts to its action has closed. Thus, in both sexes, the initiation of AMH transcription is under tight transcriptional control.

 

In the mammalian testis, but not in reptiles (413) or birds (421), SOX9 (97, 112, 113, 422, 423) -and to a lesser extent SOX8 (114)- triggers AMH expression in Sertoli cells by binding to a specific response element on the AMH promoter. Transcription factors SF1 (112, 113, 422, 424-431), GATA4 (113, 427, 432-438), WT1 (425, 439) increase, whereas DAX1 (425) and β-catenin (439) reduce, SOX9-activated AMH transcription either by binding to specific response elements or by protein-protein interaction (440, 441). In vivo, genes can affect AMH levels indirectly through their impact on testicular determination instead of acting on gene transcription.

 

Although initially gonadotropin-independent, AMH production falls under FSH control later in fetal life and after birth (113, 197, 442-444). FSH regulates AMH transcription through the FSH receptor-Gsα protein-adenylate cyclase-cyclic AMP pathway, resulting in a stimulation of protein kinase A (PKA) activity. PKA mediates phosphorylation of the transcriptional regulators SOX9, SF1 and AP2, as well as of IκB which releases NFκB. In the nucleus these factors activate AMH transcription by binding to their specific response elements on the AMH promoter (Fig. 21). LH and hCG do not have a direct effect on Sertoli cell AMH expression, but affect testicular AMH production through androgen action, as explained below.

FIGURE 21. Regulation of testicular AMH production. Left: the onset of AMH expression is gonadotropin-independent and depends on SOX9 binding to the proximal AMH promoter. Subsequently, SF1, GATA4 and WT1 enhance AMH expression by binding to specific promoter sequences or by interacting with transactivating factors. DAX1 impairs GATA4 and SF1 binding to the AMH promoters, resulting in lower AMH expression levels. Right: Later in fetal and postnatal life, FSH regulates AMH production through the FSH receptor-Gsα protein-adenylate cyclase (AC)-cyclic AMP (cAMP) pathway, resulting in a stimulation of protein kinase A (PKA) activity. PKA mediates phosphorylation of the transcriptional regulators SOX9, SF1 and AP2, as well as of IκB which releases NFκB. In the nucleus these factors bind to their specific response elements in proximal (SOX9, SF1) or distal (AP2 and NFκB) regions of the AMH promoter.
Right figure reprinted from ref. (113): Lasala C, Schteingart HF, Arouche N, Bedecarrás P, Grinspon R, Picard JY, Josso N, di Clemente N, Rey RA. SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-Müllerian gene expression in the testicular prepubertal Sertoli cells SMAT1. American Journal of Physiology – Endocrinology and Metabolism 2011; 301: E539-E547, Copyright 2011 the American Physiological Society. http://ajpendo.physiology.org/content/301/3/E539.abstract?sid=3829d833-dfdf-4310-bd6f-e7481c62be06

At puberty, FSH stimulation is antagonized by androgens resulting in a steep fall in AMH secretion by Sertoli cells (445). Androgen action requires the presence of the androgen receptor in Sertoli cells. This occurs relatively late after birth (Fig. 22) (204, 205, 446) allowing  both AMH and testosterone to reach high levels in fetuses and neonates. In androgen-insensitive patients, affected by mutations of the androgen receptor, AMH levels are abnormally elevated during the perinatal and pubertal stages (447, 448), due to unopposed stimulation by FSH. Androgens act directly on pubertal Sertoli cells to inhibit AMH promoter activity in the presence of the androgen receptor (429), even though the AMH promoter does not carry consensus androgen response elements (449). For androgens to repress AMHexpression, the existence of intact sites for binding of the transactivating factor SF1 on the AMH promoter is crucial, suggesting that the inhibition of AMH promoter activity by androgens could be due to protein–protein interactions between the ligand-bound androgen receptor and SF1 or by blockage of SF1 binding to its sites (429).

 

Gonadotropins and steroid also regulate AMH in the ovary. FSH stimulates AMH transcription in cultured granulosa cells (450) while estrogens has differential effects according to which estrogen receptor is involved (451), while LH has no effect in normal cells (452).

FIGURE 22. Ontogeny of testicular AMH production. In the mammalian fetal testis, AMH expression is triggered by the increase of SOX9 levels. It is not prevented by the rise of intratesticular levels of testosterone because fetal Sertoli cells do not express the androgen receptor (AR). After birth the number of Sertoli cells expressing the AR progressively increases. At puberty, when testosterone increases again, AR is present and AMH production is inhibited.

AMH is measurable in human serum by ELISA. Initially, the procedure was used by pediatric endocrinologists to measure testicular AMH in boys, hence the first commercially available kits were suited to the high level of AMH concentration of prepubertal males (448). Following the discovery that AMH serum concentration in women mirrors ovarian reserve (453, 454), AMH assay has become a standard procedure in assisted reproduction centers and more sensitive methods, adapted to the low concentration of AMH in female serum, were developed (455)(456)(457). In parallel, automated assays, e.g. the electrochemiluminescence Roche Elecsys assay (458) and the Beckman Coulter Access AMH assay (459), are progressively gaining ground, due to increased reproducibility and accelerated turnaround time, only 18 minutes for the Roche Elecsys assay. There is reasonable correlation between the different manual kits after manipulation of standard curves by manufacturers but not between manual assays and automated ones, which yield 20-30% lower values (458, 460). It follows that AMH values obtained with different methods are not interchangeable (461). Since clinicians are not usually aware of the problem, serious interpretation errors may arise during patient follow-up. An international standard of human recombinant AMH needs to be developed, particularly since the Immunotech assay upon which many normative values have been based (462-464) has been pulled off the market.

 

The uncleaved AMH precursor and non-covalent cleaved AMH are both detectable by commercial ELISA kits, but attempts to discriminate between the various AMH forms have not proven clinically rewarding (465-468).

 

AMH is an exceptionally stable biomarker, variations during the menstrual cycle (469, 470) and diurnal variations in men (471) are minimal. Measurement of AMH in serum has diagnostic applications in disorders of sex development (324, 472) and as a marker of prepubertal testicular function in boys (473-477). In women, AMH levels are a reliable marker of follicular reserve (453, 454) and may be used with relative accuracy to predict the onset of menopause (478) or to follow the evolution of granulosa cell tumors (479, 480). Some AMH mutations with reduced in vitrobioactivity are associated with premature ovarian insufficiency (481). In contrast, the clinical usefulness of AMH in seminal fluid in men with non-obstructive azoospermia is debatable (482). Further discussion of the diagnostic and potentially therapeutic value of AMH in the adult ovary and testis is beyond the scope of this review.

 

AMH Transduction: Type I and II AMH Receptors

 

Like other members of the TGFβ family, AMH signals through two distinct membrane-bound receptors, both serine/threonine kinases. Unlike other members of the TGFβ family truncated forms of the AMH primary receptor AMHR2 are not secreted, unless the signal sequence is replaced by the TGFβ one, suggesting that the AMHR2 signal sequence is defective (483). A three-dimensional model of extra- and intracellular domains built by analogy with crystallized receptors of the TGFβ family (Fig. 23) has served to analyze structure/activity relationship of the receptor molecule (483, 484).

 

The AMHR2 gene, located on chromosome 12q13.13, spans 8 kb pairs and is divided into 11 exons. Exons 1-3 code for the signal sequence and extracellular domain, exon 4 for most of the transmembrane domain, and exons 5-11 for the intracellular serine/threonine kinase domains (485). AMHR2 is expressed in the mesenchymal cells which surround the Müllerian duct, and also in Sertoli, granulosa (486, 487), Leydig (419) and germ cells (389), endometrium (390), neurons (391, 393) and hypothalamus (392). Expression of the receptor in the peri-Müllerian mesenchyme requires the presence of the signaling molecule WNT7A (359). The activity of AMHR2 is enhanced by WT1 (488) and by SP600125, an inhibitor of the c-Jun N-terminal kinase (489).

FIGURE 23. Molecular models of AMHR2 extracellular and intracellular domains. (A) The extracellular domain exhibits the general three-finger toxin fold of type II receptors and displays five disulfide bridges, four of which are conserved. Five amino acids (Phe62, Met76, Arg80, Asp81, and Thr108), implicated in binding AMH, are shown as spheres. (B) The intracellular domain exhibits the general fold of a two-domain kinase, with an N-lobe consisting mainly of a five-stranded β-sheet and a C-lobe, which is mainly α-helical. Some of the residues affected by PMDS mutations (Arg54, His254, Arg406, Asp426, Asp491, and Arg504) are shown as sticks. The inset shows residues affected by the p.((Gly445_Leu453del) mutation. Reprinted with permission from Elsevier, from ref. 364 (490): Josso N, Picard JY, Cate RL (2013). The Persistent Müllerian Duct Syndrome. In: New MI, Parsa A, Yuen TT, O'Malley BW, Hammer GD, eds. Genetic Steroid Disorders. New York, NY (USA): Elsevier

Binding of the receptor to its specific ligand requires proteolytic cleavage of the AMH precursor to yield the non-covalent complex AMH, but unlike other TGFβ family members, prior dissociation of this complex is not required. Dissociation is triggered by binding to AMHR2 (396) and is followed by the assembly of a tetrameric C-terminus/receptor complex with two molecules of type I receptor. Activated type I receptors then phosphorylate receptor-SMADS 1/5/8, which associate with SMAD4 and are then shuttled to the nucleus where they regulate transcription of target genes. (Fig. 24).

The AMH type II receptor is subject to processing (491). Increased expression of the receptor results in the removal of most of its extracellular domain and subsequent retention in the endoplasmic reticulum, resulting in a constitutive negative regulation.

 

The primary AMH receptor, AMHR2, is AMH-specific, a unique example of exclusive ligand-receptor pair within the TGFβ family (492). This specificity may be due to the presence of charged residues at the ligand binding interface (493). In contrast,  the downstream elements of the AMH transduction pathway are shared with the bone morphogenetic protein family, namely ALK2 (or ACVR1, Activin a receptor, type I) ALK3 (or BMPR1A, Bone morphogenetic protein receptor, type IA) and all three BMP receptor SMADS, 1, 5 and 8 (494-496). Another BMP receptor, ALK6 (or BMPR1B, Bone morphogenetic protein receptor, type IB), is engaged by ligand-bound AMHR2 (494) but has an inhibitory effect on AMH activity (497). ALK3 is the more potent AMH type I receptor in the Müllerian duct (498), in the Leydig cell (499) and in the SMAT1 Sertoli cell line (497) but in its absence, ALK2 is capable of transducing the AMH signal (496, 497).

FIGURE 24. Model showing processing of AMH, assembly of the AMH receptor signaling complex, and intracellular signaling. Cleavage of the AMH precursor results in a conformational change in the C-terminal domain, which allows binding of the AMH non-covalent complex to AMRHII. After dissociation of the N terminal proregion, the type I receptor is recruited into the complex and phosphorylated by the type II receptor kinase. The activated type I receptor can then phosphorylate Smads 1/5/8, which associate with Smad 4, translocate to the nucleus and regulate AMH responsive genes. Courtesy of Dr. Richard Cate. Data obtained from ref. (396): di Clemente N, Jamin SP, Lugovskoy A, Carmillo P, Ehrenfels C, Picard J-Y, Whitty A, Josso N, Pepinsky RB, Cate RL. Processing of anti-Müllerian hormone regulates receptor activation by a mechanism distinct from TGF-β. Molecular Endocrinology 24:2193-2206 (2010). http://mend.endojournals.org/content/24/11/2193.abstract

The Persistent Müllerian Duct Syndrome

 

Mutations of human AMH or AMHR2 (324) and gene knockout in mice (500, 501) are associated with a rare form of disorder of sex development, the persistent Müllerian duct syndrome (PMDS). These XY individuals are externally normally virilized, Müllerian duct derivatives are discovered incidentally at surgery for either inguinal hernia or cryptorchidism (Fig. 25) or following discovery of the condition in a sibling. Older patients may seek medical attention because of an abdominal tumor, hematuria or hemospermia.

FIGURE 25. Operative findings in a patient with PMDS. The Fallopian tubes are tightly attached to the testes, preventing testicular descent. Note normal male external genitalia.
Reprinted from ref. (484): Abduljabbar M, Taheini K, Picard JY, Cate RL, Josso N. Mutations of the AMH type II receptor in two extended families with Persistent Mullerian Duct Syndrome: lack of phenotype/genotype correlation. Hormone Research in Paediatrics 77:291-297 (2012). Copyright 2012 S. Karger AG, Basel, with permission. http://www.karger.com/Article/FullText/338343.

In patients with PMDS, as in the normal female, the Müllerian ducts differentiate into Fallopian tubes, uterus, and upper vagina. They retain their close apposition to Wolffian duct derivatives, epididymis, and vas deferens while remaining tied to the pelvis by the broad ligament (Fig. 25). The clinical features of PMDS are similar in AMH and AMHR2 mutations and may vary within the same sibship. The mobility of the Müllerian structures determines testicular location. Bilateral cryptorchidism is observed most frequently: the uterus remains anchored to the pelvis, and mechanically prevents testicular descent. Alternatively, one or both testes may make it into the inguinal canal or the scrotum, dragging the uterus along. This may result either in unilateral cryptorchidism with a hernia containing the uterus on the opposite side, a condition known as “hernia uteri inguinalis”. The testis on the opposite side can be drawn into the same hemiscrotum by gentle traction or may already be present there; this condition typical of PMDS is named “transverse testicular ectopia”. It may be the only sign of an AMH or AMHR2 mutation in patients with normally regressed Müllerian derivatives (502). Approximately half the cases present with bilateral cryptorchidism, the rest with hernia uteri inguinalis or transverse testicular ectopia in similar proportions. The descended testis is only loosely anchored to the bottom of the processus vaginalis by a thin gubernaculum 4. It is exposed to an increased risk of torsion and subsequent degeneration (503). Associated abnormalities such as low birth weight with or without prematurity or complex metabolic syndromes are suggestive of idiopathic PMDS unrelated to defects in the AMH pathway. Intestinal malformations have been observed in four cases, consisting of either jejunal atresia or lymphangiectasis. Skeletal malformations suggestive of defects in the BMP pathway have not been reported.

 

Testicular tumors of every denomination, mostly seminomas, are a frequent mode of presentation of PMDS in older patients, particularly in settings where cryptorchidism has been neglected in childhood. Young patients may be affected by germ cell neoplasia in situ (502, 504). Early orchidopexy is not necessarily 100% effective to preserve against testicular degeneration (505, 506). Furthermore, the incidence in PMDS adults reaches 33% (502) compared to 18% for simple cryptorchidism (507), suggesting that misplacement of the testis may not be the only factor driving testicular cancer. Evolution depends upon the histological type of the tumor; choriocarcinomas and mixed germ cell tumors share a dim prognosis.

 

Uterine tumors occur less frequently, hematuria is usually the presenting symptom (508). They should not be confused with degeneration of the prostatic utricle often mistakenly called “Müllerian” cysts (509). Farikullah et al (510)reported 11 cases of Müllerian degeneration in males, but only 3 qualified as PMDS. Exceptionally, in an elderly PMDS patient, hematospermia may be due to endocrine imbalance with low testosterone and high estrogen secretion (511).

 

Infertility is the most common complication of PMDS. Pubertal development is normal, spermatogenesis is not unheard of (512), yet few patients actually father children and stringent evidence of paternity is lacking. In all cases at least one testis was in a scrotal position (reviewed in ref. (502). There are several causes of infertility: the excretory ducts may not be properly connected to the testis or the germinal epithelium may be damaged by longstanding cryptorchidism. Paradoxically, all fertile PMDS patients fathered children before their condition was diagnosed and surgically addressed. Surgery may compromise the testicular blood supply or the vasa deferentia, particularly if hysterectomy is undertaken without proper dissection of the male excretory ducts included in the uterine wall. The prognosis may improve with modern surgical and assisted reproduction techniques. In inbred populations where fertility is a crucial issue, as in the Middle East (513), genetic counseling is recommended and molecular screening should be carried out if a consanguineous union is contemplated.

 

Treatment should aim primarily towards the prevention of the two main complications of PMDS, cancer and infertility. Both goals are served by replacing the testes in the scrotum but excising the uterus to allow abdominal testes to descend into the scrotum carries significant risks. The primary testicular blood supply is through the internal spermatic and the deferential arteries. Often the spermatic vessels are too short and must be divided to allow orchidopexy. The viability of the testis then becomes wholly dependent upon the deferential artery, which is closely associated with the Müllerian structures and may be severely damaged by attempts to remove them (514). Most authors recommend partial hysterectomy, limited to the fundus and proximal Fallopian tubes, or the simple division of Müllerian structures in the midline. If the length of the gonadal vessels is the limiting factor; a Fowler–Stephens orchidopexy or microvascular autotransplantation (515) may produce good results. Intracytoplasmic sperm injection may be helpful in the case of ejaculatory duct defects. Orchidectomy is inevitable if the testes cannot be brought down.

 

The serum level of AMH in prepubertal patients depends on the molecular origin of the syndrome. Before puberty, the level of serum AMH allows easy discrimination between AMH and AMHR2 mutations. In nearly all patients with AMH mutations, AMH levels are extremely low or undetectable. AMH gene mutations with a normal AMH serum level are very unusual and should be regarded with suspicion. We have documented only one such case, a Gln 496 His mutation, which is thought to affect binding of AMH to its type 1 receptor ALK3 (397). Menabo et al (516) reported a case of PMDS attributed to AMH variants with a normal AMH level but they did not rule out an AMHR2 mutation. AMH levels are relatively low in normal infants shortly after birth, but then repeat determinations show a progressive rise with increasing age.

 

Serum AMH levels are within normal limits for age in AMHR2 mutations and in idiopathic PMDS, unrelated to defects in known components of the AMH pathway. Obviously, serum AMH is not detectable, whatever the genotype, in the case of anorchia (503) and may be abnormally low in cryptorchid patients. Testosterone and gonadotropin levels are normal for age. After pubertal maturation, serum AMH declines physiologically, and it may be difficult to discriminate between AMH and AMHR2.

 

Approximately 80% of PMDS cases are due to AMH or AMHR2 mutations, in about equal proportions. The first AMHmutation was reported in 1991 (517) in a Moroccan family. At the time of writing, early 2020, a total of 84 families affected by AMH mutations have been published in the world literature. All exons are affected (Fig 26). Exon 1, the site of most recurrent mutations (Table 6) is hit hardest, exon 5 is next but when the number of base pairs is taken into account, the relatively short exon 2 with its 13 mutations is the runner up. Although it is shorter, the 3’ end of exon 5 that codes for the bioactive C-terminal domain of AMH is targeted nearly twice as often as the 5’ end.

FIGURE 26. Mutations of the AMH and AMHR2 genes in the Persistent Müllerian Duct Syndrome (PMDS). Mutations of the AMH (top) and AMHR2 genes (bottom) in the Persistent Müllerian Duct Syndrome (PMDS). The 3' end of the AMH gene (picture in red) codes for the C-terminal domain, responsible for bioactivity, yet mutations are spread along the whole length of the gene. Similarly, mutations of the AMHR2 affect intracellular and extracellular domains alike.

Altogether, 67 different AMH alleles bearing all types of mutations have been described in PMDS. Missense and stop mutations are the most frequent, insertions are rare (see details in ref. (502). One deletion is of particular interest, because it disrupts the SF1 response element located at -228 in the AMH promoter. Inactivation of the -102 site does not prevent Müllerian regression in transgenic mice. The greater impact of the -228 deletion detected in the PMDS patient may be due to the vicinity of the -102 SF1 site to a GATA site, to which SF1 can indirectly bind through protein/protein interaction with GATA4. This hypothesis is supported by transactivation experiments showing that destruction of the GATA site adjacent to SF1-102 results in inactivation of the AMH promoter (430).

 

A few AMH mutations have been reproduced by site-directed mutagenesis, cloned into an expression vector and transfected into COS cells to allow study of the secretion of the mutant protein into the culture medium (397). These studies confirm that most single nucleotide variations of the AMH gene act by affecting the stability and secretion of the hormone, explaining why nearly all patients with AMH mutations, regardless of the site of the mutation, have a very low level of circulating AMH.

 

TABLE 6. Recurrent (n≥4) Mutations of AMH in PMDS

Exon

cDNA

Protein

Families (n)

1

c.35T>G

p.(Val12Gly)

4

1

c.283C>T

p.(Arg95*)

6

1

c.301G>A

p.(Gly101Arg)

4

1

c.343_344delCT

p.(Leu115Thrfs*58)

5

1

c.367C>T

p.(Arg123Trp)

7

2

c.500A>G

p.(Tyr167Cys)

5

 

Up to now, 90 families with various AMHR2 mutations have been published, the first in 1995 (485). Since AMHR2mutations lead to PMDS by blocking response to AMH, the level of circulating AMH is normal for age, in contrast to PMDS due to AMH mutations.

 

A total of 75 independent mutant alleles have been described, targeting all 11 exons and 5 introns; their location within the gene are shown in Fig.26B. Most are missense or stop mutations. Two cases of classical PMDS due to a microdeletion of the chromosomal region 12q13.13, the locus of the gene for AMHR2, have been reported. One case involved a homozygous microdeletion of five exons of the AMHR2 gene. In the second case, the whole AMHR2 gene was deleted from the maternally inherited chromosome. The patient’s paternal allele carried a stop mutation, which was initially thought to be homozygous by Sanger sequencing (518).

 

The most prevalent mutation, a 27-base deletion in exon 10 (c.1332_1358del) pictured on Fig. 23 results in the deletion of 9 amino acids from an alpha helix within the kinase domain and affects 37% of families with receptor mutations. The proportion reaches 62% of Northern European families, where it probably represents a founder effect. This mutation is easily detected by PCR, without the need for sequencing.

 

Not all PMDS cases have benefited from molecular study. In many countries, genetic studies are not readily available for PMDS, and cases have been published with only clinical data (519, 520). Owing to lack of molecular characterization, it is difficult to interpret the unusual sex-linked familial transmission of PMDS reported in two families (521, 522).

 

In approximately 20% of PMDS patients, careful sequencing of AMH and AMHR2 exons and adjacent portions of introns have failed to yield an explanation. Either a mutation has escaped detection or other genes are involved. The AMH and BMP families share type I receptors and cytoplasmic effectors, which could be implicated in PMDS. Initially, BMP receptors were considered unlikely candidates because an intact BMP pathway is required for survival beyond the embryonic stage. However, this might not hold for mild missense mutations (523). Alternatively, idiopathic PMDS could be caused by mutations in other genes involved in Müllerian duct development/regression such as β-catenin (320) or patterning genes not specifically involved in reproductive development. Studies with next generation sequencing are underway to resolve this issue. Women homozygous for AMH or AMHR2 mutations are normally fertile but it is too early to know whether, similar to “AMH-null” mice (286), they will experience premature ovarian failure due to follicular depletion

 

Androgens

 

Testosterone or dihydrotestosterone (DHT), binding to the same androgen receptor (AR), are the main factors involved in maintenance of the Wolffian duct and differentiation of male sex accessory organs and external genitalia.

 

Testosterone Biosynthesis

 

Beginning at 9 weeks, testosterone is produced from cholesterol by chorionic gonadotropin stimulation of fetal Leydig cells through the coordinated action of steroidogenic enzymes (Fig. 27 and Table 7), most of which are also expressed in the adrenal gland, explaining why many steroidogenic disorders are common to the testis and adrenal. Most steroidogenic enzymes are either hydroxysteroid dehydrogenases or cytochromes P450, residing either on the mitochondrial membrane (type I) or in the endoplasmic reticulum (type II) (524). The initial step in steroidogenesis, conversion of cholesterol into pregnenolone, is mediated by the P450 side-chain cleavage enzyme (P450scc), a type I cytochrome located at the inner mitochondrial membrane. However, the inner mitochondrial membrane contains relatively little cholesterol, so the rate-limiting step of steroidogenesis is the transfer of cholesterol from the outer to the inner mitochondrial membrane. This step is dependent on steroidogenic acute regulatory protein (StAR) regulated essentially by a trophic hormone stimulated cAMP/PKA pathway (525). The exact mechanism of StAR-mediated cholesterol transport into the mitochondria is not completely understood.

 

Pregnenolone is subsequently metabolized into 17α-hydroxypregnenolone and dehydroepiandrosterone (DHEA) by P450c17. This type II cytochrome bears two distinct activities: a 17α-hydroxylase activity responsible for the conversion of pregnenolone to 17α-hydroxypregnenolone and a 17-20 lyase activity, capable of converting 17α-hydroxypregnenolone to DHEA. P450c17 receives electrons from NADPH via the flavoprotein P450 oxidoreductase (POR) (526, 527). Cytochrome b5 is required for optimal 17,20 lyase activity (528, 529). P450c17 and its partner proteins also convert the Δ4 compound progesterone into 17α-hydroxyprogesterone and Δ4-androstenedione.

FIGURE 27. Steroidogenesis. Steroidogenesis: the “classic” and “backdoor” pathways for dihydrotestosterone (DHT) synthesis. See Table 7 for enzyme nomenclature. DHEA: dehydroepiandrosterone, DHP: dihydroprogesterone. Reprinted from ref. (530): Fluck CE, Meyer-Boni M, Pandey AV, Kempna P, Miller WL, Schoenle EJ, Biason-Lauber A. Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation. American Journal of Human Genetics 89:201-218 (2011). Copyright 2011, with permission from Elsevier. http://www.cell.com/AJHG/abstract/S0002-9297(11)00262-X (top figure), and ref. (531): Wilson JD, Shaw G, Leihy ML, Renfree MB. The marsupial model for male phenotypic development. Trends in Endocrinology and Metabolism, 13:78-83 (2002), Copyright 2002, with permission from Elsevier. http://www.sciencedirect.com/science/article/pii/S1043276001005252 (bottom figure).

Two additional enzymes, 3β- and 17β-hydroxysteroid dehydrogenases are required for the synthesis of testosterone. Two isoforms of 3ß-hydroxysteroid dehydrogenases have been identified: 3ß-HSD type 1, expressed mainly in the placenta, mammary gland and skin, and 3ß-HSD type 2, expressed in the gonads and adrenal glands. Only mutations in the type 2 gene result in congenital adrenal hyperplasia and/or DSD (532, 533).

 

The final testicular enzyme in testosterone biosynthesis is 17ß-hydroxysteroid dehydrogenase (17β-HSD), formerly known as 17-ketosteroid reductase, which reduces 17-ketosteroids to 17β-hydroxysteroids, i.e. Δ4-androstenedione to testosterone and the Δ5 steroid DHEA to androstenediol. Three isoforms of 17ß-HSD have been identified. The type 3 isoform, HSD17B3, is expressed in the testis and is the only one involved in fetal male sexual differentiation (534). XY patients with impaired HSD17B3 usually develop with female or ambiguous external genitalia; however, Wolffian ducts derivatives are present in most, probably due to accumulation of the weak androgens Δ4-androstenedione and Δ5-DHEA. The type 2, HSD17B2, is expressed in the liver and has the capacity for testosterone synthesis. This could explain the virilization observed at puberty in XY patients with HSD17B3 deficiency (534, 535).

 

TABLE 7. Proteins Involved in Androgen Production

Protein

Main Role

Gene

Chromosome

Steroidogenic acute Regulatory Protein (StAR)

Cholesterol trafficking

STAR

8p11.2

P450scc (P450 side chain cleavage enzyme)

Cytochrome P450, family 11, subfamily A, polypeptide 1

Cholesterol side-chain cleavage

CYP11A1

15q24.1

P450c17 (17α-hydroxylase/17,20-lyase)

Cytochrome P450, family 17, subfamily A, polypeptide 1

Metabolizes pregnenolone

CYP17A1

10q24.32

P450 oxidoreductase (POR)

Electron donor to P450c17

POR

7q11.23

Cytochrome b5, type A

Regulation of 17,20-lyase activity

CYB5A

18q22.3

3β-hydroxysteroid dehydrogenenase 2 (3β-HSD 2)

Conversion of Δ5 to Δ4 steroids

HSD3B2

1p12

17β-hydroxysteroid dehydrogenenase 3 (17β-HSD 3)

Reduction of 17 keto to 17β-hydroxysteroids

HSD17B3

9q22.32

5α-reductase type 2

Reduction of T to DHT

SRD5A2

2p23.1

5α-reductase type 1

Reduction of progesterone and 17OH-progesterone

SRD5A1

5p15.31

Aldo-keto reductase family 1, member C2

3α-hydroxysteroid dehydrogenase, type III (3α-HSD)

Oxidoreduction of 3α-androstanediol/ DHT *

AKR1C2

10p15.1

Aldo-keto reductase family 1, member C4

3α-hydroxysteroid dehydrogenase, type I

id but less efficient

AKR1C4

10p15.1

17β-hydroxysteroid dehydrogenase 6 (17β-HSD 6)

Retinol dehydrogenase 1

3α-hydroxysteroid epimerase

Oxidizes 3α-androstanediol to DHT

HSD17B6

RODH

12q13.3

P450aro (aromatase)

Cytochrome P450, family 19, subfamily A, polypeptide 1

Aromatizes androgens to estrogens

CYP19A1

15.q21.2

Steroidogenic factor 1 (SF1)

Nuclear receptor subfamily 5, group A, member 1

Adrenal-4 binding protein (AD4BP)

Fushi tarazu factor 1 (FTZF1)

Regulates several steroidogenic enzymes

NR5A1

9q33.3

* The direction of the reaction depends on cofactor availability (530). The four last enzymes act exclusively in the alternate pathway of DHT synthesis.

 

DHT Production: Classic and Alternative (Backdoor) Pathways

 

Testosterone itself is not a very active androgen; its metabolite DHT is the main virilizing agent during male reproductive development. The conversion of testosterone to DHT amplifies the androgenic signal through several mechanisms. DHT cannot be aromatized to estrogen, and thus its effects are purely androgenic. Testosterone and DHT bind to the same androgen receptor but DHT does so with greater affinity which results in a stabilization of the hormone-receptor complex for a longer period of time (536).

 

In the classic pathway of DHT production (Fig. 27, top), testosterone is converted to DHT inside the target cell by the enzyme 5α-reductase type 2 coded by the SRD5A2 gene expressed in fetal genital skin, in male accessory sex glands and in the prostate (537). In tissues equipped with 5α-reductase at the time of sex differentiation, such as the urogenital sinus and external genitalia, DHT is the active androgen (538). During embryogenesis, 5α-steroid reductase type-2 encoded by the SRD5A2 gene plays a central role in the differentiation of the male phenotype. Patients with 5α-reductase deficiency virilize very poorly at these levels (539, 540). Another functional isoenzymes of 5α-reductase, with a different pH optimum, has been characterized (537): 5α-reductase type 1, transiently active in newborn skin and scalp and permanently expressed in liver after birth and in skin from the time of puberty, is not expressed in the fetus. Tissue distribution and ontogeny of both isoforms as well as mutation studies in humans with 46,XY DSD clearly indicate that type 2 plays the major role in sexual differentiation but the emergence of type 1 probably accounts for the pubertal virilization of the type 2-deficient patients.

 

Testosterone, however, is not an obligatory precursor of DHT (Fig. 27, bottom). Observations in a marsupial, the tammar wallaby (531), have shown that the testis itself produces biologically significant amounts of DHT through an alternate or “backdoor” pathway without using testosterone, DHEA or androstenediol as intermediates. Additional enzymes not part of the classic pathway can mediate the direct oxidation of 5α-androstanediol to DHT (524, 530). This "backdoor" pathway contributes to virilization in the human fetus as demonstrated by the genetic studies of Flück and her colleagues (530) in two families with 46,XY DSD. After they failed to demonstrate mutations in known steroidogenic enzymes, they explored genes acting in the alternate pathway of androgen synthesis. This led to discovery of mutations in the genes AKR1C2 and AKR1C4, alias 3α-hydroxysteroid dehydrogenase (or reductase) type I and type III. In the alternate pathway these enzymes catalyze the reduction of dihydroprogesterone and 17OH-dihydroprogesterone to allopregnanolone and 17OH-allopregnanolone, the precursor of androsterone and androstanediol. Their role in the oxidation of the latter to testosterone is hypothetical because they have very high affinity for NADP(H), which favors reductive reactions and low affinity for NAD(H) which favors the opposite, thus they are expected to function primarily as a reductase (541). AKR1C2 is expressed in the fetal, but not the adult testis, AKR1C4 is expressed at low levels in both tissues. The deleterious effect of AKR1C2/4 mutations proves that testicular DHT synthesis through the alternate pathway is required for normal fetal sex differentiation.

 

Gonadotropin Control of Testosterone Production

 

Testosterone production by the human fetal testis is detectable at 9 weeks, peaks between 14 and 17 weeks and then falls sharply, so that in late pregnancy the serum concentrations of testosterone overlap in males and females. Gonadotropin stimulation is not required for the initiation of steroid synthesis (220) but is necessary to maintain Leydig cell function subsequently. Testicular and serum levels of testosterone are closely correlated with human chorionic gonadotropin (hCG) concentration; the peak of fetal testicular steroidogenic activity coincides with the acme of concentrations of hCG in the circulation. In adult Leydig cells, the capacity to respond to sustained gonadotropic stimulation by increased androgen production is curtailed by the development of a refractory state, due to receptor down-regulation (542). Fetal Leydig cells apparently escape desensitization, allowing them to maintain a high testosterone output during the several weeks necessary to male differentiation of the genital tract. The fetal pituitary takes over when chorionic gonadotropin declines in the 3rd trimester (reviewed in ref. (543) (Fig. 28). Impaired LH secretion in 46,XY fetuses does not result in DSD because the most important steps of sexual differentiation, with the exception of penile growth, occur at the time Leydig cells are controlled by hCG.

 

In contrast, mutations in the LH/CG receptor of Leydig cells result in severe virilization defects (544). LH and hCG signal through a common seven-transmembrane domain receptor coupled to G proteins present on testicular Leydig cells. The human gene located on chromosome 2p21, contains 11 exons. The first ten encode a long N-terminal extracellular domain responsible for hormone binding, while the 11th exon encodes the whole transmembrane domain, involved in the cAMP/PKA signal transduction pathway. A functioning LH/CG receptor is absolutely necessary to achieve a normal development of the fetal Leydig cell population and androgen production. Loss of function mutations lead to 46,XY DSD (reviewed in ref. (545), with the exception of the deletion of exon 10, which was identified in a patient with normal male phenotype but lack of pubertal development (546, 547). This suggests that exon 10 is required for signal transduction of pituitary LH but not hCG.

FIGURE 28. Control of testosterone production in the human fetus. Note the low testosterone concentration during the last trimester, at the time that hCG production by the placenta has abated. Data obtained from ref. (548): Winter JSD, Faiman C, Reyes F (1981). Sexual endocrinology of fetal and perinatal life. In: Austin CR, ed. Mechanisms of Sex Differentiation in Animals and Man. London: Academic Press; p.205-253.

The Androgen Receptor

 

Testosterone and DHT exert their action on androgen-dependent tissues by binding to the androgen receptor, a member of the steroid receptor family (Fig. 29). Mutations of this receptor lead to the androgen insensitivity syndrome, a relatively common disorder of sex development typically characterized by a female external genital appearance in XY patients despite a normal or excessive production of testicular hormones (see ref. (549) for review). The androgen receptor is encoded by a single-copy gene located on the long arm of the X chromosome, locus Xq12 (550). It spans 75-90 kb and its open reading frame of 2.75 kb comprises 8 exons. Exon 1 is the longest and codes for the amino-terminal transactivation domain. A highly polymorphic CAG triplet containing 14-35 repeats towards the 5’-end of exon 1, is useful as a genetic marker for inheritance of X chromosomes. Interestingly, expansion of the trinucleotide repeat which encodes this long tract of glutamine residues segregates with X-linked spinal and bulbar atrophy a degenerative neuropathy characterized by the accumulation of the mutated receptor in the nucleus and cytoplasm of motor neurons (reviewed in ref. (551). Exons 2 and 3 code for sequences containing two zinc fingers implicated in DNA binding. Most mutations occur in exons 4 to 8, which encode the steroid hormone binding domain. The 5’-portion of exon 4 codes for the hinge region between the DNA- and steroid-binding domains, and plays a regulatory role (552). A complete database of androgen receptor mutations is available from McGill University in Montreal (553).

 

In contrast to receptors for other steroid sex hormones, which reside in the nucleus even in the absence of ligand binding, the androgen receptor resides mainly in the cytoplasm, associated with heat-shock and other chaperone proteins, in the absence of hormone and translocates into the nucleus in the presence of ligand (554). Nuclear localization is controlled by a nuclear localization signal spanning the second zinc finger and the hinge region competing with an androgen-regulated nuclear export signal in the ligand binding domain (555). The androgen/AR complex can also signal through non-DNA binding-dependent pathways. However, the physiological relevance of these actions remaining largely unknown (554).

 

The androgen receptor binds to specific DNA motifs, the androgen response elements (ARE), present in the promoter regions of androgen-activated genes. The consensus or classic ARE consists of two palindromic half sites spaced by three base pairs (AGAACAnnnTGTTCT).while the so-called "selective" AREs, such as the one in intron 1 of the SRD5A2 gene (556) resemble direct repeats of the same hexamer (557). After binding to AREs on the promoters of androgen-responsive genes, the androgen receptor regulates their transcriptional activity. It is aided in this task by co-regulators, partner proteins that facilitate assembly of the preinitiation complex through chromatin remodeling. These include the p160 family of coactivators, which interact selectively with the agonist-bound form of AR (558-560). Attempts at blocking the androgen receptor by preventing its interaction with co-activators are part of the therapeutic strategy in prostate cancer (554).

FIGURE 29. Androgen receptor protein, cDNA and gene.

 

The Case of the Wolffian Ducts: The Role of Local Testosterone

 

In fetal Wolffian ducts, 5α-reductase is expressed only after the ambisexual, critical, stage of male sex differentiation, thus testosterone itself, not DHT, saves them from degeneration (537, 538). Because of its close proximity to the testis, the Wolffian duct is exposed to a very high local concentration of testosterone, a source of androgen not available to organs receiving testosterone only via the peripheral circulation (Fig. 30) (297). Patients with androgen insensitivity whose androgen receptor retains very low but significant residual activity have a female phenotype but retain an epididymis or vas deferens (561). Wolffian duct differentiation is programmed during a critical time window, between 15.5 and 17.5 dpc in the rat fetus. Because the androgen receptor is expressed in the Wolffian duct stroma but not in the epithelium during this time, Wolffian duct differentiation is likely to be dependent on androgen-mediated signaling from the stroma to the epithelium (562).

 

Two phase can be described in the development of the Wolffian ducts (297). In the first phase, testosterone induces the stabilization of the ducts (in rodents, this occurs between embryonic days 13 and 16). Afterwards the Wolffian ducts undergo elongation and convolution of the cranial end, where the epididymis and vas deferens differentiate, and the seminal vesicles form at the caudal end.

 

Control of Testicular Descent

 

Androgens are required to mediate the disappearance of the cranial suspensory ligament (563, 564) and later for the inguinoscrotal phase of testicular descent. The mechanism of androgenic action on the gubernaculum is controversial. Androgens could act through the genitofemoral nerve and the neuropeptide calcitonin gene-related peptide (565, 566). Thus, any condition associated with decrease of fetal testicular production or action may impair testicular descent.

 

The first, transabdominal, phase of testicular descent is controlled by Insulin-like factor 3 (INSL3), a member of the insulin/relaxin hormone superfamily secreted by Leydig cells, signaling through its G protein-coupled receptor LGR8, now known as relaxin family peptide receptor 2 (RXFP2) (217, 567). INSL3 acts by inducing male development of the gubernaculum testis. Mutations of INSL3 have been detected in cryptorchid patients (568), similarly deletion of Rxfp2 targeted to mesenchymal gubernacular cells leads to high cryptorchidism in mice (569). Prenatal DES treatment, which is associated with cryptorchidism, impairs Insl3 expression in mouse testis and interferes with gubernacular development (570).

FIGURE 30. Respective roles of testosterone(T) and dihydrotestosterone (DHT) in sex differentiation. Normal androgen physiology in mammals. Testosterone and dihydrotestosterone are assumed to work by binding to the same receptor protein and forming hormone–receptor complexes of different allosteric configurations. Abbreviations: AR, androgen receptor; 17β-HSD3, 17β-hydroxysteroid dehydrogenase type 3; LHR, luteinizing hormone receptor; 5α-R2, steroid 5α-reductase type 2. Reprinted from ref. (531): Wilson JD, Shaw G, Leihy ML, Renfree MB. The marsupial model for male phenotypic development. Trends in Endocrinology and Metabolism, 13:78-83 (2002), Copyright 2002, with permission from Elsevier. http://www.sciencedirect.com/science/article/pii/S1043276001005252.

HORMONAL CONTROL OF FEMALE DIFFERENTIATION

 

Estrogens, Diethylstilbestrol, Xenoestrogens

 

The conclusion that ovarian hormones are not necessary to female development of the female reproductive tract (58, 59) is supported by the female phenotypic development of 45,X or 46,XY subjects with bilateral gonadal aplasia and of aromatase knockout mice unable to synthesize estrogens. Yet, inappropriate estrogen exposure is clearly detrimental. The most tragic illustration of estrogen toxicity is the « DES story ». Diethylstilbestrol (DES), a synthetic estrogen, was widely administered to pregnant women in the early 1940s in the hope of preventing abortion. It was later discovered that female progeny exhibited severe abnormalities of the reproductive tract: vaginal clear-cell adenocarcinoma, vaginal adenosis and squamous metaplasia, transverse vaginal ridges and structural malformations of the cervix and uterus (571, 572).

 

Environmental chemicals that exert deleterious effects upon the endocrine axis are called endocrine disruptors. By binding to nuclear hormone receptors, they may affect sexual differentiation. Unregulated exposure to xenoestrogens such as bisphenol A is now incriminated in the occurrence of cryptorchidism and hypospadias (573-575). Phthalates also adversely affect male differentiation by increasing the expression of COUP-TF2, a transcription factor which represses steroidogenic enzymes (576). Evidence from animal studies show that environmental exposure to endocrine disrupting chemicals is at least partially responsible (reviewed in (577, 578). Phthalates may act as pseudo-estrogens (biphenol A, alias BPA) or as antiandrogens (diethylhexylphthalate, alias DEHP) (579); however caution is required for interpretation of animal studies because of species differences. In human testes, germ cells appear the most susceptible to damage by phthalates (580). Atrazine, a herbicide widely used in the United States, demasculinizes male gonads and reduces sperm count by interfering with phosphodiesterase enzymes and SF1 (581).

 

CONCLUSION

 

A bewildering number of hormones and growth factors is involved in sex determination and differentiation, making it one of the best studied developmental processes. The uncovering of an active genetic pathway towards ovarian development has overturned the dogma of a default pathway towards female gonadal differentiation. For the moment, testicular hormones retain their primacy in modeling the reproductive tract but who knows what surprises the future holds in store?

 

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The Management of Type 1 Diabetes

ABSTRACT

 

Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive pancreatic beta-cell loss resulting in insulin deficiency and hyperglycemia. Exogenous insulin therapy is essential to prevent fatal complications from hyperglycemia. The Diabetes Control and Complications Trial and its long-term follow up, the Epidemiology of Diabetes and its Complications study, demonstrated that stringent glycemic control with intensive insulin therapy can prevent or postpone progression of microvascular disease and reduce risk for macrovascular disease and all-cause mortality. In addition, data obtained from the T1D Exchange, a registry of T1D patients founded in 2010, has become an invaluable resource for scientists worldwide, facilitating collaboration and accelerating understanding of prevailing diabetes practices. Insulin therapy using rapid- and long-acting insulin analogs is the mainstay of management of T1D. Insulin delivery is achieved subcutaneously using multiple daily injections or subcutaneous insulin infusion using insulin pumps. Effective management also involves use of self-monitoring of blood glucose using improved blood glucose meters, continuous glucose monitoring (CGM) devices, and newer insulin pumps with integrated sensor-augmented systems. Addressing psychosocial aspects of T1D plays a crucial role in effective disease management. Strategies to manage T1D are rapidly evolving. In addition to newer insulins, adjunctive non-insulin therapies such as use of incretin agents and SGLT-2 and combination SGLT-1/2 inhibitors are being actively pursued. CGM technology combined with glucose prediction algorithms has allowed for the development of artificial pancreas delivery systems. Cellular replacement options include pancreas and islet cell transplantation which can restore euglycemia but are limited by donor availability and the need for chronic immunosuppression. Newer strategies under development include islet cell encapsulation techniques, which might obviate the need for immunosuppression. Smart-insulin delivery systems, capable of releasing insulin depending on ambient glucose, are also being evaluated.

HISTORY OF TYPE 1 DIABETES TREATMENTS

 

Insulin Therapy

 

The discovery of insulin in 1921-22 was one of the greatest medical breakthroughs in history (1) (Figure 1). Initial work at the University of Toronto allowed for pancreatic extracts to be used to decrease blood glucoses in diabetic dogs. Developments by the pharmaceutical industry allowed for the large-scale commercial insulin production in 1923 (2). Individuals, mostly children with type 1 diabetes (T1D), whose life expectancies were measured in months were now able to prevent fatal ketoacidosis by taking injections of crude “soluble” (later known as regular) insulin. However, problems were soon noted. Hypoglycemia, occasionally life-threatening, was encountered as diabetes monitoring by urine testing for glycosuria was crude at best during those first decades after the discovery of insulin. The insulin itself was often impure and varied in potency from lot to lot. Allergic reactions were common and occasionally anaphylaxis would occur. Even more concerning was the appreciation that these patients often succumbed to chronic vascular complications which either dramatically reduced quality of life or resulted in a fatal cardiovascular event.

 

Tools to manage individuals with T1D improved over the decades since the discovery of insulin. Initial insulins were manufactured from bovine or porcine pancreata and production techniques became more efficient. Insulins with longer duration of action were first introduced in the 1930s, and over time purity and consistency of potency of these insulins improved (3). Nevertheless, “standard” animal insulins prior to 1980 contained 300-10000 parts per million of impurities, and elicited local and systemic effects when injected. Present day insulins sold in the United States today all contain less than 1 part per million of impurities.

 

Major improvements in insulin were developed in the late 1970s and early 1980s. First, not only was “purified” insulin introduced, but in 1982 the first human insulin was marketed both by Eli Lilly (recombinant DNA technology) and Novo (semi-synthetic methodology).  These insulins were available as short-acting (regular) and longer-acting (Neutral protamine Hagedorn (NPH), lente, and ultralente) preparations. The other major advance with insulin therapy was with the delivery by the first continuous subcutaneous insulin infusion (CSII) pumps. While pumps were initially touted as providing less variable insulin absorption, the use of CSII had a greater impact: both patients and clinicians used this tool to teach themselves how to best use “basal bolus” insulin therapy, a strategy that would become a standard of care after the beginning of the next century with the development of insulin analogs.

Figure 1. Time line of the evolution of insulin therapy. Figure source ref 3.

Monitoring Tools

 

At the same time as the development of human insulin and insulin pumps, improvements in glucose monitoring were introduced. Although there was initial skepticism if home blood glucose monitoring would be accepted by patients with diabetes, history has confirmed that this technology has revolutionized diabetes management and has allowed patients to titrate blood glucose to normal or near-normal levels. While self-monitoring of blood glucose (SMBG) allowed immediate evaluation of diabetes management, the introduction of hemoglobin A1c (HbA1c, or glycated hemoglobin, A1C) around the same time was used as a marker of objective longer-term (about 90 days) glucose control. When hemoglobin is exposed to glucose in the bloodstream, the glucose slowly becomes nonenzymatically bound to the hemoglobin in a concentration-dependent manner. The percentage of hemoglobin molecules that are glycated (have glucose bound to it) indicates what the average blood glucose concentration has been over the life of the red blood cell. Perhaps as importantly, A1C made it possible for researchers to study the effects of long-term glucose control and the development of vascular complications. New students of diabetes may now find it difficult to appreciate that one of the greatest medical controversies between the discovery of insulin and the early 1990s was the relationship between glucose control and diabetes complications. Improved insulins, pumps, SMBG, and A1C finally allowed this question to be properly studied.

 

THE DIABETES CONTROL AND COMPLICATIONS TRIAL

 

In 1993, all controversy regarding the impact of glucose control and vascular complications was dramatically answered with the publication of the Diabetes Control and Complications Trial (DCCT) (4). The trial showed definitively that stringent blood glucose control (for an average of 6.5 years) could slow or postpone the progression of retinal, renal, and neurological complications in individuals with T1D (Figure 2). In patients treated with “intensive therapy”—that is, therapy aimed at maintaining blood glucose levels as close to normal as possible—the risk of developing diabetic retinopathy was reduced by 76%, diabetic neuropathy by 60%, and diabetic nephropathy by 54%, compared with conventionally treated patients. Other benefits of intensive diabetes management include improved lipid profiles, reduced risk factors for macrovascular disease, and better maternal and fetal health.

 

Since the DCCT was completed in 1993, the research subjects have been followed in an observational study calledEpidemiology of Diabetes and its Complications (EDIC) (5).  It was soon observed that the impact of this improved diabetes therapy for an average of 6.5 years (maintaining a A1C of approximately 7% with multiple injections or CSII compared to once or twice daily insulin and a A1C of approximately 9%) had long-lasting effects. Termed “metabolic memory”, there continued to be improvements in microvascular complications four years after the DCCT ended (Figure 3) (6-8).  Despite the fact that A1C levels remained about 8% for both groups after the DCCT, the risk reduction for nonfatal myocardial infarction, stroke, or death were reduced by 57% eleven years after the conclusion of the formal study. The conclusions of this are profound since this was the first study to report a reduction of macrovascular disease with glucose control. Furthermore, these data confirmed the need to control blood glucose as meticulously as possible early in the course of the disease (9).

Figure 2. Relationship between microvascular complications and A1C in T1D

Figure 3. Cumulative incidence of further 3-step progression of retinopathy from DCCT closeout to EDIC study year 10 (adjusted for retinopathy level at DCCT end, cohort, entry A1C, baseline diabetes duration). From reference (10).

TYPE 1 DIABETES EXCHANGE 

 

Compared with treatment methods used in the DCCT over 20 years ago, many new tools and technologies have now become available that enable patients and clinicians to attain target A1C levels more safely. Rapid- and long-acting insulin analogs, improved blood glucose meters, newer insulin pumps with integrated sensor-augmented systems and with automatic threshold suspend capabilities and continuous glucose monitoring (CGM) devices now play an integral part of T1D management. To evaluate how these advances in diabetes technology have impacted glycemic control in T1D, a broad-based, large-scale, multisite registry that includes patients at all ages across the life span in the U.S. was established in 2010 through a grant from the Leona M. and Harry B. Helmsley Charitable Trust. Called the T1D Exchange, this registry aims to provide an expansive data set to address important clinical and public health issues related to T1D. It comprises three complementary sections: i) a clinic network of adult and pediatric diabetes clinics; ii) a Web site called Glu, serving as an online community for patients; and iii) a biobank to store biological human samples for use by researchers. A statistical resource center provides statistical support to the Exchange as well as other T1D researchers. The data have provided information about various aspects of T1D, including metabolic control and management, in the United States and the opportunity to compare this data with registries from Europe and Australia (11). The clinic registry has provided valuable information regarding the state of T1D management and outcomes and allowed for addressing important clinical and public health issues. Registry data also have helped identify knowledge gaps leading to further advancements in clinical trials and epidemiologic research with over 47 publications as of March 2019 (12).

 

Currently there are over 35,000 patients enrolled in the registry, ranging in age from 1 - 93 years, with a duration of diabetes ranging from 1.5 to 83 years, 50% female, 82% were non-Hispanic white (13). Most recent data from the registry revealed that mean A1C in adults over age 30 ranged from 7.5-7.8%, which is lower than the value of 8% observed in the DCCT (14). However mean A1C levels increased in teens and emerging adults from 8.5% to 9.3%. Insulin pump use was observed in 63% of individuals. CGM use increased exponentially from 2010-12 to 2016-18 from 7% to 30%, with most participants using the Dexcom system (77%).  CGM use increased significantly in the pediatric population. Many patients in the registry were able to achieve target A1C levels without an increase in the frequency of serious hypoglycemia as was observed in the DCCT. Use of adjunctive non-insulin glucose-lowering therapies was low overall and primarily included metformin, in 6% of adult participants over age 26 years.

 

CURRENT TECHNOLOGY IN TYPE 1 DIABETES

 

Glucose Meters

 

Current blood glucose monitoring systems (BGMS) are small electronic devices capable of analyzing glucose levels in capillary whole blood. To test blood glucose levels, patients are required to prick a finger using a lancing device to obtain a small drop of blood. The patient then places the drop of blood onto a glucose test strip, which has been previously inserted into the glucose meter. Typically, just a few seconds are required for the device to provide a blood glucose value.

 

BGMS use enzymatic reactions to provide estimates of blood glucose levels and the enzymes utilized include glucose oxidase, glucose dehydrogenase and hexokinase. The specific enzyme is usually packaged in a dehydrated form in a glucose test strip. Once blood is applied to the test strip, glucose in the patient’s blood sample rehydrates the enzyme activating a reaction. The product of this reaction can then be detected and measured by the glucose meter (15).

 

Notably, the advent of point-of-care BGMS has revolutionized diabetes care by allowing patients and practitioners to obtain real-time estimates of blood glucose values. These portable devices enabled patients to perform self-monitoring of blood glucose (SMBG), an integral component of effective diabetes self-management. The benefits of SMBG were confirmed during the DCCT which showed that intensive insulin therapy, requiring SMBG≥4 times/day with concomitant insulin dose titration, delayed the onset and slowed the progression of microvascular complications (4). Later, it was shown in the T1D Exchange that a higher frequency of testing (up to 10 times daily) is inversely associated with A1C levels in all age groups (16).

 

SMBG allows patients to guide management decisions (e.g., adjusting food intake, insulin therapy, and exercise) and determine whether glucose targets are being achieved. Further, it can help patients in monitoring and preventing asymptomatic hypoglycemia (17).

 

Patients with T1D should perform SMBG at a minimum of 4 times a day (before meals and at bedtime), as this will allow adjustments to prandial and basal insulin doses. In addition, SMBG should be considered prior to snacks, before and at completion of exercise, in the event of symptoms suggestive of hypoglycemia, and after treating hypoglycemia until blood glucose levels have normalized. Lastly, patients should test their blood glucose before performing critical tasks such as driving a motor vehicle or operating heavy machinery. Ultimately, frequency of SMBG will largely depend on patients’ individual needs (17).

 

An important point to make, however, is that patients should also be educated on avoiding “overuse” of SMBG. Testing too frequently may lead to administration of multiple correction doses within short periods of time, particularly if patients are anxious about their glucose levels not returning to target “fast enough”, leading to insulin “stacking” and resulting in iatrogenic hypoglycemia.

 

The technology of BGMS has evolved over the years and current devices are relatively easy to use and require minimal amounts of blood (Figure 4). Some instruments are able to capture events affecting glucose control (e.g., exercise, meals, insulin administration), provide customized reports, and calculate insulin bolus needs according to glycemia and intake of carbohydrate based on pre-established settings (i.e., insulin sensitivity factor and insulin-to-carbohydrate ratios). However, despite these unique advances in self-monitoring of blood glucose, independent analytic testing has shown that various BGMS do not fulfill the accuracy requirements set by the International Organization for Standardization (ISO) 151917 which requires for ≥95% of results to fall within ± 15 mg/dL of the reference result for samples with glucose concentrations <100 mg/dL and ±15% for samples with glucose concentrations ≥100 mg/dL (18). In addition, the FDA has stated that the ISO 15197 criteria are not sufficient to adequately protect lay-users of SMBGs because, for example, the standard does not adequately address the performance of over-the-counter blood glucose testing systems in the hypoglycemic range or across test strip lots. In view of this, the FDA has developed the “Self-Monitoring Blood Glucose Test Systems for Over-the-Counter use” guidance document which is intended to guide manufacturers in conducting appropriate performance studies and preparing 510(k) submissions for these device types (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/self-monitoring-blood-glucose-test-systems-over-counter-use). Thus, there is a pressing need for high quality standards to ensure improved accuracy and precision from BGMS.

 

SMBG has important drawbacks since blood is only sampled intermittently and therefore only glimpses of blood glucose concentrations are provided. SMBG does not offer information on glucose fluctuations even if performed frequently. Thus, there is potential for missing episodes of hyperglycemia and hypoglycemia.

Figure 4. Examples of a few blood glucose monitoring systems.

Glucose Downloads

 

The vast majority of currently available BGMS allow the generation of downloadable reports. These reports are a unique component of the patients’ evaluation allowing the identification of areas that require special attention in diabetes management. However, technical difficulties often compromise the usefulness of these data. For instance, it is not unusual for the date and/or time of the glucose meters to be inaccurate. Simple errors such as these have a huge impact on patient management as the data downloaded becomes largely uninterpretable. In addition, as each glucose meter usually has its own proprietary software, if a clinic does not have the specific software installed on their local computers, then the data may not be downloaded. The clinician is left with trying to review the data directly from the device, which is time consuming and does not offer the detailed overview from a customized printable report. There are platforms that are currently available which allow downloading various glucose meters, insulin pumps and CGM data and provide standardized reports (e.g., Clinipro®, Diasend®, Carelink®, Glooko®). However, there needs to be a unified effort by BGMS, insulin pump, and CGM companies in order to generate a universal download protocol as this would simplify data analysis and interpretation by practitioners (19).

 

Continuous Glucose Monitoring

 

Perhaps the most innovative technology for the treatment of T1D is the introduction of CGM (Figure 5). CGM technology allows for the measurement of glucose concentrations in the interstitial fluid (ISF) which correlates with plasma glucose values. However, when interpreting CGM values it is important to understand that ISF glucose consistently lags plasma glucose. A study in healthy adults analyzing glucose tracers following an overnight fast showed that it takes 5-6 minutes for glucose to be transported from the vascular to the interstitial space (physiological delay) (20). This is particularly relevant when glucose levels are trending up or down quickly as CGM data will not be as reliable in such scenarios and thus patients should confirm the direction of their glucose concentration by SMBG.

 

The components of CGM consist of a sensor that is inserted subcutaneously, a small electronic device that serves as the platform for the sensor, a transmitter, and a receiver device, which can be a standalone device or a smartphone (Figure 5). CGM Sensors can measure glucose levels up to every minute allowing for a glucose tracing to be generated and displayed in real-time (RT-CGM) on a receiver device, greatly improving the understanding of patients’ glucose profiles. Further, with the exception of the GuardianTM Connect system (Medtronic) which is pending approval, currently available CGM devices have obtained FDA approval for non-adjunctive use which means that patients can rely on their CGM values in order to guide management decisions (21).

 

Patients can customize alarms to activate for hypoglycemia or hyperglycemia. Understanding the trend allows patients to decide whether an increase or decrease in mealtime insulin dose is necessary. CGM thus also allows patients to intercept hypoglycemia (or hyperglycemia) prior to it occurring. Patients can also “flag” events thereby improving interpretation of glucose control associated with meals, insulin administration, and exercise. Also, most CGM devices allow users to share their RT-CGM data with others (e.g., family members or friends) which can then be monitored on a smartphone or other internet-enabled devices. This is of particular interest in the pediatric population as it allows parents to remotely monitor their child’s glucose profile when away from home or while exercising (e.g., participating in sports). Features of currently available CGM devices are listed in Figure 6.

 

Based on how the CGM data is delivered to the user, current CGM devices fall under 2 categories: Flash glucose monitoring (or intermittently scanned glucose monitoring) and Real-time glucose monitoring.

 

FLASH GLUCOSE MONITORING

 

Flash glucose monitoring requires the user to hold a reader device (which can be a smart phone) close to the subcutaneously inserted sensor (the patient “scans” the sensor with the reader) to have the real-time interstitial glucose value displayed. During a scan, the reader displays the real time glucose value, glucose alerts, a historic glucose trend of values recorded and a trend arrow indicating the glucose direction (22). There are currently 2 approved Flash CGM devices for patient use, the FreeStyle Libre 14 day and the FreeStyle Libre 2 (Figure 5). The Libre 14 days allows for real-time data sharing but is limited by the lack of alarms in case glucose values are dangerously high or low. Nonetheless, this device may be appealing to those patients who want to minimize capillary blood glucose measurements and complain of CGM sensor alarm fatigue (23, 24). On the other hand, the Libre 2 has optional real-time glucose alarms but currently it requires a dedicated stand-alone receiver (data cannot be sent to a smartphone) and it does not have the capability of real-time data sharing.

 

REAL-TIME GLUCOSE MONITORING

 

Real-time glucose monitoring allows for data to be continuously sent to a receiver device and apart from viewing the display to check glucose levels and the direction of glucose profile, no additional action is required by the patient. Further, real-time CGM systems provide real-time alerts which can be customized to prevent or treat hyper or hypoglycemia. In addition, all currently approved real-time CGMs allow for data sharing.

 

Another advantage of CGM is the amount of data that can be generated and downloaded in customizable reports (Figure 7). Health care professionals are not only able to download daily glucose profiles in a graphic display but can also obtain several statistics including means, medians, standard deviations, interquartile ranges, and minimum and maximum values. This provides a better assessment of glycemic variability (Figure 8). Most importantly, time in glucose ranges can be identified and evaluated. This is particularly helpful in patients who have hypoglycemia unawareness and allows for adjusting the treatment plan by both the patient and practitioners to eliminate occurrence of hypoglycemia.

 

KEY CGM METRICS

 

Key CGM metrics include: Time in target range (TIR) defined as the percentage of readings and time per day within the recommended target glucose range of 70-180 mg/dL; time below target glucose range (TBR); and time above target glucose range (TAR) (see Figures 7 and 9 for examples). Current recommendations are to achieve TIR >70% (>16 h, 48 min), TBR <4% (<1 h) and TAR <25% (<6 h). However, recommendations are different for older adults/high-risk populations and during pregnancy (25). In addition to time in glucose ranges, CGM data has also allowed to generate a formula to estimate the laboratory A1C based on CGM mean glucose levels. This estimated A1C has been named “Glucose Management Indicator” and offers the advantage of being unaltered by limitations inherent to the laboratory A1C measurement (e.g., anemia, iron deficiency, glycation abnormalities, drug interference).  The enormous amount of data generated by CGMs can be overwhelming and difficult to follow and interpret and the need for a standardized report is critical for data interpretation and medical decision making. The Ambulatory Glucose Profile is a standardized report which incorporates all the core CGM metrics and recommended targets along with a 14-day composite glucose profile and is the recommended report by the International Consensus on Time in Range (Figure 9) (25, 26).

Figure 5. Examples of real-time continuous glucose monitoring systems.

Figure 6. Features of currently approved CGM devices in the United States.

Figure 7. A 14-day DEXCOM CGM overview report showing sensor glucose data over a 24-hour period including mean (dotted line), standard deviation, glucose management indicator, interquartile range (grey bars), upper and lower glucose thresholds (orange and red lines, set by the user), percent time in range, sensor usage, top patterns, and average daily calibrations.

Figure 8. A 7-day DEXCOM CGM overlay report showing daily profiles allowing for the identification of trends and patterns.

Figure 9. Ambulatory Glucose Profile (AGP) sample.

INSURANCE COVERAGE AND BILLING OF CGM DEVICES

 

Insurance coverage in the United States for devices is highly variable and challenging to navigate, and maybe unaffordable for some patients due to high copays or coverage issues. (These coverage requirements vary depending upon geographic area; practitioners are urged to follow guidelines in their country of practice). Understanding requirements for prescribing any CGM device is necessary and appropriate documentation is necessary. For individuals on Medicare to receive approval for a CGM device, documentation must include the following (as of 2021):

 

  1. The patient has diabetes mellitus and requires a therapeutic CGM.
  2. The patient is performing SMBG at least 4 times daily (Medicare only provides 3 test strips daily).
  3. The patient is treated with insulin and is injecting insulin at least 3 times daily or is on an insulin pump.
  4. The patient’s insulin treatment regimen requires frequent dose adjustment based on SMBG/CGM results.
  5. The patient had an in-person visit within 6 months prior to ordering the CGM with the treating practitioner to evaluate their diabetes and determine that criteria 1 to 4 are met. Subsequently, the patient must have an in-person visit every 6 months following the initial prescription to assess adherence to CGM and diabetes treatment plan.

 

There are billing codes for analyzing data from CGM devices. The patient visit should include certain key elements that need to be clearly documented in the chart as follows:

 

  1. A brief statement or narrative that the glucose sensor data were evaluated
  2. What patterns were noted
  3. Action steps and plan based on data interpretation provided to the patient
  4. Electronic or print of data report should be attached to the patient chart

 

CGM Integrated Insulin Pumps

 

As seen in Figure 10, some sensors are already integrated with insulin pumps (“sensor-augmented pumps”) so that the pump and receiver are in the same device. In addition, development of an integrated sensor and infusion set is currently being pursued, as this will simplify the incorporation of sensor technology into insulin pumps. Eventually, it is expected that all insulin pumps will be integrated with sensors. Yet, it should be appreciated that CGM is an equally important tool for MDI patients, and probably a more important diabetes management tool than using an insulin pump (21). Even after short periods of time, many patients can learn how to best use this technology to improve both mean glucose and glycemic variability. In a meta-analysis, comparing SMBG with RT-CGM, the latter achieved a lower A1C (between-group difference of change, -0.26%, (95% CI, -0.33% to -0.19%)) without increasing hypoglycemia (27). In the Juvenile Diabetes Research Foundation’s CGM trial, those individuals starting with baseline A1C levels under 7% overall had less hypoglycemia with CGM (28). A recent analysis of the T1D registry data suggests that CGM users, irrespective of insulin delivery method – i.e. multiple daily injections vs. pump therapy – had lower A1C levels than non-CGM users even after adjustment for confounding factors (29).

 

The American Association of Clinical Endocrinologists and American College of Endocrinology recommend the use of CGM for patients with T1D particularly for those with a history of severe hypoglycemia, hypoglycemia unawareness, and to assist in correction of hyperglycemia in patients not at goal. It may also be considered in pregnancy as it can help fine-tune insulin dosing, monitor for overnight hypoglycemia or hyperglycemia, and assess occurrence of postprandial hyperglycemia (30). The Endocrine Society guidelines on CSII Therapy and Continuous Glucose Monitoring in Adults recommend the use of RT-CGM for adult patients with T1D who either have A1C levels above target or well-controlled T1D and are willing and able to use these devices on a nearly daily basis (31).

Figure 10. Examples of modern-day insulin pumps.

OVERVIEW OF THERAPY FOR TYPE 1 DIABETES

 

Glycemic Targets

 

A1C is a measure of average glycemia over ~3 months and is a strong predictor of complications of diabetes (32). Current glycemic targets for adults from the American Diabetes Association (ADA) include a target A1C of <7%. However, it should be noted that this recommendation is a general target and the goal for the individual patient is as close to normal as possible (A1C of < 6%) without significant hypoglycemia. In addition, patients with T1D and hypoglycemia unawareness, long duration (> 25-30 years) of disease, limited life expectancies, very young children, or those with co-morbid conditions will require higher A1C targets. Individualized A1C targets need to be reviewed with each patient (17).

 

Thus, A1C testing should be performed routinely in all patients with diabetes as part of ongoing care. Frequency of A1C testing is determined based on the clinical situation, the treatment regimen used, and the clinician’s judgment. A1C measurements every 3 months help in the assessment of whether a patient’s glycemic targets have been reached. Although convenient, there are drawbacks to A1C measurements, as glycation rates may vary with patients’ race/ethnicity. Similarly, in patients with hemoglobinopathies, hemolytic anemia or other conditions that shorten the red blood cell life span, the A1C may not accurately reflect glycemic control or correlate with SMBG testing results. In such conditions, fructosamine may be considered as a substitute measure of long-term (average over 4 weeks) glycemic control. Clinicians should routinely compare downloaded SMBG or CGM averages with A1C as there are many reasons A1C may be altered due to a non-glycemic etiology and thus fructosamine or the downloaded glucose data itself would be a better metric to follow (33).

 

Non-Glycemic Treatment Targets

 

It should also be pointed out that in addition to glycemic targets, specific non-glycemic targets have also been recommended (34). Non-glycemic targets should also be tailored according to the individual with less stringent treatment goals for individuals with multiple coexisting illnesses and/or poor health and limited life expectancy. Recent real-world data from the T1D Exchange revealed that the incidence of cardiovascular disease (CVD) over 4.6 years was ~3.7% (35). Age, longer duration of diabetes, glycemic control, obesity, hypertension, dyslipidemia, and diabetic nephropathy were all associated with increased risk for CVD.

 

BLOOD PRESSURE

 

Good quality data to guide blood pressure management in T1D is lacking and most data are extrapolated from type 2 diabetes (T2D) clinical trials. The ADA recommends treatment to a goal of <140/90 mmHg for individuals with diabetes and hypertension at lower risk for CVD. Lower targets of <130/80 mmHg, should be considered for individuals who have higher cardiovascular risk or pre-existing ASCVD. Antihypertensive therapy should be initiated using a drug class that has demonstrated cardiovascular benefit such as angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), thiazide-like diuretics, or dihydropyridine calcium channel blockers. ACE inhibitors or ARBs are the preferred first line treatment for individuals with albuminuria.

 

LIPIDS

 

Very limited data exists for lipid management in patients with T1D of any age. Limited evidence suggests that primary prevention with lipid-lowering medications decreases the incidence of CVD (36). The ADA has adopted the approach of the 2018 American College of Cardiology/ American Heart Association multi-society cholesterol guidelines and recommends similar statin approaches for individuals with T1D (34). All patients with T1D and CVD should be treated with high intensity statins. Addition of non-statin therapies such as ezetimibe and PCSK9 inhibitors should be considered based on overall risk and achieved LDL-C thresholds. Patients with T1D over the age of 40 should be offered statin therapy. In individuals younger than age 40 with T1D and additional risk factors (such as albuminuria, HTN, strong family history, long duration of diabetes >20 years), moderate intensity statin therapy should be considered after clinical discussion. Recently, a prediction model for CVD events in T1D to help decision making for primary prevention that has been developed and shows promise but needs further validation (37). There is new evidence of the contribution of cardiac autoimmunity to CVD in T1D in the DCCT/EDIC cohort that warrants further investigation (38). 

 

INSULIN THERAPY

 

Insulin therapy is the cornerstone of management of T1D as beta cell dysfunction or destruction progressively leads to absolute insulin deficiency. Physiologic insulin replacement that aims to mimic normal pancreatic insulin secretion is the preferred method of treatment of T1D patients. Basal insulin is the background insulin required to suppress hepatic glucose production overnight and between meals. Prandial (bolus or meal-time) insulin replacement, provides enough insulin to dispose of glucose after eating. Such a therapeutic insulin regimen providing both basal and bolus insulin allows flexibility of dosing. Older twice-daily combination of regular and NPH regimens generally should not be used in T1D as they are less effective since the time-action profile of these two standard insulins do not readily allow for the clear separation of basal and prandial insulin action. However, it may be necessary to use such regimens in patients who cannot otherwise afford insulin. It also should be pointed out that for newly diagnosed patients with T1D, transient use of once- or twice-daily basal injections is sometimes adequate.

 

Principles of Management of T1DM

 

Management of T1D involves a multidisciplinary framework that includes the following:

 

  1. Physiologic insulin replacement using basal-bolus therapy, either as MDI or CSII
  2. Blood glucose monitoring with SMBG and/or CGM with development of individualized A1c goals
  • Patient education
  1. A supportive team of providers including endocrinologists, nurses, certified diabetes care and education specialists (CDCES)s, pharmacists, psychologists, dietitians, social workers, other specialists such as cardiologists, nephrologists, psychiatrists as well as family members, social support groups etc.

 

Types of Insulin

 

Selecting the appropriate insulin depends largely on the desired time course of insulin action. Table 1 shows the pharmacokinetic characteristics—time to onset of action, time of peak action, effective duration of action, and maximum duration of action—of currently available insulins; however, these can vary considerably among individuals.

 

Insulin products are categorized according to their action profiles:

 

  • Rapid-acting: e.g., insulin lispro, insulin aspart, and insulin glulisine (genetically engineered insulin analogs)
  • Short-acting: regular (soluble) insulin
  • Intermediate-acting: NPH (isophane)
  • Long-acting, e.g., insulin glargine, insulin detemir, and insulin degludec (genetically engineered insulin analogs)
  • Pre-mixed insulin
  • Inhaled insulin

 

Insulin analogs are insulin molecules modified by genetic engineering and recombinant DNA technology. The amino acid structure of insulin is altered to change the properties of insulin – i.e., time to onset, peak, and duration of action, compared to human regular insulin. However, the biological properties and stability of the insulin molecule are intact. A general principle to bear in mind is the longer the time to peak, the broader the peak and the longer the duration of action. Additionally, the breadth of the peak and the duration of action will be extended with increasing dose. Figure 11 should therefore be considered a conceptual representation of insulin action curves.

 

Mealtime (Prandial) Insulins

RAPID-ACTING INSULIN

 

These are insulin analogs with a rapid onset in 15-30 minutes, peak in 30-90 minutes, and an effective duration of 4 to 5 hours when injected subcutaneously.  They have a shorter time action profile compared to human (regular) insulin because they do not self-aggregate in solution. All rapid-acting insulin analogs have a 1 - 2 amino acid difference from the primary structure of human insulin. Insulin lispro differs from human insulin by an amino acid exchange of lysine and proline at positions B28 and B29 (39). The substitution of aspartic acid for proline at position B28 characterizes insulin aspart (40). Insulin glulisine differs from human insulin in that the B3 asparagine is replaced by lysine, and B29 lysine is replaced by glutamic acid (41). These modifications in the primary structure of human insulin increase the rapidity of breakdown of insulin hexamers in the analogs and thus result in more rapid absorption. When administered before meals, rapid-acting insulins used as part of multiple daily injections (Figure 11) or with CSII, resemble physiologic insulin increases stimulated by food. Doses can be adjusted proportionate to food consumed; in patients with gastroparesis or poor appetite, insulin can be injected halfway through or after the meal. A follow-on biologic to insulin lispro (biosimilar lispro) is now available as Admelog. 

 

 Ultra-rapid acting insulin aspart (Fiasp) available since 2018 is insulin aspart with added niacinamide. This results in quicker absorption with faster onset of action after injection and therefore can be injected right before the start of a meal (or within 20 minutes after the start of a meal). This allows for some flexibility of dosing. Safety and efficacy data in adults and children is similar to insulin aspart (42). Fiasp has recently also been approved for use in insulin pumps. Data in pregnant women is lacking. Recently, ultra-rapid acting lispro (lispro-aabc) has become available in several countries including the United States. This insulin has been shown to appear in the bloodstream within 1 minute of injection (43). Ultra-rapid acting lispro was found to be non-inferior to rapid-acting lispro and superior for postprandial blood glucose control in T1D and T2D (44, 45).

 

INHALED INSULIN

 

Currently, one form of inhaled insulin is available in the market. Afrezza was approved by the FDA in 2014. This is a drug-device combination that contains powdered human insulin in single use dose cartridges delivered via a small inhaler. When inhaled, it dissolves immediately on contact with the alveolar surface of the lung and is rapidly absorbed into the systemic circulation, reaching a peak within 15 minutes. Thus, Afrezza acts similar to rapid-acting insulin analogs but with a much faster peak of action, and shorter duration of action. Prior to initiation of its use, patients should be screened for underlying lung disease with spirometry. Follow-up spirometry is recommended after 6 months’ use, and annually thereafter. The main advantages of inhaled insulin are avoidance of injections, faster onset of action, less weight gain, and less hypoglycemia (46). Dosing is not flexible as cartridges are available in fixed doses (4, 8 and 12 units). Afrezza is contraindicated in patients with chronic lung disease such as asthma or chronic obstructive pulmonary disease (COPD).

 

SHORT-ACTING INSULIN

 

Regular insulin is structurally similar to endogenous human insulin. It consists of dissolved zinc-insulin crystals which self-aggregate in the subcutaneous tissue and results in a delayed onset of action of 30 to 60 minutes, a peak of 2 to 3 hours, and an effective duration of 6 to 8 hours. Proper use requires injection at least 20 to 30 minutes prior to meals to match insulin availability and carbohydrate absorption. Use of regular insulin is associated with greater hypoglycemia risk (47). Regular insulin acts almost instantly when injected intravenously.

 

Basal Insulins

 

INTERMEDIATE-ACTING INSULIN

 

Neutral protamine Hagedorn (NPH) insulin, developed in the 1950s, is a combination of recombinant human insulin with protamine which results in crystal formation. When injected subcutaneously, precipitated crystals of NPH insulin are released slowly resulting in a longer duration of action compared to regular insulin. Action of NPH varies quite widely within the same patient as well as between patients.  Its onset of action occurs 2 to 4 hours from the time of injection, with a peak effect lasting 6 to 10 hours, and an effective duration of 10 to 16 hours.  Due to this peak effect, NPH insulin acts as a basal and a prandial insulin, necessitating that patients eat a meal at the time the insulin is peaking. NPH typically requires twice a day dosing (48).

 

LONG-ACTING INSULIN ANALOGS

 

Long acting insulin analogs were created by modifying the amino acid sequence on the beta chain of insulin (49). They exhibit much improved pharmacokinetics and pharmocodynamics without a peak effect and maintain a longer duration of action. Improved absorption rates result in significantly decreased inter-individual and intra-individual variability with improvement in glycemic control and reduced hypoglycemia risk. 

 

Insulin glargine is a modified human insulin produced by the substitution of glycine for asparagine at position A21 of the insulin molecule and by the addition of two arginine molecules at position B30 (48). These changes result in an insulin molecule that is less soluble at the injection site forming a precipitate in the subcutaneous tissue to form a depot from which insulin is slowly released after injection and is slowly released into the circulation. It has no pronounced peak and a longer duration of action of about 20 to 24 hours in most patients, allowing for once daily dosing. In clinical practice, many patients with T1DM may benefit from twice-daily injections.  Insulin glargine is solubilized in acidic pH and should not be mixed with rapid-acting insulins as the kinetics of both insulins will be altered. Insulin glargine shows a greater reduction in A1C and decreased hypoglycemia in patients with T1DM compared to NPH insulin (50).

 

Insulin detemir is a soluble basal insulin analog. It is covalently acylated with fatty acids on the lysine at position B29, which allows for reversible binding to albumin (51). This delays its absorption from subcutaneous tissue and prolongs its time in the circulation. Although the mean duration of action of insulin detemir has been shown to be 24h, one study showed shorter duration of action (about 17h), which suggests that most patients with T1D may require twice-daily dosing of insulin detemir (52).

 

ULTRALONG-ACTING INSULIN ANALOGS

 

Insulin degludec is an ultra-long acting basal insulin available in the US since 2015 that has the same amino acid sequence as human insulin, apart from the deletion of the threonine amino acid residue at B30 and the addition of a fatty acid to the lysine at B29 (53).  The fatty acid moiety causes self-aggregation of insulin molecules into soluble multihexamers. Slow dissociation of zinc from the insulin allows for gradual and stable absorption of insulin monomers resulting in a long half-life and a prolonged duration of action of 42 hours at steady state. In patients with T1D, similar A1C reduction with lower rates of nocturnal hypoglycemia have been reported with insulin degludec compared with insulin glargine (54, 55). The extended duration of insulin degludec allows for more flexibility of day-to-day dose timing without compromising glycemic control or safety (56).

 

U-300 glargine (Gla-300) is a formulation of insulin glargine that delivers the same number of insulin units as insulin glargine 100 units/mL (Gla-100), but in a third of the volume. The compact depot renders a smaller surface area of insulin glargine for a given dose, leading to a slower release of insulin glargine over time. This translates into a more constant PK/PD profile, with a prolonged duration of action (up to 30 hours) with Gla-300 compared with Gla-100 in patients with T1DM (57). Gla-300 has been shown to provide similar glucose control compared to Gla-100 with less weight gain and hypoglycemia (58).

 

Pre-Mixed Insulins

 

Premixed insulins are mixtures of prandial and intermediate acting insulins (the same prandial insulin attached to protamine so that it becomes intermediate acting). Insulin mixtures are available as human insulin mixtures (NPH and regular mixture) as well as analog mixtures. In the US, insulin lispro protamine mixtures are available in two forms: 75% insulin lispro protamine suspension and 25% insulin lispro injection (75/25) and 50% insulin lispro protamine suspension and 50% insulin lispro injection (50/50). Available preparations of insulin aspart protamine mixtures include 50/50 and 70/30 suspensions. A variety of other ratios are available in Europe. There is only one mixture of analog-analog without protamine (aspart 30% +degludec 70%, Ryzodeg). These insulin mixtures are typically administered before breakfast and dinner. This alleged twice daily dosing is the primary advantage of these insulins. In general, use of premixed insulins restricts adjustment of doses and meal timing. Therefore, premixed insulins are not recommended for adult patients with T1D, where intensive regimens with ability to make adjustments in the premeal short-acting insulin bolus are better suited for glycemic control. Premixed insulin in T1D could have benefit for some patients who do not adhere to an intensive insulin regimen, and with consistent food intake and timing of meals.

 

Concentrated Insulins

 

U-500 INSULIN

 

U-500 insulin is highly concentrated regular insulin, administered 2-3 times a day without basal insulin. Due to its concentration, the action is prolonged and variable. In T1D, use is primarily limited to individuals with significant insulin resistance (requiring >200 units of insulin a day). Caution should be used while prescribing this insulin as confusion may occur among clinicians, pharmacists, nurses, and patients who are unfamiliar with its use. U-500 insulin is also available in a pen delivery system allowing patients to administer insulin by 5 units increments up to a maximum of 300 units at a time. Units to be delivered are clearly readable through the pen “dose window” which should minimize or eliminate confusion when administering this highly concentrated insulin formulation.

 

CONCENTRATED INSULIN ANALOGS

 

U-200 formulations of insulin lispro and insulin degludec are also available and allow for delivery of lower volumes and therefore better absorption. U-300 glargine is available in pen form and holds up to 900 units of insulin with dosing capability up to 160 units per dose.

 

CONVERSION FROM U-100 TO CONCENTRATED INSULIN

 

Switching from U-100 insulin to concentrated insulin may occasionally be necessary in the setting of severe insulin resistance and use of large amount of U-100 insulin. U-200 lispro is bioequivalent to U-100 lispro, and U-200 degludec is bioequivalent to U-100 degludec. This means that the dose can be converted 1:1 on a unit basis when switching from U-100 to U-200 formulation. The insulin is delivered at 50% less volume. U-300 glargine, on the other hand is not bioequivalent to U-100 glargine. Individuals with T1D often require 15-20% higher dose of U-300 glargine. Similarly, a dose reduction of 20% is essential when switching from U-300 glargine back to U-100 glargine to avoid hypoglycemia. When initiating U-500R, dosing should be determined based on current and targeted glycemic goals to optimize efficacy and safety. U-500R provides mealtime coverage and its extended duration of action provides basal coverage also.

 

Biosimilar Insulins/Follow-on Biologics

 

According to the FDA, a “biosimilar” is a biological product that is highly similar to a US-licensed reference biological product not withstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. As of 2020, there are 4 follow-on biologics approved. These include Basaglar (US, Europe - insulin glargine), Basalin (China- insulin glargine), Semglee (EU, Australia, insulin glargine) and

 

Table 1. Currently Available Insulin Preparations

Insulin Preparation

Onset of action (h)

Peak          Action (h)

Effective duration of action (h)

Maximum duration(h)

Rapid-acting analogs

 

 

 

 

     Insulin lispro (Humalog, Admelog) 

¼ - ½  

½-1 ½

3-4      

4-6

     Insulin aspart (NovoLog)

¼ - ½

½ -1 ¼

3-4                   

4-6

     Insulin glulisine (Apidra)

¼ - ½

½ -1 ¼

3-4         

4-6

     Insulin aspart (Fiasp)

¼ -1/3

 1.5-2.5

3-4

5-7

     Insulin lispro-aabc (Lyumjev)

1/8

2

 

4-6

Inhaled insulin (Afrezza)

seconds

12-17 min

2-3

2-3

Short-acting

 

 

 

 

     Regular (soluble)

½ - 1

2-3

3-6

6-8

Intermediate-acting

 

 

 

 

     NPH (isophane)

2-4

6-10

10-16

14-16

Long-acting analog

 

 

 

 

     Insulin glargine (Lantus, Basaglar)

0.5-1.5

8-16

18-20

20-24

     Insulin glargine U-300 (Toujeo)

0.5-1.5

none

24

30

     Insulin detemir (Levemir)

0.5-1.5

6-8

14

~20

     Insulin degludec (Tresiba)

0.5-1.5

none

24

40

 

Figure 11. Available basal insulins and duration of action. Figure source Ref (59).

Factors Influencing Insulin Absorption

 

Insulin absorption variability is one of the greatest obstacles to replicating physiologic insulin secretion. Among the many factors that affect insulin absorption and availability (Table 2) are injection site, the timing, type or dose of insulin used, and physical activity. Day-to-day intra-individual variation in insulin absorption is approximately 25%, and the variation between patients may be as high as 50%. This occurs more commonly with larger doses of human insulin which form a depot and can unpredictably prolong duration of action; however, this is less of an issue with rapid-acting insulin analogs. In general, any strategy that increases the consistency of delivery should decrease glucose fluctuations; and insulin regimens that emphasize rapid-acting insulin are more reproducible in their effects on blood glucose levels. Insulin pumps using a rapid-acting insulin analog can significantly reduce glucose variability. Like multiple-injection regimens, use of an insulin pump requires frequent blood glucose monitoring. In addition, pump users need a back-up method of insulin administration, and attention to mechanical and injection site issues.

 

Reducing Variability of Insulin Absorption

 

INJECTION SITES

 

Subcutaneous insulin is absorbed most rapidly when injected into the abdomen, followed by the arms, buttocks and thighs. These differences are likely due to variations in regional blood flow. A single region should be utilized for injections without rotation between regions, as this may result in day-to-day variation of insulin absorption. However, while using a region, site rotation (i.e. – rotating injections systematically within the abdomen) is important to avoid development of lipohypertrophy or atrophy due to repeated injections at the same site. Injection into lipohypertrophic areas results in erratic, slower absorption of insulin. Exercise increases the rate of absorption from injection sites, likely by increasing blood flow to the skin; local effects may also be involved.

 

TIMING OF PRE-MEAL INJECTIONS

 

Gauging the appropriate interval between preprandial injections and eating, known as the “lag time,” is essential for coordinating insulin availability with glycemic excursions following meals. The timing of the injections should also be adapted to the level of premeal glycemia. Insulin lispro, insulin aspart, and insulin glulisine have rapid onset of action and, ideally, should be given approximately 10-20 minutes before mealtime when blood glucose is in the target range, keeping in mind that if the meal is delayed, hypoglycemia may ensue. When blood glucose levels are above a patient’s target range, the lag time should be increased to permit the insulin to begin to have an effect sooner. In this case, rapid-acting acting insulin analogs can be given 20-30 minutes before the meal, depending upon the degree of hyperglycemia. If premeal blood glucose levels are below target range, administration of rapid-acting insulin should be postponed until after some carbohydrates have been consumed. Use of frequent home glucose monitoring or CGM can assist in determining appropriate lag times. It is important to emphasize the effect of administering prandial insulin up to 20 minutes before a meal. Pre-bolusing has been shown to reduce post-prandial glucose spike by up to 50 mg/dL.

 

OTHER FACTORS

 

Exercise, as discussed earlier, results in increased blood flow to muscle groups and can increase rate of insulin absorption. Heat can also increase the rate at which insulin is absorbed from the skin. For example, being out in the sun or injection before going into a hot tub may lead to hypoglycemia. Intra-muscular injections result in a more rapid onset of action compared to subcutaneous tissue. This route can be utilized under certain situations such as ketoacidosis, insulin pump failure or in the event of profound hyperglycemia.

 

Table 2. Factors Affecting the Bioavailability and Absorption Rate of Subcutaneously Injected Insulin

Factor

Effects

Site of injection

Abdominal injection (particularly if above the umbilicus) results in the quickest absorption; arm injection results in quicker absorption than thigh or hip injection.

Depth of injection

Intramuscular injections are absorbed more rapidly than subcutaneous

injections.

Insulin concentration

U-40 insulin (40 units per mL) is absorbed more rapidly than U-100 insulin (100 units per mL).

U-40 insulin is an old insulin formulation not available in the United States for patient use. Currently, it is used for treating canine and feline diabetes mellitus

Insulin dose

Higher doses have prolonged duration of action compared with lower doses.

Insulin mixing

Regular insulin maintains its potency and time-action profile when it is

mixed with NPH insulin

Exercise

Exercising a muscle group before injecting insulin into that area

Increases the rate of insulin absorption.

Heat application or Massage

Local application of heat or massage after an insulin injection increases

the rate of insulin absorption.

 

Role of Insulin Analogs in Management of T1D

 

Most of the problems of insulin replacement in T1D arise from the fact that subcutaneous injection or pump infusion remains a relatively poor route of administration. From the subcutaneous site of injection, insulin is absorbed into the systemic, not portal circulation. More importantly, subcutaneous injection leads to variable absorption from one injection to another, due largely to the non-physiologic pharmacokinetics of standard insulins. Insulin analogs were developed to overcome this problem.

 

Currently there are three rapid-acting insulin analogs: insulin lispro, insulin aspart, and insulin glulisine, all of which have a rapid onset of action and peak, thereby improving 1- to 2-hour postprandial blood glucose control compared with regular insulin. These rapid-acting analogs must be used in conjunction with a basal insulin to improve overall glycemic control (Figures 11 and 12). Importantly, the rapid-acting analogs have consistently outperformed regular insulin in terms of post-absorptive hypoglycemia. This finding should not be surprising since the duration of regular insulin is much longer than the gut absorption of a typical mixed meal.

Figure 12. Idealized insulin curves for prandial insulin with a rapid-acting analog (RAA) with basal insulin glargine or insulin detemir. Each insulin preparation is responsible for either the prandial or basal component. Many patients find the basal insulins do not last the entire 24 hours and they give the basal insulin twice daily. B=breakfast; L=lunch; S=supper; HS=bedtime

Clinical trials have demonstrated lower fasting glucose levels and less nocturnal hypoglycemia with insulin glargine than with NPH insulin, advantages that are especially relevant in patients aiming for meticulous control (A1C <7%) or those with hypoglycemia unawareness. Trials with T1D have shown similar results with insulin detemir which compared with NPH insulin was equally effective in maintaining glycemic control, although detemir was administered at a higher molar dose. The newest basal insulin preparations, insulin degludec and U-300 insulin glargine are claimed to show less nocturnal hypoglycemia than insulin glargine or insulin detemir.  In general, hypoglycemia is reduced with any of these basal analog insulins compared to NPH insulin. Since hypoglycemia is clearly one of the treatment-limiting aspects of T1D therapy, the use of these analogs has gained wide-spread acceptance.

 

Multiple Daily Injection (MDI) Insulin Therapy

 

A simpler conceptual approach preferred by most patients with T1D is using a prandial insulin analog for each meal (i.e., insulin lispro, insulin aspart, or insulin glulisine) and a separate basal insulin analog (i.e., insulin glargine, insulin detemir, or insulin degludec). Although these true basal-prandial regimens require more shots than conventional twice-daily regimens, they are considerably more flexible, allowing greater freedom to skip meals or change mealtimes. Moreover, use of the long-acting basal and rapid-acting insulin analogs, allows strategies to achieve individual, defined blood glucose targets more easily. Such modifications might include changing the timing of insulin injections in relation to meals, changing the portions or content of food to be consumed, or adjusting insulin doses or supplements for premeal hyperglycemia.

 

The basic treatment principles of insulin dosing include establishing a total daily dose, an insulin to carbohydrate ratio and an insulin sensitivity or correction factor. 

 

ESTABLISHING A TOTAL DAILY DOSE (TDD) OF INSULIN

 

This is the first step in starting treatment in a patient with newly diagnosed diabetes. This dose can vary based on the individual and can range from 0.3- 1.5 units/kg/day.  A good starting dose is ~0.5 units/kg/day. Once the TDD is determined, this number is divided by half to establish the basal and bolus requirements.  As a general rule of thumb, half the insulin is used as basal insulin, while the other half is used as prandial or mealtime insulin.For example, in a person weighing 75 kg, a typical total daily insulin dose might be 75 kg X 0.7 units/kg = roughly 37 units/day. The basal insulin dose would be roughly 18 units and bolus insulin total would be 18 units (divided amongst meals, see below).

 

Long-acting insulin analogs U-100 glargine and detemir can be administered once or twice daily. Insulin degludec or U-300 insulin glargine can be administered once a day.

 

USING PRANDIAL INSULIN

 

Establishing an Insulin to Carbohydrate (Carb) Ratio

 

Patients with T1D derive the greatest therapeutic benefit when basal and prandial analogs are used together, because the physiologic pharmacokinetics and pharmacodynamics of these analogs make separating the basal and prandial components of insulin replacement easier. In general, administering the appropriate amount of pre-meal insulin requires that the patient know at least their current blood glucose level and the estimated amount of carbohydrates for a meal. Initially, the amount of prandial insulin can be determined by approximating the percentage of calories consumed at each meal. As patients become more educated, however, they may alter the prandial dose by estimating the carbohydrate component of each meal or snack. As patients become more sophisticated, they may note that the same carbohydrate quantity may have a different effect on their blood glucose level depending upon the specific type of meal consumed.

 

The carb ratio provides the dose of rapid acting insulin (lispro, aspart, glulisine) to cover the carbohydrate content of a meal. A typical starting point in patients with T1D is to give 1 unit of rapid acting insulin for every 15 grams of carbohydrates. This ratio is variable ranging from 1 unit for every 5g to 30 g of carbohydrate. To estimate the carb ratio, the “500 rule” can be used:

 

500/total daily dose (TDD) = grams of carbohydrate covered by 1 unit of insulin.

 

Example: A person who takes a total of 50 units of insulin per day (both basal and prandial combined) will need 1 unit of rapid acting prandial insulin for every 10g carbohydrate (500/50 = 10g of carbohydrate covered by 1 unit of insulin, using above formula).

 

Alternative way to calculate the carb ratio – Add all carbohydrates consumed in a day and divide this by the total units of prandial insulin taken that day, using an average over 3 days.

 

Prandial insulin may be reduced/skipped when:

 

  • Extra carbohydrates are used to raise low blood sugars or cover increased physical activity
  • Recent dose of correction insulin within past 1-2 h
  • Nausea or vomiting preventing oral intake

 

Determining the Correction Dose or “Insulin Sensitivity Factor” (ISF)

 

In addition to covering the carbohydrate load of a meal, individuals will also need to correct hyperglycemia, called the “correction dose”. The method commonly used for this is the “1800 Rule”. This estimates the point drop in glucose for every unit of rapid-acting insulin administered:

 

1800/TDD = Point drop in glucose for 1 unit of rapid-acting insulin

This ISF (also called the correction factor) can be used for between-meal elevations in blood glucose. Thus, in general this correction dose can be utilized anytime provided the patient has not taken an injection of rapid acting insulin over the past 2-4 hours (insulin on board, Figure 12).  

 

Target glucose: The ISF enables achieving appropriate individualized blood glucose targets.

 

For example: A person who takes a total of 60 units of insulin per day will require 1 unit of rapid acting insulin to drop the glucose by 30 points. If the patient’s glucose is 180 mg/dL and the glucose target has been set at 120 mg/dL, a correction dose of 2 units would be required to bring the glucose down to target:

 

  1. ISF = 1800/60 (TDD) = 30; 1 unit of rapid-acting insulin will decrease glucose by 30 points
  2. 180 mg/dL (actual glucose level) – 120 mg/dL (target glucose level) = 60; this is the excess glucose, that is, the value that is above target and that needs to be corrected
  3. 60/30 (ISF) = 2; dividing the excess glucose by the ISF will provide the amount of correction insulin units that are required to bring down the glucose to target, in this case it will be 2 units.

 

Putting it All Together - Combining the Carb Ratio and ISF

 

Combining the carbohydrate load and ISF will enable patients to appropriately target their pre-meal glucose. 

 

For example: An individual with a carb ratio of 1:15 and ISF of 1 unit/50mg/dL, prior to a meal of 60g carbohydrates and a pre-meal blood glucose of 220mg/dL and target of 120mg/dL would take the following steps to administer the appropriate amount of prandial insulin as follows:

 

  1. To cover carbohydrate intake: 60g/15g per unit =4 units
  2. Correction dose: 220 mg/dL (actual glucose)– 120mg/dL (target glucose) = 100mg/dL. ISF is 100/50 = 2 units to correct.
  3. Total amount of prandial insulin: 4+2= 6 units

 

Insulin Titration and Pattern Adjustments

 

Reviewing blood glucoses and recognizing patterns is one of the most important aspects of diabetes management, allowing for timely and appropriate adjustments in insulin dose, food intake, and managing physical activity. Pattern management is aided by valuable tools such as SMBG with information obtained through download software (see above) or logbooks and CGM data. These tools can be used in order of priority, for assessment of hypoglycemia, hyperglycemia, glycemic variability, frequency of SMBG readings, etc.

Figure 13. The appearance of insulin into the blood stream (pharmacokinetics) is different than the measurement of insulin action (pharmacodynamics). This figure is a representation of timing of insulin action for insulin aspart from euglycemic clamp data (0.2 U/kg into the abdomen). Using this graph assists patients to avoid “insulin stacking”. For example, 3 hours after administration of 10 units of insulin aspart, one can estimate that there is still 40% X 10 units, or 4 units of insulin remaining. By way of comparison, the pharmacodynamics of regular insulin is approximately twice that of insulin aspart or insulin lispro. Currently used insulin pumps keep track of this “insulin-on-board” to avoid insulin stacking. Adapted from reference (40).

INSULIN DELIVERY SYSTEMS

 

Significant improvement in pharmacokinetics and pharmacodynamics of insulin analogs and advances in technology has allowed for insulin delivery systems to resemble endogenous insulin secretion as closely as possible.

 

Insulin Pens

 

Insulin pens were first introduced in 1981 as injection devices. These pens contain a cartridge holding insulin which is injected into the subcutaneous tissue through a fine, replaceable needle. Insulin pens are convenient, portable and are widely used as a part of MDI therapy. Currently, insulin pens are available as disposable pens containing prefilled cartridges or reusable insulin pens with replaceable insulin cartridges. Several insulin pens allow the convenience of ½ unit dosing, a critical need for pediatric patients and those adults with high insulin sensitivity and low insulin requirements.

 

Insulin smart pens - The first insulin smart pen was approved for use in the United States (InPen, Companion Medical, California, USA) in 2017 (Figure 14). Smart pens can record timing and amount of each administered insulin dose, display the last dose and insulin onboard and also make dosing recommendations based on pre-specified information (Figure 15). This information is wirelessly transmitted via Bluetooth to a dedicated mobile application on a smartphone device. Other similar devices are in development including the NovoPen 6 and NovoPen Echo Plus reusable insulin pens equipped with near-field communication technology (Novo Nordisk, Denmark), recently approved in the European Union.

Figure 14. InPen Smart Insulin Pen

Figure 15. InPen Insight report. Report provides missed doses, bolus calculator dosage, long-acting insulin assessment and CGM data (if paired with the InPen app).

Continuous Subcutaneous Insulin Infusion Therapy (CSII)

 

While not a new tool, insulin pump therapy remains the gold standard of insulin delivery for T1D (Figure 9). CSII is the most precise way to mimic normal insulin secretion because basal insulin infusion rates can be programmed throughout a 24-hour period. Essentially, the CSII pump may be thought of as a computerized mechanical syringe automatically delivering insulin in physiologic fashion. Patients can accommodate metabolic changes related to eating, exercise, illness, or varying work and travel schedules by modifying insulin availability. Basal rates can be adjusted to match lower insulin demands at night (between approximately 11 PM and 4 AM) and higher requirements between 3 AM or 4 AM and 9 AM.

 

Various studies comparing glycemic control during CSII versus intensive insulin injection regimens have been published. A meta-analysis of 12 randomized controlled trials of CSII versus multiple injection regimens showed a weighted mean difference in blood glucose concentration of 16 mg/dL (95% CI 9-22) and a difference in A1C of 0.5% (0.2-0.7) favoring CSII (60). The slightly but significantly better control in patients on CSII was accomplished with a 14% average reduction in daily insulin dose.

 

A meta-analysis funded by the Agency for Healthcare Research and Quality showed that in adults with T1D A1C levels decreased more with CSII than multiple injections, but one study heavily influenced this finding  (27). For both children and adults, there was no difference in severe hypoglycemia. The common misconception that CSII leads to more hypoglycemia is not valid.

 

Modern insulin pumps are much smaller and easier to use than the pumps of the past (Figure 10).

 

With the exception of insulin lispro-aabc (Lyumjev), all rapid-acting analogs are approved in the United States for use in insulin pumps. The basal rate of the insulin pump replaces the use of daily injections of basal insulin. The boluses given before each meal are essentially the same as normal insulin injections of rapid acting insulin. The pump allows programming of several different basal infusion rates at increments that can range from 0.025 up to 35.0 units/hour (usually ranging from 0.4 to 2.0 units/hour) to meet non-prandial insulin demands, though it is unlikely that the average patient will require more than 2 or 3 different rates (Figure 16). As with MDI, correction doses can be provided before or between meals. Figures 17 and 18 show data that is typically downloaded from a pump.

Figure 16. Idealized insulin curves for CSII with either insulin lispro, insulin aspart, or insulin glulisine. Note the basal insulin component can be altered based on changing basal insulin requirements. Typically, insulin rates need to be lowered between midnight and 0400 h (predawn phenomenon) and raised between 0400 h and 0800 h (dawn phenomenon). The basal rate the rest of the day is usually intermediate to the other two. Modern-day pumps can calculate prandial insulin dose by the patient entering the blood glucose concentration and the anticipated amount of carbohydrate to be consumed. The pump calculates how much previous prandial insulin is still active and provides the patient a final suggested dose which the patient may activate or override.

There are many fundamental differences between CSII and MDI. These include:

 

TITRATION OF BASAL RATES

 

From a practical point of view, the first and most important insulin dose to provide in a correct amount is the basal rate. If the basal dose is set incorrectly, neither the bolus doses nor the correction doses will be appropriate. A common mistake observed in CSII therapy is that the basal dose is set too high, making the administration of even small insulin correction doses result in hypoglycemia. The greatest advantage of CSII is it allows more flexibility and titration of the basal doses.

 

The basal dose can be titrated throughout the day to meet patients’ individual needs and this should be done in a systematic manner by performing “basal checks.” Prior to starting a basal rate assessment (basal check), the following conditions should be met for the day of the test: last meal and/or insulin bolus should have occurred at least 4 hours prior to starting the assessment; last meal should preferentially be low in fat and not have too much protein; avoid exercise and alcohol; do not perform the assessment if hypoglycemia has occurred earlier in the day or there is an inter-current illness. Of note, it is recommended to repeat the assessment on several occasions to identify a pattern prior to making adjustments to the basal rate. 

 

Nighttime Basal Rate

 

It is usually best to start by addressing the overnight basal rate. An overnight basal assessment is performed on a night the patient has a bedtime glucose level within target. The patient is asked not to have anything to eat during the assessment. The patient then measures glucose levels at bedtime, midnight, 3AM and upon awakening to assess for changes in glucose profile (the use of a CGM obviously makes this exercise much easier). Glucose should also be checked in case of hypoglycemic symptoms. If hypoglycemia ensues or glucose level rises above target, the assessment is stopped and the patient treats the glucose level accordingly. Rises or falls of ≤ 30 mg/dl from bedtime to morning (upon awakening) are usually acceptable. By contrast, glucose changes > 30 mg/dl will require adjustments in basal rates usually consisting of 10-20% changes in insulin dose (as deemed clinically appropriate) starting 2 hours before the observed rise or fall in glucose levels. In general, a change in a basal dose takes two to four hours to result in a change in blood glucose.

 

Daytime Basal Rates

 

Daytime basal rates are checked by assessing the glucose profile across a skipped-meal time segment (i.e., pre-breakfast to pre-lunch, pre-lunch to pre-dinner, and pre-dinner to bedtime). To check the “pre-breakfast to pre-lunch” time segment, breakfast is skipped and glucose level is checked at 1-2 hour intervals for the duration of the time segment (prior to lunch). Glucose levels should also be checked in the event of hypoglycemic symptoms. The same recommendations regarding changes in glycemic levels requiring insulin dose adjustments described for the overnight basal assessment apply here. 

 

TRACKING OF INSULIN-ON-BOARD

 

Another major difference between CSII and MDI is the pump can accurately track the insulin-on-board for safer use of correction doses (Figure 13). As noted above, doing this accurately can have a major impact in preventing insulin stacking.

 

INSULIN DOSE CALCULATOR

 

Insulin-to-carbohydrate ratios and insulin sensitivity factors with corresponding target glucose values can be set and modified as needed in insulin pumps. Patients are only required to enter their glucose level and/or anticipated carbohydrate amount to be consumed and the insulin pump will calculate the insulin dose and recommend a bolus dose. So, the complicated mathematics to best utilize MDI are done automatically with CSII.

 

MODIFICATIONS TO BOLUS DELIVERY

 

Pumps can be programmed for individual boluses to be administered over an extended period of time (“extended” or “square wave” bolus). This feature may be particularly helpful for very high-fat meals or those patients with delayed gastric emptying, seen with gastroparesis or in those receiving pramlintide (see below).

 

TEMPORARY BASAL RATES

 

The other major advantage of CSII is that it allows the use of “temporary basal rates.” This is extremely helpful in situations where metabolic demands have “temporarily” changed such as during illness (requiring an increase in insulin dose) or during exercise (requiring a dose reduction). Again, due to the time action of the rapid-acting analogs, sufficient time must be incorporated when using a temporary basal rate.

 

DOWNLOAD CAPABILITY

 

Pump data can be downloaded, and the data obtained is extremely helpful in understanding patients’ glycemic responses to an established insulin regimen (Figure 17). Also, it can assist in evaluating patients’ behaviors pertaining to their glucose management. Downloads provide information regarding the total daily insulin use broken down into percentages corresponding to basal and bolus delivery. This allows determining if patients are consistently administering boluses or whether they are essentially “running on basal.” Some of the additional data that can be downloaded includes average glucose levels, frequency of glucose monitoring, days between site changes, amount of time patients are suspending the pump or using temporary basal rates, frequency of boluses (which allows to identify non-compliance or insulin stacking behaviors), and average daily carbohydrates consumed (Figure 18).

Figure 17. A patient’s insulin pump download showing comprehensive data for one day including basal rates, boluses and use of bolus calculator, glucose monitoring, carbohydrate intake, and percentage of glucose at target.

Figure 18. A patient’s pump download showing glucose measurements, bolus events, fill events (denoting frequency of site and set changes), as well as insulin pump suspension duration for a 14-day period).

However, despite the multiple benefits of CSII therapy there are also several risks. The first is an abrupt stoppage of insulin delivery either from an occlusion or dislodging of the catheter. For most patients who measure glucose levels at least 4 times daily the problem can be discovered and rectified quickly. However, for the occasional patient who tests infrequently or misses several glucose tests the discontinuation of the insulin infusion can result in ketoacidosis. Fortunately, this is rare. When glucose levels are found to be elevated for no apparent reason, it is appropriate to bolus the appropriate correction dose and if after 1 to 2 hours glucose levels are not improved, an injection of insulin is recommended, and the infusion site should be changed.

 

Another potential complication is infection, often an abscess, at the infusion site. This is also rare and can be minimized with meticulously cleaning the pump site prior to insertion. Although not as severe, inflammation from pump sites can be problematic. This can be improved by changing the infusion set every 24 to 72 hours and rotating pump sites. Similarly, some patients develop lipohypertrophy from infusing the insulin in the same area. This can result in extreme variability in insulin absorption. Again, frequent rotation of pump sites can alleviate this problem which is under-reported. Clinicians should therefore make pump site observation a part of every clinic visit.

 

CLASSIFICATION OF INSULIN PUMPS

 

Insulin pumps can be classified by the way insulin is delivered into:

 

Pumps with Tubing

 

These insulin pumps require an infusion set for insulin delivery. They house an insulin-filled cartridge connected to a tubing with a prespecified length, allowing patients to select the length that better accommodates to their needs.  At the end of the tubing is a needle or soft Teflon cannula that can be inserted into the subcutaneous tissue at a 30- to 45- or 90-degree angle, depending on the type of infusion set used. The abdomen is the preferred infusion site because placement of the catheter there is convenient and comfortable and insulin absorption is most consistent in this region. However, the upper outer quadrant of the buttocks, upper thighs, and triceps fat pad of the arms may also be used.

 

Infusion sets allow removal of the insertion needle, leaving only the soft cannula in place subcutaneously. Patients who experience frequent soft cannula kinking or those with Teflon allergies can opt for infusion sets that use a small stainless-steel needle to infuse insulin instead of a Teflon cannula. Infusion sets have a quick-release mechanism, allowing them to be temporarily disconnected from the insertion site. This quick-release feature makes dressing, swimming, showering, and other activities more convenient.

 

Tubeless Pump

 

Patients may also choose the convenience of a tubeless or patch pump. This pump consists of disposable “pods” which are discarded every three days. The pods are essentially small self-contained insulin pumps with an internal insulin cartridge, an insertion needle and cannula, and the necessary hardware required for insulin administration. Insulin is infused directly from the pod through a catheter without the use of any tubing. Both basal and bolus insulin dosing is communicated to the pod through either audio frequency or Bluetooth technology via a separate “personal diabetes manager” device.

 

Artificial Pancreas Device Systems

 

Improvements in CGM sensor technology have allowed for the integration of CGM systems with insulin pumps and the development of artificial pancreas device systems (APDS), also known as closed-loop (CL) systems. An APDS consists of an insulin pump, a CGM device, and an insulin infusion algorithm designed for safety and glucose control optimization.

 

In 2009, the JDRF developed an artificial pancreas road map defining 6 stages of APDS technology based on the level of automation (61):

  • First generation:
    • Stage 1: Very-Low-Glucose Insulin Off Pump. Pump shuts off when user not responding to low-glucose alarm.
    • Stage 2: Hypoglycemia Minimizer. Predictive hypoglycemia causes alarms, followed by reduction or cessation of insulin delivery before blood glucose gets low.
    • Stage 3: Hypoglycemia/Hyperglycemia Minimizer. Same product as #2 but with added feature allowing insulin dosing above high threshold.
  • Second Generation:
    • Stage 4: Automated Basal/Hybrid Closed Loop. Closed loop at all times with mealtime manual assist bolus.
    • Stage 5: Fully Automated Insulin Closed Loop. Manual mealtime bolus eliminated.
  • Third Generation
    • Stage 6: Fully Automated Multihormone Closed Loop.

 

First generation devices focused primarily on prevention of hypoglycemia. Second generation devices have introduced automation of basal insulin delivery with or without automatic correction boluses. Lastly, third generation devices are expected to fully close the loop while providing a multi-hormonal (e.g., insulin, glucagon, amylin) delivery approach.

 

It is important to note that the development of any of these specific stages is not dependent on the previous one being completed and can occur in tandem.

 

APDS can also be classified according to the type of control algorithm used to determine insulin delivery (62):

 

  • Proportional Integral Derivative (PID). This algorithm responds to measured glucose levels where: “proportional” refers to the difference between the measured sensor glucose and the target glucose; “integral” refers to how long the sensor glucose has been away from the target; and “derivative” refers to how rapidly the sensor glucose is changing.
  • Model Predictive Control (MPC). This algorithm allows prediction of glucose levels at a specific point in the future and based on this data, modulation of insulin delivery.
  • Fuzzy logic. The calculation of insulin doses is similar to what a diabetes specialist would recommend based on CGM data.
  • Bio-inspired. Uses a mathematical model of beta cell insulin production in response to changes in blood glucose.

 

A list of currently approved APDS and features is listed in Figure 19 (63-67)

Figure 19. Features and CGM outcomes from pivotal studies on currently available artificial pancreas device systems.

ADJUNCTIVE NON-INSULIN THERAPIES IN TYPE 1 DIABETES

 

Intensive insulin therapy for T1D is associated with increased risk of hypoglycemia. Additionally, glycemic variability and weight gain with resultant non-adherence to insulin are commonly encountered. Weight gain also contributes to increased cardiometabolic risk such as hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Insulin therapy also does not address glucagon excess and altered gastric emptying that is seen in patients with T1D. Hence adjunctive therapies could be of potential benefit in management of T1D.

 

Amylin Analog - Pramlintide

 

Amylin is a neuroendocrine hormone co-secreted with insulin by the pancreatic beta cells in a fixed ratio (68); T1D is a state of deficiency. Amylin reduces postprandial hyperglycemia by reducing mealtime glucagon secretion. It also delays gastric emptying, increases satiety and enables weight loss. Overall, amylin complements the action of insulin by targeting postprandial hyperglycemia.

 

Pramlintide is an injectable amylin analog approved for use in T1D as an adjunct to prandial insulin. Pramlintide has similar physiological effects as amylin, such as decreased food intake, and decreases mean A1C by 0.3-0.5% with modest weight loss (69). A recent crossover study of pramlintide infusion co-administered with human regular insulin via a pump over 24h improved glycemic variability and postprandial hyperglycemia in adults with T1D (70). Pramlintide is injected just prior to meals at an initial dose of 15 mcg and increased as tolerated to a final dose of 60 mcg. It should be administered only prior to major meals consisting of 250 calories or 30 grams of carbohydrate. Prandial insulin doses of insulin (in MDI or CSII therapy) should be reduced as food intake decreases and gastric emptying is delayed. For those receiving insulin via a pump, using an “extended bolus” (see above) works best to avoid postprandial hypoglycemia. For those using MDI, some patients administer their insulin just prior to eating (without a lag time) or after eating. Use of pramlintide is limited by nausea, often mild and self-limited. Severe insulin-induced hypoglycemia has also been noted with the use of pramlintide if insulin doses are not sufficiently reduced on initiation of pramlintide therapy.  However widespread use of pramlintide as a therapeutic adjunct in T1D has been limited due to concerns of nausea, hypoglycemia and additional injection burden. Long-term use of pramlintide is unclear at this time.

 

Metformin

 

Metformin, a biguanide, is used as first-line therapy in patients with T2D. It decreases hepatic gluconeogenesis and improves insulin sensitivity (71). Metformin may have some benefit in reducing insulin doses and possibly improve metabolic control in obese/overweight individuals as observed in small studies in patients with T1D. An early meta-analysis of 5 studies suggested that addition of metformin resulted in a decrease in insulin requirement (6.6 units/day), and a decrease in weight with minimal change in A1C (72). A randomized placebo-controlled trial in 140 overweight adolescents with T1D evaluated the addition of metformin to insulin (73). There was no improvement in glycemic control after 6 months but use of metformin resulted in decreased insulin dose and improved measures of adiposity, despite increased gastrointestinal adverse events. A meta-analysis of 19 RCTs suggests short term improvement in A1C that is not sustained after 3 months and associated with higher incidence of GI side effects (74). Although metformin has been shown to decrease CVD morbidity in T2D, data in T1D is lacking.  Recent evidence suggests that metformin decreases insulin resistance and improves vascular health in adolescents with T1D (75). The REMOVAL trial assessed benefit of metformin in T1D and cardiovascular risk and showed no evidence of sustained A1C reduction, and no benefit in carotid intima-media thickness (the study’s primary endpoint); however, reductions in body weight, LDL-C and total insulin requirements was observed (76).  Therefore, based on current evidence, concomitant use of metformin in patients with T1D and is not recommended in current published guidelines.

 

Sodium Glucose Cotransporter 2 (SGLT2) Inhibitors

 

SGLT2 is a protein expressed in the proximal convoluted tubule (PCT) of the kidney and is responsible for re-absorption of filtered glucose.  Inhibition of SGLT2 prevents glucose reabsorption in the PCT and increases glucose excretion by the kidney. SGLT1 is the major intestinal glucose transporter. SGLT1 inhibition also increases postprandial release of the gastrointestinal hormones GLP-1 and polypeptide YY, probably by increasing delivery of glucose to the distal small intestine, thereby regulating glucose and appetite control. Notably, the action of these agents is insulin-independent, therefore this class of drugs has potential as adjunctive therapy for T1D. Additionally recent clinical trials have also demonstrated improvements in cardiovascular outcomes trials as well as reductions in renal outcomes in T2D; therefore, there is significant interest for use in T1D. Early small studies of SGLT2 inhibitors in T1D showed promising results with evidence of decreased total daily insulin dosage, improvement in fasting glucose and A1C, measures of glycemic variability, rates of hypoglycemia and body weight (77-79).

 

Common side effects associated with this class of drugs include genital and urinary infections. Euglycemic diabetic ketoacidosis has been recognized in patients with T1D due to glycosuria masking hyperglycemia but with a catabolic state (due to insulin deficiency and hyperglucagonemia) with ketonemia (80, 81).

 

A dual inhibitor of SGLT1 and 2 sotagliflozin is under development and shows promise in T1D patients (82). Currently in the US, SGLT2 inhibitors are approved for use in T2D only. SGLT2 and mixed SGLT1/2 inhibitors are approved for use in T1D by the European Medicines Agency.

 

All four available SGLT2 inhibitors have been studied in T1D. When added to insulin therapy, all SGLT2 inhibitors appear to decrease A1C levels, averaging 0.35-0,5% within 6 months of initiation; however, this effect does not appear to be sustained at 1 year in clinical trials and effects appear to wane with time (83). Insulin dosing should be adjusted with caution to avoid hypoglycemia. There is no data on efficacy comparing the different agents currently. It is estimated that these agents increase risk of diabetic ketoacidosis by 8-fold, and therefore are not approved for use in T1D in the US.

 

Incretin Therapies

 

Endogenous glucagon-like peptide-1 (GLP-1) is secreted from L cells (present in the small and large intestine) in response to food ingestion. GLP-1 enhances glucose-induced insulin secretion, inhibits glucagon secretion, delays gastric emptying, and induces satiety. GLP-1 secretion in T1D patients is similar to that seen in healthy individuals. In vitro studies suggest that incretin-based therapies can expand beta cell mass, stimulate beta cell proliferation and inhibit beta cell apoptosis, although this has not been demonstrated in humans. Thus, due to their putative effects on beta cell integrity and function, GLP-1 receptor agonists and oral dipeptidyl peptidase-4 (DPP-4) inhibitors are of interest in T1D.  GLP-1 receptor agonists delay gastric emptying, suppress postprandial glucagon secretion, and increase satiety. Studies suggest that these agents may decrease insulin requirements and facilitate weight loss (84, 85). Early RCTs of liraglutide in T1D revealed weight loss and some A1C lowering benefit (85, 86). Recent data suggests benefit of liraglutide 1.8 mg in individuals with T1D and higher BMI in decreasing A1C, weight and no increased hypoglycemia risk (87). However, these effects may not be sustained, based on results from a weekly exenatide study (88). At this time, GLP-1 receptor agonists are not a recommended treatment option in T1D.

 

The DPP-4 enzyme degrades endogenous GLP-1 and removes it from the circulation. DPP-4 inhibitors lower blood glucose by preventing breakdown of endogenous GLP-1, thereby increasing concentration in the circulation. In patients with T2D, DPP-4 inhibitors potentiate glucose-dependent insulin secretion and inhibit glucagon release without effect on gastric emptying or bodyweight. Patients with T1D have inappropriately raised glucagon secretion and DPP-4 inhibitors added to insulin could potentially enhance insulin secretion in patients with residual endogenous insulin secretion and improve glycemic control.  However, observed effects in patients with T1D are limited with modest improvements in A1C that are short-term and not sustained (89). Therefore, these agents cannot be recommended for use in T1D.           

 

Bariatric Surgery

 

Bariatric and other metabolic surgeries are effective weight loss treatments in severe obesity. In T1D individuals with morbid obesity, bariatric surgery has been shown to result in significant weight loss, decrease in insulin requirements and an overall improvement in metabolic profile. However, DKA and hypoglycemia occur in the post-operative period. Longer term and larger studies are required to further evaluate the role of bariatric surgery in T1D (90).

 

OTHER ASPECTS OF MANAGEMENT

 

Psychosocial Aspects

 

Assessment and management of psychosocial issues are an important component of care in individuals with T1D throughout their life span (91). While the individual patient is the focus of care, family support should be encouraged when appropriate. Evaluation and discussion of psychosocial issues and screening for depression screening should be included as part of each clinic visit. Many patients experience “diabetes distress” related to the multitude of self-care responsibilities to optimize glycemic control. Diabetes distress is frequently associated with suboptimal glycemic control, low self-efficacy and reduced self-care. Depression, anxiety from fear of hypoglycemia, and eating disorders can develop and are associated with poor glycemic control. In young adults, comprehensive management of diabetes that addresses these psychosocial issues can improve glycemic control and reduce hospitalization due to diabetic ketoacidosis. Strategic interventions such as cognitive restructuring, goal setting and problem solving can help individuals particularly adolescents and young adults reduce diabetes distress (92). Thus, early identification and treatment including referral to a mental health specialist can help aid management of diabetes.

 

Management in Exercise

 

The benefits of exercise and physical activity in patients with type 1 diabetes have been well documented (93, 94). However, achieving adequate glycemic control during and after completion of exercise remains a rather challenging aspect of type 1 diabetes management. Glycemia at the initiation of exercise, sensor glucose trend (if using a CGM), timing from the previous meal, carbohydrate content in the meal preceding exercise, type and duration of exercise, are all but a few of the factors that need to be considered to ensure that glycemic control remains stable during and after cessation of exercise.

 

In 2017, an international consensus statement for exercise management in type 1 diabetes was published (95). This consensus is a unique resource which provides detailed glucose management strategies. Recommended adjustments to basal and prandial insulin, for both insulin pump and multiple daily insulin injection users, as well as carbohydrate intake requirements depending on the intensity and duration of activity are clearly presented. Quite importantly, the consensus also covers factors that would preclude exercise including the presence of elevated ketones, recent hypoglycemia, and diabetes-related complications which may be exacerbated in the context of vigorous exercise and/or competitive endurance events. We encourage the reader to refer to this publication for additional guidance.

 

For those patients on hybrid closed loop systems, a way to minimize the occurrence of exercise-induced hypoglycemia is the use of a higher glucose target for exercise. For the Medtronic 670G, the standard Auto-Mode target is 120 mg/dL which can be temporarily changed to 150 mg/dL. For the Tandem X2 with Control IQ, the standard target for regular activity is between 112.5 and 160 mg/dL and can be temporarily changed to 140-160 mg/dL.

 

A study in open loop insulin pump users found that a basal rate reduction starting 90 min before exercise was superior to pump suspension at exercise onset for reduction of hypoglycemia risk during exercise and did not compromise the post-exercise meal glycemic control (96).

 

Another strategy that may be more effective than basal rate reduction for prevention of exercise induced hypoglycemia is the use of a subcutaneously administered mini-dose of glucagon. A small study including 15 subjects with type 1 diabetes on insulin pump therapy who exercised in the fasting state in the morning for 45 min, found that a dose of 150 µg of subcutaneous glucagon, compared to a 50% basal insulin reduction or 40-g oral glucose tablets, resulted in no hypoglycemia (vs. basal insulin reduction) and no hyperglycemia (vs. oral glucose tablets) (97). However, larger and long-term studies are required before determining if a mini-dose of glucagon is safe and effective for prevention of exercise induced hypoglycemia in subjects with type 1 diabetes. 

 

Management of Special Populations

 

OLDER ADULTS

 

Adults with T1D now span a very large age spectrum—from 18 to 100 years of age and beyond. These individuals are unique in that they usually have lived with a complex disease for many years (91).  An understanding of each individual’s circumstances is vital and management often requires assessment of medical, functional, mental, and social domains. The ADA emphasizes that glycemic targets should be individualized with the goal of achieving the best possible control while minimizing the risk of severe hyperglycemia and hypoglycemia (98).

 

Glycemic goals in older adults vary. Most older adults with T1D have long-standing disease (unlike individuals with T2DM where diabetes can be long-standing or new onset). Additionally, there is a wide range of health in older individuals, with some patients enjoying good functional status and no comorbid conditions, while others are limited by multiple comorbidities as well as physical or cognitive impairments. Older T1D patients may develop diabetes related complications which pose a challenge in disease management. Insulin dosing errors, hypoglycemia unawareness, and inability to manage hypoglycemia when it occurs may result from physical and cognitive decline. Special attention should be focused on meal planning and physical activities in this population.  Severe hyperglycemia can lead to dehydration and hyperglycemic crises (91). Issues related to self-care capacity, mobility, and autonomy should be promptly addressed.

 

Thus, treatment goals should be reassessed and individualized based on patient factors. Older patients with long life expectancy and little comorbidity should have treatment targets similar to those of middle-aged or younger adults. In patients with multiple comorbid conditions, treatment targets may be relaxed, while avoiding symptomatic hyperglycemia or the risk of diabetic ketoacidosis (91). Therefore, it is important to assess the clinical needs of the patient, setting specific goals and expectations that may differ quite significantly between a healthy 24-year-old and a frail 82-year-old with retinopathy and cardiovascular disease.

 

There are few long-term studies in older adults demonstrating the benefits of intensive glycemic, blood pressure, and lipid control (98). As with younger adults, glycemic control should be assessed based on frequent SMBG levels (and CGM data, if available) as well as A1C to help direct changes in therapy. More stringent A1C goals (~6.5-7%) can be recommended in select older adults if this can be achieved without hypoglycemia or other adverse effects. This is appropriate for older individuals with anticipated long-life expectancy, hypoglycemia awareness and no CVD. Less stringent A1C goals (for example A1C<8.5%) may be appropriate for patients with a history of severe hypoglycemia, hypoglycemia unawareness, limited life expectancy, advanced microvascular/macrovascular complications, or extensive comorbid conditions (91, 99).  

 

INPATIENT MANAGEMENT AND OUTPATIENT PROCEDURES

 

The challenges involved in management of individuals with T1D in the hospital and in preparation for scheduled outpatient procedures include difficulties associated with fasting, maintaining a consistent source of carbohydrate, and facilitating inpatient blood glucose management while modifying scheduled insulin therapy. Individuals with T1D may have difficulty fasting for long periods of time (more than 10 h) prior to a procedure. Patients with T1D should be prepared with a treatment plan for insulin dose adjustments and oral glucose intake prior to any procedure that requires alterations in dietary intake and/or fasting.

 

In general, goals for blood glucose levels in individuals with T1D are the same as for people with T2D or hospital-related hyperglycemia (100) . It is imperative that the entire health care team, including anesthesiologists and surgeons as well as other specialists who perform procedures, understands T1D and how it factors into the comprehensive delivery of care. First, the diagnosis of T1D should be clearly identified in the patient’s record.  Second, the awareness that people with T1D will be at high risk for hypoglycemia during prolonged fasting and are at risk for ketosis if insulin is inappropriately withheld. Under anesthesia, individuals with T1D must be carefully monitored for hypoglycemia and hyperglycemia. Third, a plan for preventing and treating hypoglycemia should be established for each patient.

 

SMBG should be ordered to fit the patient’s usual insulin regimen with modifications as needed based on clinical status. Self-management in the hospital may be appropriate for some individuals with T1D including those who successfully manage their disease at home, have cognitive skills to perform necessary tasks such as administer insulin and perform SMBG, count carbohydrates and have a good understanding of their condition (100). For some individuals, once the most acute phase of an illness has resolved or improved, patients may be able to self-administer their prior multiple-dose or CSII insulin regimen under the guidance of hospital personnel who are knowledgeable in glycemic management.  Individuals managed with insulin pumps and/or multiple-dose regimens with carbohydrate counting and correction dosing may be allowed to manage their own diabetes if this is what they desire, once they are capable of doing so.

 

The need for uninterrupted basal insulin to prevent hyperglycemia and ketoacidosis is important to recognize. Insulin dosing adjustments should also be made in the perioperative period and inpatient setting with consideration of oral intake and blood glucose trends.

 

The use of CGM in the inpatient setting is an area of ongoing research. Currently, the Endocrine Society recommends against the use of real-time CGM (RT-CGM) alone in the intensive care unit or operating room settings due to limited available data on accuracy (101). A study in T2D patients on basal bolus insulin therapy admitted to the general ward evaluated the use of retrospective CGM versus point of care capillary glucose testing for inpatient glycemic control (102). Although average daily glucose levels were comparable between CGM and capillary blood glucose testing, CGM detected a higher number of hypoglycemic episodes (55 vs 12, P < 0.01) suggesting that CGM may be beneficial for identification of hypoglycemia in the general ward particularly in patients with hypoglycemia unawareness. We feel it is reasonable to allow T1D patients who already benefit from use of RT-CGM to continue the use of this technology in the non-ICU inpatient setting under the supervision of the care team. Large prospective randomized trials will be required to establish benefit or lack thereof of RT-CGM use on inpatient glycemic control.

 

BETA-CELL REPLACEMENT STRATEGIES

 

Pancreas Transplantation

 

Pancreas transplantation is a currently available therapeutic option for patients with diabetes who meet specific clinical criteria. Patients with end-stage renal disease are eligible to undergo simultaneous pancreas kidney (SPK) transplantation. Also, pancreas transplantation may be offered as a separate procedure after a patient has already received a kidney transplant (pancreas after kidney (PAK)). In addition, solitary pancreas transplantation may also be offered to those individuals presenting with severe metabolic complications attributed to poor glycemic control (pancreas transplant alone (PTA)). Pancreas transplantation procedures have been performed since the 1960’s. A 2011 update on Pancreas Transplantation from the International Pancreas Transplant Registry reported improvements in patient survival and graft function over a course of 24 years of pancreas transplantation (103). These improved outcomes were related to changes in surgical technique and immunosuppressive regimens as well as tighter donor selection criteria. At 5-years post-transplantation, pancreas graft survival is now reported at ~70% for SPK and at ~ 50% for PAK and PTA. Further, patient survival at 10 years exceeds 70% with the highest survival rate observed in PTA recipients (82%).

 

Islet Transplantation

 

Islet transplantation provides a less invasive surgical alternative for beta-cell replacement in patients with labile diabetes and has the potential to restore normoglycemia, eliminate severe hypoglycemia and restore hypoglycemia awareness. However, this procedure is still considered experimental in the United States. Marked improvements have also been noted in the field of islet transplantation over the past decade which have led to insulin independence rates at 5 years being comparable to pancreas transplantation outcomes (104). A pivotal study of islet transplantation in patients with T1D showed that at 1-year post transplant, 87% of study participants achieved the primary endpoint of a A1C <7.0% and freedom from severe hypoglycemia (from day 28 to 365) (105). Further details about islet transplantation can be found in the Endotext chapter on this topic.

 

FUTURE DIRECTIONS IN MANAGEMENT OF TYPE 1 DIABETES

 

Artificial Pancreas Device Systems - Closed Loop Systems

 

In addition to insulin-only CL-systems, bi-hormonal closed loop systems are also being actively explored. Additional manufacturers utilizing insulin-only CL-systems are expected to launch their devices in the near future. The introduction of faster-acting insulins (biochaperone lispro and faster-acting insulin aspart (FIAsp)) could potentially make these strategies more effective. As this technology advances, we are getting closer to the goal of a fully automated device which will be able to predict with high accuracy changes in glucose profiles and respond accordingly with stringent modulation of infusion of hormones (e.g., insulin, glucagon, amylin) to maintain glycemia within normal ranges.   

 

Implantation of Encapsulated Islets

 

Some of the limitations of islet transplantation currently include the limited availability of donors and the need for long term immunosuppression to prevent rejection of the transplanted graft. Protecting the islets from the immunologic environment may allow both the use of non-human islets for transplantation and minimize or eliminate the need for systemic immunosuppression. Thus, the encapsulation of islets to attain these goals has been sought for several years but unfortunately this technology is still not at the stage to make it to the clinical arena. Although initial attempts at encapsulation of islets resulted in damage of the capsule by local tissue responses, newer techniques allowing for conformal coating of human islets have shown promising results in pre-clinical models and are currently being explored (106).

 

Islet Xenotransplantation

 

An alternative to human pancreas and islet transplantation which is currently being explored is the use of pig islets. Pig islets have major physiologic similarities to human islets. Notably, pig insulin differs from human insulin by only one amino acid. Donor pigs may be genetically engineered to be protected from the human immune system thus reducing the need for potent immunosuppression. Studies in non-human primates using encapsulated pig islets have resulted in graft survival for more than 6 months (107). Research in this field in actively ongoing.

Stem Cell Based Therapies

 

Stem cell research has allowed the generation of insulin-producing pancreatic β-cells from human pluripotent stem cells (108). Further, scientists can now also generate alpha and delta cells from stem cells therefore more closely mimicking a fully functional human islet. This technology has the potential to generate vast amounts of glucose-responsive β-cells and allow for the development of customizable islets containing predetermined amounts of specific cell lines. Results in preclinical models are encouraging and a clinical trial is expected in 2021.

Glucose Responsive Insulins (Smart Insulins)

 

Another area of ongoing research is the development of “smart” drug delivery systems able to respond to environmental or external triggers greatly improving therapeutic performance. Conceptually, “smart” insulins should be able to respond to changes in ambient glucose which would dictate activation or cessation of insulin delivery.  Several efforts have been made to generate glucose-responsive insulin delivery systems and some have shown promising results in pre-clinical studies including the utilization of enzymatic triggers, glucose-binding proteins, and synthetic molecules able to bind to glucose. However, current limitations include the potential for immunogenicity and poor glucose selectivity (109). Continued progress in this field in the coming years to reduce the burden of diabetes is anticipated. 

 

CONCLUSIONS

 

No disease has had such an evolution of therapy in the past 100 years as T1D. From certain death to the discovery of insulin, from impure animal insulin preparations to purified human insulins, from once daily long-acting insulin to CSII, from urine glucose testing to real-time continuous glucose sensors and closed loop insulin pumps, treatments continue to emerge that improve the lives of people with T1D. Our current challenges remain teaching the providers how to best use these new tools, directing our medical systems to allow us to best utilize these therapies, and perhaps most importantly, transferring diabetes technologies to the patients who can best apply them. Although the future is exciting, we need to continually master the use of our current tools before we can successfully move forward. Hopefully, soon the successful management of T1D will become a reality for all with this disease.

 

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Endocrinology of The Gut and the Regulation of Body Weight and Metabolism

ABSTRACT

 

Obesity prevalence continues to increase globally, leading to ill-health and reduced life expectancy in those affected and an urgent need for effective preventative and therapeutic strategies. Until recently obesity was viewed simplistically as an imbalance between energy expenditure and consumption that could be easily corrected by lifestyle changes. However, obesity is now recognized to be a chronic progressive disease, with bodyweight controlled by a complex interplay between the central nervous system, peripheral signals of energy balance from adipose tissue and the gastrointestinal tract, environmental food cues, and a powerful biological drive to defend the highest weight achieved. Currently, bariatric surgery represents the most effective treatment for people with severe obesity, leading to marked sustained weight loss as a consequence of altered eating behavior with improved health and life expectancy. Bariatric surgical procedures were initially envisaged to limit calorie intake by physically restricting food passage and inducing malabsorption. However, it is now clear that the success of bariatric surgery lies rather in the impact of these procedures on the biological regulation of energy homeostasis.  In this review we summarize the complex bi-directional communication system known as the gut-brain axis with special focus on gut hormones, bile acids and gut microbiota. We discuss the impact of obesity, lifestyle interventions and bariatric surgery upon the gut-brain axis. Finally, we discuss the progress being made to pharmacologically mimic the beneficial hormonal milieu of bariatric surgery.

 

INTRODUCTION

 

Obesity, defined as the accumulation of excess adipose tissue that impairs health, is now recognized as a chronic progressive disease. Its prevalence continues to increase unabated (1). Globally in 2016, approximately 39% of the adult population were overweight (1.9 billion) and 13% had obesity (> 650 million) (1). Increased fat deposition is the result of an imbalance between energy expenditure and consumption, which in turn is due to an alteration of the homeostatic and/or hedonic systems that regulate energy homeostasis (2). This simplistic definition does not consider how complex obesity is, being the consequence of interactions between genetic, environmental, dietary, psychological and socio-economic factors (3, 4). Eating behavior is governed by specific brain areas that integrate peripheral signals regarding nutrient intake and energy stores (5).

 

The obese state is a very difficult condition to treat because of the coexistence of low-grade inflammation, dysbiosis, hormonal and neurogenic imbalances (3, 4, 6) (Figure 1). These factors also make major contributions to obesity-related diseases, such as type 2 diabetes (T2D), cardiovascular disease, and some types of cancer, impacting adversely upon health, socio-economic factors and life expectancy (1, 7, 8). Weight loss can improve these co-morbidities and increase life expectancy. However, current treatments that emphasize dietary (especially low-calorie diets) or lifestyle approaches for obesity lack long-term efficacy. A meta-analysis of weight loss clinical trials mediated by lifestyle interventions showed an average weight loss of 5% to 9% in the first 6 months, which back-tracked to 3% to 6% in those studies where 48-month data were available (9). Another review assessing the long-term outcomes of calorie-restricted diets showed that up to two-thirds of dieters regain more weight than they lost during their weight loss programs (10). The data for the impact of anti-obesity medication (AOM) on total weight loss percentage after 1 year are highly variable, ranging from 3% with lorcaserin to 9.4% with phentermine/topiramate (11).

Figure 1. Schematic diagram comparing the simplistic definition of obesity, thought to be the result of an imbalance between energy expenditure and consumption (1), with the very complex physiopathology of the obese state (2). This is the result of genetic, inflammatory, microbiota, endocrine, neurogenic and other factors. This pathophysiological complexity underlies the difficulty in finding effective treatments to combat obesity.

Bariatric/metabolic procedures are currently the most effective treatments for people with severe obesity both in terms of weight loss amount and sustainability and the resolution of complications (12). The mechanisms behind the success of bariatric/metabolic surgeries remain to be fully elucidated but post-surgical changes in gut-derived hormonal peptides, bile acids (BA), gut microbiota, and vagal tone are suggested to be involved (13, 14). Importantly, research studies undertaken in animal models and patients with obesity undergoing bariatric surgery have significantly advanced our understanding of the important interplay between the central nervous system (CNS) and the gastrointestinal (GI) tract in regulating energy and glucose homeostasis. Several brain regions integrate continuous information provided by chemical messengers and neural networks arising from the periphery that reflect nutrient availability, the body's energy status, and play a key role in regulating energy homeostasis. The GI tract is responsible for generating the majority of inputs communicated to the CNS regarding both the quality and quantity of a meal. This complex bi-directional communication system between the GI tract and the CNS has come to be known as the gut-brain axis (4, 15) (Figure 2).

Figure 2. Schematic diagram illustrating the gut-brain axis. The entire gastrointestinal tract (GIT) is responsible for generating multiple signals that inform the central nervous system (CNS) regarding quality and quantity of a meal. Key components include neural signals, gut hormones, bile acids, and gut microbiota.

In this review we will explore the gut-brain axis in detail, focusing on the role of gut hormones, Bas, and gut microbiota. We will concentrate our attention on the perturbations of the gut-brain axis in the obese state, and the compensatory response to weight loss induced by lifestyle interventions. Finally, we will discuss the impact of bariatric surgery upon gut hormones, Bas, and gut microbiota and the evidence supporting a role for these factors in mediating the beneficial weight and metabolic effects of bariatric surgeries.

 

THE PHYSIOLOGY OF BODY WEIGHT REGULATION AND THE GUT—BRAIN AXIS

 

During the majority of human evolution food has been scarce. It is therefore not a surprise that endogenous systems have evolved to prioritize food-seeking behaviours when necessary to ensure adequate nutrition for reproduction and survival.  Neuronal circuits within the brain control energy homeostasis, integrating peripheral signals of energy availability originating from the GI tract, adipose tissue mass, muscle mass, and bone density, together with information from higher cognitive centers and external environmental food cues (16). Upon food consumption, sensory information reflecting nutrient availability is transferred from the GI vagal and/or somatosensory (spinal) afferent fibers to the nucleus tractus solitarius (NTS) that, in turn, are integrated and transferred to several other brain centers, including the hypothalamus (17).

Figure 3. Schematic diagram illustrating the central effects of hormones that control eating. Leptin and insulin are secreted in proportions to body fat mass and decrease appetite by inhibiting neurons that produce the NPY and AgRP, while stimulating melanocortin-producing neurons in the ARC region of the hypothalamus, near the third ventricle of the brain. NPY and AgRP stimulate eating, and melanocortins inhibit eating, via higher-order neurons. Activation of NPY/AgRP-expressing neurons inhibits melanocortin-producing neurons. The gastric hormone acyl-ghrelin stimulates appetite by activating the NPY/AgRP-expressing neurons. Gut hormones released from the GI tract in response to eating, including PYY, inhibit these neurons and thereby suppress appetite and decrease energy intake. Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; CART, cocaine and amphetamine-regulated transcript; NPY, neuropeptide Y, PVN, paraventricular nucleus; PYY, peptide tyrosine-tyrosine 3-36; POMC, pro-opiomelanocortin: Lepr, Leptin receptor; GHSR, Ghrelin receptor, MC3R, Melanocortin 3 receptor, MC4R, Melanocortin 4 receptor, Y1r, NPY receptor; Y2r, NPY/PYY3-36 receptor.

The hypothalamus is the key integrative brain site that governs reciprocal orexigenic and anorexigenic behavioral responses, as well as adaptive metabolic changes in response to alterations in food availability and activity levels (18)(Figure 3). The arcuate (ARC), paraventricular (PVN), ventromedial and dorsomedial nuclei, as well as the lateral hypothalamus, are the most important hypothalamic areas involved in energy homeostasis (19). The ARC responds to peripheral and central signals reflecting nutrient availability and energy expenditure by releasing neurotransmitters from two separate and reciprocally connected neuronal populations: pro-opiomelanocortin (POMC)/cocaine-and-amphetamine-regulated transcript (CART) and neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons. NPY/AgRP neurons are situated in the medial ARC and release AgRP and NPY, which stimulate hunger, appetite, and decrease energy expenditure (17, 19-21). Neighboring POMC and CART-containing neurons located in the lateral ARC release α-melanocortin-stimulating hormone (α-MSH) and CART respectively (17). These neurotransmitters are antagonistic of AgRP and NPY and act via the melanocortin-4 receptor (MC4R) to decreased hunger and increased energy expenditure (22) (Figure 3). In addition to vagal signaling, gut hormones can also directly influence these hypothalamic circuits. For example, injected peptide YY (PYY) can inhibit food intake by binding to Y2 receptors localized to the ARC (23) (Figure 3).

Figure 4. Schematic diagram illustrating the mechanisms involved in regulating feeding behavior. Nutrient entry into the GI tract causes gastric and intestinal distension, secretion of pancreatic enzymes and BA, altered enteric and vagal nerve signaling and exposure of EECs to nutrients with altered circulating gut hormone levels (e.g. decrease in orexigenic hormone acyl-ghrelin and increase in anorectic hormones PYY3-36 and GLP-1). Gut-derived signals (nutrients, hormones, and neural) and adipokines (e.g. leptin and others) act directly and indirectly upon the brainstem and hypothalamic areas (see Figure 3 for a detailed description of hypothalamic nuclei controlling energy homeostasis). All of these factors are involved in the regulation of homeostatic hunger. Social factors, emotion, reward, pleasure, increased food availability and sensory cues can influence brain reward and higher cognitive brain regions leading to altered feeding behavior (hedonic influences on hunger and appetite control). Taste and olfactory signals can also influence energy intake acting on both homeostatic and brain reward systems. Insulin leptin, GLP-1, PYY and ghrelin are present in saliva with cognate receptors on taste buds and olfactory neurons. Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; CART, cocaine and amphetamine-regulated transcript; EEC’s, enteroendocrine cells; FGF-19, fibroblast growth factor-19; GLP-1, glucagon-like peptide 1; LHA, lateral hypothalamic area; NPY, neuropeptide Y, peripheral nervous system, PNS; PVN, paraventricular nucleus; PYY, peptide tyrosine-tyrosine 3-36; POMC, pro-opiomelanocortin, sympathetic nervous system, SNS.

Several other brain regions have key roles in energy homeostasis. `The area postrema (AP) is proximally located to NTS and, along with the ARC, are unique in that they have incomplete blood-brain-barriers, thus allowing them to be directly accessed and influenced by blood-borne gut hormones and other circulating factors (4). In animal models, AP lesions have been shown to result in diminished central effect of  several gut hormones (24). Signals from the GI tract also interact with the brain reward systems that constitute dopaminergic neurons located in the ventral tegmental area, nucleus accumbens, and other sites.  These neuronal pathways are thought to mediate effects of exposure to hedonic food cues present in obesogenic environments of Western societies, possibly contributing to the creation of the desire to eat in the absence of an energy requirement in what has been termed “hedonic obesity” (18) (Figure 4).

 

More broadly, the gut-brain axis includes bidirectional communication between the CNS and the enteric nervous system (ENS), autonomic nervous system (ANS) (with sympathetic and parasympathetic branches), neuroendocrine system, gut immune system, BAs, and gut microbiota (25, 26). Both acute and chronic alterations in these systems can arise in response in changes in energy expenditure and consumption (6). Peripheral energy-regulating signals are traditionally classified as long-term signals of energy balance, such as leptin and insulin levels reflective of body adipose stores (“adiposity signals”), and short-term signals, which convey information regarding nutrient and meal-derived energy availability (5) (“satiety signals”), whereas the role of the immune system of the GI tract is still under investigation  (Figure 4).

Figure 5. Schematic diagram illustrating the regulation of an L-cell, one of the several EECs present in the GI tract. Nutrients and their interaction with gut microbiota and BAs in the intestinal lumen activate luminal receptors located on the apical cell membrane, which then activate intracellular metabolism leading to calcium influx to induce the synthesis and release of gut hormones into the sub-epithelial space. Luminal receptors includes receptors for short-chain fatty acids (SCFAs) (e.g., GPR41, GPR43), long chain fatty acids (e.g., GPR40, GPR120), proteolytic products (e.g., CaSR) and BAs (e.g., TGR5). Various gut-derived hormones glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and oxyntomodulin (OXM) are synthesized, secreted and released from L-cells systemically to induce an effect on various tissues throughout the body such as the brain. These hormones can act systemically or cooperate with the enterocytes via local paracrine action. Their systemic effects could be endocrine but also neural through the activation of afferent neurons located in the GI wall. The secretion of gut hormones can be stimulated, in turn, by circulating hormones and glucose or by stimulation from enteric nerves.

Most of these communications related to energy homeostasis involve hormonal and neural signals, which are quite substantial given that the GI tract releases more than 100 different hormonally active peptides (27) and contains approximately 500 million neurons (28). In response to nutrient ingestion, stretching of stomach mechanoreceptors generates the first ENS feedback signals to the CNS mediating meal cessation (4, 29). Subsequently, digested luminal nutrients come in contact with the microvilli of apical cell membranes of enteroendocrine cells (EECs) located in the epithelium of the GI tract to stimulate gut hormone release. Therefore, the majority of signaling and communication within the gut-brain axis is initiated in response to pre-absorptive nutrients (15) (Figure 5 illustrates in detail the biology of an L-cell as a model of an EEC). Following gut hormone receptor activation, nutrient-derived signals exert local control over intestinal function and are conveyed directly and indirectly to the brain via vagal and spinal afferents (30-32) (Figure 5).

 

Digestion and absorption take place predominantly in the stomach and small intestine where dense innervation originating from splanchnic and vagal nerves transmit neurological signals generated during the process of nutrient sensing (29). Additional roles of the ENS include regulation of gastric emptying by vagal activation (17, 33) and mediation of GI endocrine signaling via vagal nerve afferents that project into the lamina propria of the gut ending at the basolateral cell membrane of EECs. It is through these nutrient-specific sensory signals that the GI tract informs the CNS about a meal’s energy and macronutrient content (34).

 

LINKING THE GUT AND BRAIN: GUT HORMONES, BILE ACIDS AND THE GUT MICROBIOTA

 

Gut Hormones

 

As previously described, the gut-brain neuronal-signaling axis is initiated by nutrient-induced gut hormone secretion (32). EECs distributed throughout the entire GI tract length respond to luminal nutrients and release a panoply of gut hormones that act as endocrine, autocrine and paracrine regulators of energy and glucose homeostasis (35) (Figure 5). Although nutrient ingestion triggers the secretion of numerous gut hormones, in this review we will focus on glucagon-like peptide 1 (GLP-1), oxyntomodulin (OXM), and PYY (all of which are released from L-cells and have central appetite-suppressing effects (35, 36)), pancreatic polypeptide (PP), cholecystokinin (CCK), ghrelin, and anandamide. 

 

The enteroendocrine L-cells responsible for secreting PYY, GLP-1 and OXM reside throughout the GI tract with the highest concentrations in the ileum and colon. In response to nutrient intake, circulating PYY, GLP-1 and OXM  levels show a biphasic increase with an initial early peak within 15 minutes and then a later peak around 90 minutes post-ingestion (4, 37, 38). The early increase is thought to be mediated by neural (vagal) and/or hormonal mechanisms whilst the later peak, which is in proportion to the energy intake, is thought to result from the direct contact of nutrients with L-cells located in the distal small intestine and large intestine (39).

 

GLUCAGON-LIKE PEPTIDE 1

 

Glucagon-like peptide 1 results from post-translational processing of the preproglucagon gene (37, 40). Glucagon-like peptide 1 exerts its metabolic effects by activating the GLP-1 receptor, which is widely expressed in the GI tract, pancreas, and CNS (41, 42). Actions mediated by the GLP-1 receptor include enhancing glucose-dependent insulin release (incretin effect) (43), inhibition of glucagon secretion (44), and stimulation of  satiety centers in the ARC, NTS and AP leading to decreased hunger and increased satiation (45). In addition, GLP-1 limits energy intake by reducing the rate of gastric emptying, which in turn increases gastric distension (46). As a result of these functions, GLP-1 receptor agonists are currently used for the treatment of both T2D and obesity (47).

 

Glucagon-like peptide 1 is rapidly inactivated by the enzyme dipeptidyl peptidase-IV (DPP-IV) with only about 10% of GLP-1 reaching the systemic circulation (48-50). Thus, GLP-1 is thought to mainly act in a paracrine fashion. The vagus nerve is particularly important for the action of GLP-1 as demonstrated by vagotomy studies altering the effects of this hormone (51). Glucagon-like peptide 1 also stimulates the brainstem to enhance motor output and/or thermogenesis (52).

 

OXYNTOMODULIN

 

Like GLP-1, OXM is synthesized by post-translational processing of the preproglucagon gene (53, 54). Other similarities of OXM to GLP-1 include binding to GLP-1 receptors within the GI tract, the pancreas, and the ARC; subsequent reductions in gastric acid secretion, blood glucose concentrations and food intake (53, 55, 56); and degradation by DPP-IV (57). In addition, OXM administration enhances satiety and increases energy expenditure in both animal models and humans (53, 54).

 

PEPTIDE YY

 

Circulating levels of PYY are low in the fasted state (58, 59) and increase during nutrient ingestion in proportion to the caloric content (60), exhibit differential responses according to the specific macronutrient composition of the meal (36, 59), and remain elevated for several hours after a meal with sustained endocrine effects (61).

 

Peptide YY circulates in two native forms: PYY1−36 (minor form) and PYY3−36 (major form) (36, 59).  Peptide YY3−36 results from the N-terminal cleavage of PYY1−36 by the enzyme DPP-IV (59). Interestingly, PYY1−36 and PYY3−36 have divergent actions on appetite, glucose homeostasis and differential binding affinities of each form for the five neuropeptide Y receptor (YR) subtypes (59). Y2 receptors are located on the vagus nerve, in the NTS and in the ARC (23, 36, 60). Peptide YY1−36 has equivalent affinities for Y1R and Y2R, whereas PYY3−36 is a high-affinity ligand for Y2R (59).  By binding to the Y2 receptor, PYY3-36 decreases energy intake by inhibiting the orexigenic effects of NPY neurons and activating the anorexigenic POMC neurons in the ARC (4, 20, 62) (Figure 3), physiological effects supported by studies showing that PYY knockout mice become hyperphagic and obese (36, 60). Increased PYY levels have been associated with prolonged appetite loss and food aversion during exogenous administration and following bariatric surgery (6, 63-65).

 

PANCREATIC POLYPEPTIDE

 

Pancreatic polypeptide is secreted by specialized F-cells located in the islets of Langerhans (66) during the pre-absorptive (cephalic) phase of nutrient metabolism and for up to 6 hours post-prandially in proportion to energy intake (67, 68). Pancreatic polypeptide acts centrally upon the Y4 receptor within the AP, NTS, and the ARC, reducing energy intake. Peripherally, it induces gallbladder relaxation, inhibits pancreatic secretion and delays gastric emptying thus inducing satiety (69, 70). Pancreatic polypeptide is a potent appetite suppressant (71) and studies have demonstrated a difference in PP concentrations in anorexic and obesogenic states, where it is increased and diminished respectively (61). Moreover, studies in people with Prader-Willi syndrome and obesity suggest that circulating post-prandial PP levels are reduced in comparison to healthy individuals. Furthermore, intravenous PP injection to these patients led to a significant decline in energy intake (72).

 

CHOLECYSTOKININ

 

Cholecystokinin is secreted from EECs located mainly in the duodenum and jejunum (73). Cholecystokinin release is stimulated by fat and protein ingestion and its circulating concentrations increase within 15 minutes after meal ingestion (6, 74). Cholecystokinin has a short half-life and it acts upon CCK-1 and CCK-2 receptors located throughout tissues of the GI tract and the CNS, including the vagal nerve, NTS and hypothalamus (6). Cholecystokinin increases gallbladder and GI motility and secretion but also has an active role in controlling food intake, energy expenditure and glucose utilization (17). Cholecystokinin reduces energy intake in a dose-dependent manner and it is a specific mediator of fat and protein satiation (6, 75). It has been suggested that it acts synergistically with leptin and amylin, a pancreatic hormone co-secreted with insulin (17). However, repeated doses can induce tolerance to CCK (6), potentially explaining why attempts to use CCK-derivatives as a medication to induce weight loss have failed (76).

 

GHRELIN

 

Ghrelin is a 28-amino-acid orexigenic hormone and is secreted by P/D1 cells located primarily in the gastric fundus in the absence of nutrient intake, leading to increased appetite and food intake (77). Ghrelin is also produced by the pituitary gland (77) and within the ARC and PVN area of the hypothalamus. Ghrelin is secreted as the inactive des-acyl-ghrelin. The active orexigenic form, acyl-ghrelin, is synthesized by the action of ghrelin O-acyltransferase enzyme (GOAT) (77) and can bind to growth hormone secretagogue receptor (GHS-R) to increase food intake in rodents (77)and humans (77, 78). The extremely complex ghrelin/GOAT/GHS-R system has a crucial role in the regulation of energy and metabolism as well as in the adaptation of energy homeostasis to environmental changes (77).

 

Acyl-ghrelin administration to humans has been used as an orexigenic agent in patients with anorexia that accompanies cachexia (79). A rising circulating ghrelin level precedes nutrient ingestion and decreases rapidly after a meal (78) which has led to the speculation that this is the first discovered “hunger hormone” (80). Plasma levels of ghrelin increase after diet induced weight loss, thought to be part of the body’s homeostatic adaptation response that restores lost weight, and are very high in patients with anorexia nervosa (81). Nutrient intake but not water ingestion is the main regulator of ghrelin leading to a decrease of its plasma levels (82). Peripheral ghrelin exerts its orexigenic actions through the stimulation of NPY/AgRP co-expressing neurons (83).

 

ANANDAMIDE

 

Anandamide and other bioactive lipids belonging to the endocannabinoid system contribute to the gut-brain axis. These molecules are secreted in the GI tract and activate endocannabinoid receptors 1 and 2 (CB1/CB2) (3, 4) which are expressed in the CNS, peripheral nervous system, liver, pancreas, adipose tissue, and immune cells (84). Exogenous cannabinoids convey orexigenic effects and so it is not a surprise that the endocannabinoid regulates gut motility and appetite (3, 84). Endocannabinoid receptor 1 antagonists were used to induce weight-loss in subjects with obesity before being withdrawn for their severe psychological side-effects (3, 85), including increased suicidality (86).

 

Bile Acids

 

Bile acids (BAs) are endogenous steroid molecules synthesized from cholesterol in the liver, stored in the gallbladder and secreted into the duodenum upon nutrient ingestion. These amphipathic molecules facilitate micelle formation, promoting the digestion of dietary fat and fat-soluble vitamins. More recently, BAs have also been shown to play a role in regulating glucose and lipid metabolism and energy expenditure via the activation of BA receptors in the liver, gut, and peripheral tissues (87, 88). Interactions between BAs, their receptors, and the gut microbiota determine synthesis, metabolism, and distribution of bile acids in the body (88).

 

There is complex cross talk between BAs, gut hormones and the microbiome (Figure 6). Bile acids stimulate GLP-1 secretion via activating G protein‐coupled receptors (TGR5) on L-cells and fasting total circulating BAs levels are positively correlated with post-prandial GLP-1 levels (89). TGR5 receptors are also located on skeletal muscle and in brown adipose tissue where they increase energy expenditure by promoting the conversion of inactive thyroxine (T4) into active thyroid hormones (T3) (90). Bile acids have been shown to act on farnesoid X receptors (FXR). During BA binding of FXR on pancreatic β cells, insulin release is increased (91). Bile acid activation of intestinal FXR-containing cells stimulates the secretion of fibroblast growth factor-19 (FGF-19), a protein that contributes to improved peripheral glucose disposal and lipid homeostasis, increased metabolic rate, and reduced weight (92, 93). In animal studies, BA supplementation has been shown to reduce weight gain (90). In humans, postprandial BA levels are also inversely related with body fat mass (94). Thus, the physiologic effects of BA likely extend beyond the gut and pancreas to include actions that improve body weight and glucolipid metabolism.

Figure 6. Schematic diagram illustrating the complex cross talk between BAs, gut hormones, FGF-19, and the microbiome. BAs are important regulators of energy balance and glucose metabolism, primarily via the FXR and the TGR5. The trans-intestinal BAs flux activates intestinal FXR, inducing synthesis and secretion into the circulation of the ileal-derived enterokine FGF-19. FGF-19 can improve glucose tolerance by regulating insulin-independent glucose efflux and hepatic glucose production. FGF-19 can also increase energy expenditure with its central and peripheral effect in the adipose tissue. BAs acting via TGR5 stimulate L-cell secretion of GLP-1 (and PYY) then enhancing insulin secretion acting on β-cells. TGR5 activation in muscle and brown adipose tissue promotes the conversion of inactive thyroxine into active thyroid hormone inducing thermogenesis. BAs can reduce food intake centrally through FGF-19 and anorectic gut hormones. BAs also regulate gut microbiota composition. BAs are actively reabsorbed from the terminal ileum and returned via the portal circulation to the liver. A small percentage of BAs are deconjugated by gut bacteria, forming secondary BAs, which are reabsorbed or excreted in feces. Red dotted lines: FGF-19 effects. Blue dotted lines: IGF-1 effects. Green lines: BAs circulation.

The Gut Microbiota

 

The healthy human gut hosts trillions of microorganisms with a ratio of bacterial-to-human cells of 1.3:1, comprising a complex ecosystem referred to as the gut microbiota (95, 96).  These microbes exist within a symbiotic relationship with their human host, who provides a nutrient-rich environment. The microbiota, in turn, provides metabolic processing of these nutrients that the host's genome does not possess (97, 98). With more than a thousand different bacterial species, the diversity and function of the microbiota is dynamic depending on the host’s diet, antibiotic exposure and other environmental factors (98, 99).

 

The extent of the symbiotic relationship between the host and the microbiota is highlighted by studies showing that mice lacking a microbiota (germ-free) have reduced adiposity, energy intake, and energy extraction from a standard rodent diet compared to conventionally-raised mice (100). More recent studies including germ-free rats transplanted with the microbiota of either obesity-prone or obesity-resistant rats confirmed the importance of the microbiome for the production of enzymes involved in energy harvesting from indigested carbohydrates (75). Both the Westernized-diet and obesity fecal transplant models are associated with an increased ratio of bacteria belonging to the Firmicutes phylum compared to the Bacteroidetes phylum, which is reversed upon surgical and dietary interventions (29, 101, 102).

 

Gut microbiota have been demonstrated to affect adiposity and weight-gain through several pathways.  A typical Western diet contains indigestible carbohydrates, such as resistant starches and plant cell wall polysaccharides that are hydrolyzed by gut microbiota generating small-chain fatty acids (SCFA) (3). Short chain fatty acids in the form of butyrate, acetate, and propionate provide approximately 10% of the host's daily energy requirements (103). In the obesogenic state, feces contain an increased quantity of SCFA, especially propionate (103). Short-chain fatty acids are not always correlated with increased weight-gain as some may possess beneficial properties (4, 29). In animal models, prebiotics (indigestible compounds that can modulate the composition and activity of the gut microbiota) and oral or intestinal SCFA infusions lead to a reduction in food consumption and a decrease in body weight. This occurs when prebiotics and supplements promote the growth of favorable microbial species or when SCFA activates signaling pathways that ultimately increase gut hormone synthesis (104-106). For example, SCFA’s have also been shown to activate free fatty-acid receptors 2 and 3 (FFAR2/FFAR3) in the GI tract, immune cells, liver and adipose tissue (107). Intestinal FFAR2/FFAR3 receptors are expressed by EECs, in particular, L-cells and, when activated, can facilitate release of gut hormones, such as GLP-1 and PYY (107). Moreover, FFAR3 expressed within the ENS and ANS can stimulate the sympathetic tone in the adipose tissue regulating fat storage as well as glucose utilization in muscle and liver tissues (3, 108). The gut microbiota may also moderate the endocannabinoid tone affecting colonic CB1 expression and anandamide concentrations (3, 109). Finally, gut microbiota can enhance energy expenditure by intracellular thyroid hormone activation via FXR signaling (3, 90, 99), which may help to explain the observation that germ-free mice are resistance to adiposity despite an increased food intake (110).  

 

Obesity is characterized by the presence of chronic low-grade inflammation. In another pathogenic pathway involving the microbiota, high-energy dense diets can lead to obesity and obesity-related diseases through changes in bacterial species composition that favors an increase in systemic lipopolysaccharide (LPS) concentrations (111, 112). Lipopolysaccharide is the pro-inflammatory component located in the cell wall of gram-negative bacteria that can enter the circulation when the permeability of intestinal epithelium is altered in a process called metabolic endotoxemia (111, 112).  Leakage of LPS into the systemic circulation is a proposed trigger of a cascade of pro-inflammatory events in adipose tissue and throughout the whole body  mediated by LPS stimulation of the toll-like receptor 4 (TLR4), which enhances the synthesis of inflammatory cytokines linked with reduced host insulin sensitivity (113, 114). Lipopolysaccharide can also inhibit the interstitial cells of Cajal, which are responsible for smooth muscle contraction in the gut and regulation of the ENS. This inhibition has been associated with disorders of both GI motility and gut hormones (4, 115).

 

The gut microbiota may also directly influence CNS-mediated stress and anxiety behaviours and the regulation of energy homeostasis (4).  For example, germ-free mice have been found to have a resistance to adiposity despite an increased food intake (127). Germ-free mice have been found to have higher gene expression of food intake-regulating peptides like GLP1 and OXM within the brainstem and hypothalamus, when compared to normally-reared mice (116).   On the other hand, a reduced leptin-mediated suppression of orexigenic peptides NPY and AgRP in the conventionally-raised mice has been noted, suggesting how the gut-microbiota could directly affect energy homeostasis leading to an increase in adiposity (116, 117).  Further studies are needed, however, to understand if specific manipulations of the gut microbiota phenotype could be used as obesity treatments (Figure 7).

Figure 7: Schematic diagram illustrating the possible causative links between an altered gut microbiota and obesity. The hydroxylation of indigestible carbohydrates and the altered intestinal permeability could lead to increased energy harvest and weight gain. The production of certain types of SCFA can reduce the sympathetic tone favoring fat accumulation in the adipose tissue and dysregulation of glucose utilization in muscles and the liver. Gut microbiota can induce a leakage of LPS into the systemic circulation (endotoxemia) and chronic low-grade inflammation. This is in turn responsible of insulin resistance and weight gain. The increased endocannabinoid tone could induce food intake. SCFAs, inflammation and BAs perturbations may all lead to a reduced activation of anorectic pathways. All these mechanisms could be responsible for weight gain, inflammation and obesity-related comorbidities. Abbreviations: FGF-19, fibroblast growth factor-19; GLP-1, glucagon-like peptide 1; PYY3-36, peptide tyrosine-tyrosine 3-36; SCFA; short chain fatty acid; LPS, Lipopolysaccharide; FFAR2 and FFAR3, free fatty-acid receptors 2 and 3; T2D, type 2 diabetes.

THE COMPLEXITIES OF ENERGY HOMEOSTASIS

 

The Physiology of Weight Regulation

 

As mentioned above (Figure 3), the adipokine leptin acts as a signal of long-term energy availability (fat mass), promoting satiety via its inhibitory action on orexigenic neurons located in the ARC of the hypothalamus (118). A recent report of a patient with leptin deficiency highlights key interactions between leptin and gut hormones. Leptin supplementation resulted in significant rises in meal-stimulated insulin, GLP-1, and PYY levels (61). In the same study, ghrelin levels were decreased, highlighting the regulatory effect of leptin on ghrelin secretion and the interplay between leptin, GLP-1 and PYY.

 

An important physiologic insight that has implications for pharmacological weight management is that gut hormones act synergistically. For example, GLP-1 and PYY in combination are more potent in reducing energy intake compared to either of the two hormones alone (119, 120). Oxyntomodulin, CCK and other gut hormones also act synergistically with GLP-1 to enhance its effects on appetite behaviours (120-123). An additional layer of complexity is added when considering that MC4Rs have been localized on L- and P/D1-cells and could, in turn, regulate GLP-1, PYY and ghrelin secretion (124). Furthermore, gut hormones influence energy homeostasis through interactions with the microbiome and BAs.

 

Hedonic factors can generate meaningful physiological responses that interact with homeostatic signals of energy availability in the regulation of body weight. This could lead to excess energy intake with possible weight gain. In humans, brain functional imaging studies, have shown that several gut hormones modulate neural activity in brain reward regions altering the reward value of food (2, 58, 125) by food cues, memory and social factors, and strongly influencing eating behavior (18). Exposure to food-related stimuli can induce changes in circulating gut hormone levels. Those in turn act upon brain reward pathways, either decreasing in the case of PYY or, increasing in the case of ghrelin, the reward value of food (23, 126).  Those hormones are also present in saliva and their cognate receptors are present on taste buds and the olfactory bulb (127, 128).  The taste and smell of food are key influencers of food selection with impacts on energy intake (127).

 

Adding to the complexity of the gut-brain regulation of body weight, studies have demonstrated that energy expenditure can increase without changes in activity but through the action of gut-derived neurohumoral signals that increase thermogenesis and basal metabolic rate (4). The existence of the gut-brain-brown adipose tissue axis has been hypothesized after studies showing how intestinal lipid-sensing activates vagal afferent fibers to enhance brown adipose tissue thermogenesis through a CCK-dependent pathway (129). Table 1 summarizes key gut hormone actions including their perturbations in the obese state and the changes induced by bariatric surgery procedures.

 

Table 1. GI Tract Hormones Involved in the Control of Energy Balance and Changes in their Circulating Levels Induced by Obesity and Bariatric Surgery

Hormone

EEC (Type)

Location

Receptor

Food

Intake

Other Effects

Obese State

Bariatric

Surgery

PYY

Ileum

(L cell)

Y2-R

↓ Gastric acid secretion and emptying

↓ Pancreatic and intestinal secretion

↓ Gastrointestinal motility

↑ Insulin secretion and vagus stimulation

GLP-1

Ileum

(L cell)

GLP-1R

↑ Insulin secretion

↑ β-cell proliferation and gene expression

↓ β-cell Apoptosis

↓ Gastric acid secretion and emptying

Ghrelin

Stomach

(P/D1cell)

GHS-R

↑ Growth-hormone secretion

↑ Gastric acid secretion and emptying

↑ Vasodilatation

↓ Insulin secretion

CCK

Duodenum, jejunum and pancreas

(I/L cell)

CCK 1, 2

↓ Gastric emptying

↑ Pancreatic secretion

↑ Gallbladder contraction

?

PP

Pancreas

(F-cell)

Y4, Y5

↓ Gastric emptying

↓ Leptin levels

↑ Insulin secretion

↓ β-cell Apoptosis

GIP

Duodenum and

jejunum

(K-cell)

GIP-R

?

↑ Insulin secretion and β-cell proliferation

↓ β-cell Apoptosis

↑ Lipoprotein lipase activity and fat deposition

↑ Fatty acid synthesis

↓?

OXM

Ileum

(L cell)

GLP-1R

↓ Gastric emptying and acid secretion

↓ Blood glucose

↑ Insulin secretion

↑ Energy expenditure

↑?

Glucagon

Pancreas

(α-cell)

GCGR

↑ Energy expenditure

↑ Blood glucose

?

Amylin

Pancreas

(β-cell)

AMY1-3

↓ Gastric emptying and acid secretion

↓ Postprandial glucagon secretion

↓ Glucose elevation

Insulin

Pancreas

(β-cell)

Insulin receptor

↑ Absorption

↑ Glycogen synthesis

↓ Blood glucose

↑ Lipid synthesis

↓ Lipolysis and proteolysis

Leptin

Adipose Tissue and gastric EECs

Leptin (Ob-R)

↓Glucose production and steatosis in the liver

↑ Glucose uptake and fatty acid oxidation in muscles

↓ Insulin and glucagon secretion

↑ Sympathetic nervous system tone

↑Thyroid hormones

Modulates immunity and fertility

FGF-19

Ileum

(FXR activation from BA)

FGFR 1, 2, 3, 4

Regulates glucose and lipid metabolism,

↑Hepatic protein and glycogen synthesis

↑ energy expenditure

NT

Jejunum

(L-cell)

NTR1, NTR2, NTR3

↓ Reduces GI motility and gastric secretion,

↑ Pancreatic and biliary secretion

?

↑?

Abbreviations: FGF-19, fibroblast growth factor-19; GLP-1, glucagon-like peptide 1; PYY3-36, peptide tyrosine-tyrosine 3-36; PP, pancreatic polypeptide, GIP, gastric inhibitory polypeptide; OXM, Oxyntomodulin; BA, Bile Acids: NT, Neurotensin. ? = effect not certain or not valid for every bariatric procedure.

 

The Obese State: Pathophysiologic Changes

 

Obesity is the result of a period of uncompensated chronic positive energy balance (130) when energy intake exceeds energy requirements. Dysregulation of the metabolic mechanisms controlling energy homeostasis includes an impaired gut hormone secretion response to nutrient ingestion (131). People with obesity have reduced circulating baseline and meal-stimulated levels of PYY and GLP-1 levels compared to lean subjects (131-133).  Lower circulating ghrelin levels and a reduced suppression of circulation ghrelin levels after nutrient intake have been demonstrated in people with obesity, suggesting that dynamic changes more than absolute values are important in appetite regulation (7, 40). Animal models with diet-induced obesity show reduced circulating concentrations together with impaired circadian secretion profiles of PYY and GLP-1 (134), in addition to an increase in ghrelin-producing cells (135). However, reduced diurnal variability in circulating ghrelin is thought to contribute to the lack of a regular meal pattern and the frequent snacking behavior often observed in individuals with obesity (77, 136).

 

The directionality of the association between obesity and altered gut hormone profile remains to be fully elucidated. For example, high energy intake per se may affect gut hormone responsiveness to ingested nutrients. Moreover, intestinal EEC population differentiation and responsivity is reduced in people with obesity, which may underlie their blunted gut hormone secretion (137). Obesity has also been shown to blunt the rise in circulating post-prandial BAs levels (33). Paradoxically, most studies have found increased total BAs levels in subjects with obesity suggesting that BAs composition could shift unfavorably with detrimental metabolic effects (138). Interestingly, while obesity is thought to be due to resistance to the effects of insulin and leptin within key weight regulatory centers in the hypothalamus, sensitivity to the effects of PYY, GLP-1 and OXM during exogenous administration is preserved, suggesting these hormones and their receptor systems offer a viable therapeutic target for the treatment of obesity (139).

 

As detailed in the previous section, a dysbiotic relationship between host and gut microbiota has been suggested to contribute to the development of obesity (140), with profound differences found between the composition of the gut microbiota of obese and lean individuals (141). Obesity is associated with the relative increase or reduction of certain bacterial species and the importance of the relative proportions of those species remains an area of active investigation. Transplantation of gut bacteria from obese mice to normal-weight germ-free mice results in weight gain in the recipients (142). Conversely, fecal transplantation from lean human donors to recipient patients with metabolic syndrome led to improvements in insulin sensitivity (143). A dysbiotic relationship may affect host energy and nutrient metabolism by altering intestinal mucosal permeability, and promoting increased fat storage in adipose tissue (110). The mechanism for this could be by enhancing the absorption of SCFA derived by otherwise indigestible luminal polysaccharides and by triggering inflammatory responses through a process referred to as “metabolic endotoxemia” (144, 145) (Figure 7). Altered neural responses to food cues in people with obesity compared to people with normal weight have been confirmed by brain-imaging studies showing an increased stimulation of central reward pathways in response to eating or food cues (2). In addition, there is evidence that eating behavior in people with obesity becomes dissociated from perceptions of satiety and hunger (146, 147). Furthermore, in the obese state, enhanced endocannabinoid tone, CB1 expression, and plasma and adipose tissue endocannabinoid concentrations coexist (3). All these complex pathophysiological changes create an internal environment conducive to expression of unwanted weight gain, maintenance of the obese state, and resistance to diet-induced weight loss, providing an explanation as to why treating people with obesity can be challenging (Figure 7).

 

BARIATRIC/METABOLIC SURGERY

 

Bariatric/metabolic surgery is recognized as the most effective weight loss treatment for people with severe obesity (148).  Procedures with the best outcomes involve surgical modifications of the anatomy of the GI tract that alter nutrient flow, thus affecting GI tract biology (83). Many clinical trials have demonstrated the superiority of bariatric surgery in terms of sustainability of weight loss and resolution of obesity-related comorbidities, especially diabetes, when compared with intensive medical interventions (12, 149, 150). Mechanisms other than restriction and/or malabsorption are responsible for this superiority and this has resulted in a marked increase in the number of procedures undertaken worldwide (151). Currently, the most commonly performed bariatric/metabolic procedures globally are sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB), whereas purely restrictive procedures, like gastric banding, are now performed less frequently (151) (Figure 8). However, post-operative weight loss can be highly variable (152), an important consideration total amount of weight loss plays a major role in determining post-operative remission of comorbidities (153).

Figure 8. Schematic diagram illustrating the normal upper GI anatomy (a) and the two most commonly performed bariatric surgical procedures. The metabolic procedures: (b) RYGB and (c) SG (Refer to the main text for a detailed description of surgical techniques). Abbreviations: RYGB, Roux-en-Y gastric bypass, SG, Sleeve gastrectomy.

Roux-en-Y gastric bypass involves division of the stomach into two parts, generating a small gastric pouch (20-30 mL), which is then anastomosed with the mid-jejunum, creating the alimentary limb or Roux limb. Nutrients bypass most of the stomach, duodenum, and the proximal jejunum. In the common limb, after the anastomosis of the biliopancreatic limb with the jejunum, nutrients, BAs and pancreatic secretions mix and the absorption of nutrients occurs (154). In a SG, a transection along the greater curvature is performed, removing the fundus and body and creating a tube-like stomach (155). The transit of gastric contents into the duodenum is rapid. The SG was initially performed as a first-stage procedure followed by a second more invasive malabsorptive step (biliopancreatic diversion), but the significant weight loss results observed with this procedure led to its adoption as a standalone approach. Because it is a simpler operation compared with RYGB and has fewer complications with similar short-term weight-loss, SG have become the most common bariatric procedure worldwide (151).

 

Biological Changes Favoring Sustained Weight Loss and Metabolic Improvement Following Bariatric/Metabolic Surgery

 

A negative energy balance is a key component of many lifestyle interventions. Unfortunately, weight regain is very common after initial weight loss. Multiple powerful adaptive biological changes occur in response to weight loss from lifestyle alone that lead to increased hunger, enhanced neural responses to food cues and heightened drive to consume energy-dense foods. These include decreased total energy expenditure secondary to reduced lean muscle mass, sympathetic activity (156), circulating leptin, GLP-1 and PYY levels, along with increased ghrelin levels (147). Other changes following lifestyle-induced weight loss that have been described and may contribute to weight recidivism include impaired circulating BAs levels, an altered gut microbiome, and decreased vagal signal transmission (10, 157).

Figure 9. Schematic diagram illustrating the different biological changes induced by weight loss achieved through dieting (upper part) compared to bariatric/metabolic surgery (lower part). Powerful compensatory biological changes contribute to the high rate of weight recidivism observed following lifestyle intervention weight management. Many homeostatic mechanisms act to restore a higher body weight and these includes hormonal alterations and a decreased energy expenditure leading to increased hunger and energy consumption. By contrast, bariatric surgery leads to a favorable biology that includes increased satiety hormones, reduced ghrelin, enhanced BA secretion and a “lean” microbiota. Together these mechanisms lead to reduced hunger and a shift towards healthier food options with a resetting of body weight “set point” to a lower level facilitating meaningful and sustained weight loss. References for this figure: (149, 158, 159). Abbreviations: GLP-1, glucagon-like peptide 1; PYY3-36; peptide tyrosine-tyrosine 3-36. *Suggestion that leptin sensitivity may improve

Weight loss following RYGB and SG are the result of multifactorial mechanisms and not from malabsorption or restricted stomach size alone (160, 161) (Figures 9 and 10). Reduced energy intake, as a result of altered eating behavior, is recognized as the main driver for weight loss following these procedures, and increased exposure of EECs to ingested nutrients is thought to play a key mediating role in the expression of these appetitive behaviours (83) (Table 1 and Figure 10). In contrast to lifestyle approaches to weight loss, favorable changes in these behaviours following bariatric/metabolic procedures include reduced hunger and neural responsiveness to food cues. Multiple studies have shown that bariatric surgery causes marked elevations in nutrient-stimulated levels of several anorectic hormones including PYY and GLP-1, along with decreased ghrelin levels, which have been reported post-RYGB but are more pronounced post-SG (162, 163). Following RYGB, increased nutrient-stimulated circulating levels of PYY and GLP-1 are most likely as a result of increased nutrient stimulation of L-cells as a consequence of anatomical rearrangement. Sleeve gastrectomy leads to rapid gastric emptying and enhanced exposure of L-cells to nutrients with increased nutrient-stimulated PYY and GLP-1 levels, but to a lesser extent than following RYGB. Sleeve gastrectomy leads to sustained and greater reduction in circulating acyl-ghrelin levels than RYGB because of the removal of the fundus of the stomach where most ghrelin-producing cells are located (164). These changes are present immediately after surgery and sustained up to 10 years post-operatively (165, 166). Oxyntomodulin levels are increased after RYGB (167) and a rise in CCK levels has been demonstrated following both RYGB and SG (163). Emerging evidence also suggest that the number of EECs changes after bariatric surgery. The total numbers of EECs in the stomach and duodenum of people with obesity are reduced when compared to lean individuals (31) and this has been found to normalize 3 months post-SG (158).

Figure 10. Schematic diagram illustrating RYGB and SG and the mechanisms leading to weight loss and resolution of comorbidities. For every mechanism the effect of the procedure is represented with a “” when stimulating or “” when suppressing. A “+” means that the proposed mechanism is present only after surgery when compared to the pre-operative period. When the effect is stronger for one of the two procedures there is a double arrow compared with a single one. When the effect is missing for one procedure it means that the mechanism is procedure specific. Abbreviations: RYGB, Roux-en-Y gastric bypass; SG, Sleeve gastrectomy; GLP-1, glucagon-like peptide 1; PYY3-36; peptide tyrosine-tyrosine 3-36; GIP, gastric inhibitory polypeptide; FGF-19, fibroblast growth factor-19, CCK; cholecystokinin.

Variability in EEC secretion response may underlie differences in weight loss responses to bariatric/metabolic procedures. Profound anorexia and excessive weight loss post-SG have been associated with markedly elevated circulating fasted and post-meal PYY levels (65). Patients with poor weight loss after surgery have been found to have increased appetite coupled with lower meal-stimulated GLP-1 and PYY and higher ghrelin levels when compared with good responders (168). Additional support for the importance of EEC in weight loss responsiveness in the post-op period comes from data showing that administration of octreotide (a general inhibitor of EEC secretion), or selectively blocking GLP-1 and PYY, promotes appetite and weight gain (14, 65, 169).

 

Following SG and RYGB, food becomes less rewarding and there is a shift in preference from energy dense food rich in fat and sugar to healthier options enabling patients to adopt more favorable eating behaviours (158). These changes in eating behavior are the result of multiple mechanisms, some of which are common to both SG and RYGB and others that are procedure specific (Figure 10).

 

Studies of the physiological changes following bariatric/metabolic have also elucidated novel effectors of changes in weight and metabolism, many of which are gut-related and discussed above. For example, following RYGB and SG, changes in circulating BAs levels and composition are reported that may contribute to weight loss and improved glucose metabolism. Despite their anatomical differences, RYGB and SG exert similar effects on BA composition and circulating concentrations, although the changes observed following SG are more modest (87, 170). The exact mechanism responsible for elevated BAs following RYGB and SG is unclear, but animal studies suggests that an accelerated nutrient flow to the distal small intestine is a key mechanism (171). Indeed, in animal models, rerouting bile to the distal small bowel by transposing the common bile duct increases plasma BA levels similarly those seen after RYGB and results in weight loss, improved glucose metabolism, and reduced hepatic steatosis. The rise in circulating BAs appears even greater several months post-operation and may be due to intestinal cellular adaptations (172), increased hepatic synthesis, altered enterohepatic recirculation of bile, or a combination of these possibilities. Post-surgically increased BAs diversity might also impact on GLP-1 secretion and energy expenditure.  Binding of BAs to TGR5 receptors in skeletal muscle and brown adipose tissue may contribute to enhanced action of thyroid hormones, thereby increasing energy expenditure (173). Therefore, BAs could contribute to weight loss and metabolic improvements after bariatric surgery through direct and indirect regulatory mechanisms.

 

Weight‐loss surgery can also affect the interplay between BAs and gut microbiota, which can have favorable metabolic effects in the post-operative period (174) (175). For example, in RYGB subjects, bacterial overgrowth in the biliopancreatic limb may generate secondary BAs species with altered affinity for FXR or TGR5 receptors (176). In rodent models, the importance of the FXR receptor in mediating weight loss and metabolic improvements after SG was demonstrated when FXR knockout mice regained lost weight following this procedure (173), although whether FXR signaling and/or FGF-19 contributes to the beneficial effects of bariatric surgery in humans is uncertain at present. Finally, a study that measured serum BAs levels before and after bariatric surgery showed that they were significantly increased only at one-year post‐surgery, whereas, the substantial increase in PYY and GLP-1 levels could be observed as soon as 1-week post-surgery. This finding suggest that increased plasma BAs may be less important in early metabolic improvements observed after bariatric surgery (170) but more so for long-term effects.

 

Alterations in intestinal microbiome following RYGB and SG have been described. Animal studies of fecal transplants from RYGB-treated to germ-free mice showed significantly greater weight loss in the germ-free mice, suggesting that the altered microbiome per se contributes to weight loss (177). RYGB can produce greater and more favorable changes in gut microbiota functional capacity and species than SG despite similar weight loss (178) (179, 180). Although the specific procedure-related mechanisms responsible for post-surgery gut microbiota changes remain to be delineated (181), potential explanations include differences in the physical manipulation of the GI tract and final anatomy, dietary changes, and weight loss differences between procedures. In rodents, these changes can be detected as early as 7 days after RYGB (175), with similar patterns observed in humans (102). Because of significant differences between the rodents and the humans, it is not possible to firmly conclude that gut bacteria are essential for the effects of metabolic procedures. However, it is evident in the rodent models that changes in gut microbiota induced by RYGB are sufficient to produce weight loss (174).

 

Other appetite-related post-surgery effects that may influence weight loss include changes in taste and smell that could, in turn, influence food preference (83). Interestingly, early data suggest that RYGB and SG may differently impact subjective changes in appetite, taste, olfaction and food aversion post-operatively (182). Finally, neurophysiological studies suggest that vagal nerve signaling also increases post-RYGB (157) and these changes may affect food intake in a procedure-specific fashion (183, 184).

 

Developing “Knifeless Surgery”

A multitude of compounds mimicking gut hormone actions are currently under development, opening a new era of pharmacotherapy for obesity. At present, GLP-1 analogues are broadly used in the management of people with T2DM and obesity (185). The longer acting GLP-1 analogue semaglutide has shown promising results for weight loss in early phase studies with both a weekly subcutaneous injection (186) and an oral compound form (187, 188). Intravenous administration of supra-physiological levels of native gut hormones like PYY, GLP-1 and others lead to reduced appetite and decreased energy intake (23, 189-191).  Strategies aimed at reducing acyl-ghrelin and/or increasing des-acyl-ghrelin are also being developed and show promise. The inhibition of GOAT has been shown to reduce energy intake and bodyweight (192).

 

In order to mimic bariatric/metabolic procedures, the effects of combinations of hormones are under investigation with the aim of circumventing compensatory adaptive changes associated with energy restriction. For example, the co-infusion of GLP-1, PYY and OXM induced a 32% reduction in energy intake when compared to placebo (193). Animal models suggest a potential role of CCK as an adjunct to GLP-1 based therapies (194) or monomeric GLP-1/GIP/glucagon triagonism to reduce food intake and obesity (195).

 

CONCLUSION

 

Obesity is a complex disease where genetic, environmental, dietary, psychological and socio-economic factors interact complicating treatments for this life-threatening condition. Peripheral signals such as gut hormones, BAs and gut microbiota inform the CNS regarding the quality and the quantity of any ingested meal and are part of the complex bi-directional communication system known as the gut-brain axis. During the recent years many studies have identified perturbations of this system as a cause of weight gain. Current lifestyle approaches to weight loss lack efficacy because multiple powerful adaptive biological responses promote weight regain. Bariatric surgery, which reduces energy intake as a consequence of favorable gut-brain axis signaling, is currently the most successful treatment for people with severe obesity, leading to marked sustained weight loss and improved health.  Understanding the hidden mechanisms responsible for this success is an exciting area of current research and holds promise to identify novel effective obesity pharmacotherapies.

 

ACKNOWLEDGEMENTS

 

The authors would like to thank Chiara Bullo for the illustrations, Janine Makaronidis and Cormac McGee for their critical review of the manuscript.

 

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Ultrasonography of the Thyroid

ABSTRACT

Thyroid ultrasonography (US) is the most common, extremely useful, safe, and cost-effective way to image the thyroid gland and its pathology. US has largely replaced the need for thyroid scintigraphy except to detect iodine-avid thyroid metastases after thyroidectomy and to identify hyper-functioning (toxic) thyroid nodules. This chapter reviews the literature; discusses the science and method of performing US; examines it’s clinical utility to assess thyroid goiters, nodules, cancers, post-operative remnants, cervical lymph nodes, and metastases; presents it’s practical value to enhance US-guided aspiration biopsy of thyroid lesions (FNA); and endorses it’s importance in medical education. US reveals, with good sensitivity but only fair specificity very important and diagnostically useful clues to the clinician and surgeon about the likelihood that a thyroid nodule is malignant. Color flow Doppler enhancement of the US images, that delineates the vasculature, is essential. Comprehensive understanding of the local anatomy, the specific disease process, technical skill and experience are essential for proper interpretation of the US images. Features that favor the presence of a malignant nodule include decreased echogenicity, microcalcifications, central hypervascularity, irregular margins, an incomplete halo, a tall rather than wide shape (larger in the anteroposterior axis compared to the horizontal axis, the nodule is growing in one direction and not growing concentrically), documented enlargement of the solid portion of the nodule and associated lymphadenopathy. Several of these attributes enhance the diagnostic probability. The patient’s history, physical examination, and comorbidities refine the diagnosis. FNA and cytological examination of thyroid nodules and adenopathy in adults, children, and adolescents has become a major, specific, and highly diagnostic tool that is safe and inexpensive. In addition, the aspirate may be analyzed by biochemical measurements and especially by evolving molecular genetic methods.

 

INTRODUCTION

 

Ultrasonography (US) is the most common and most useful way to image the thyroid gland and its pathology, as recognized in guidelines for managing thyroid disorders published by the American Thyroid Association (1) and other authoritative bodies. In addition to facilitating the diagnosis of clinically apparent nodules, the widespread use of US has resulted in uncovering a multitude of clinically imperceptible thyroid nodules, the overwhelming majority of which are benign. The high sensitivity for nodules but inadequate specificity for cancer has posed a management and economic problem. This chapter will address the method and utility of clinically effective thyroid US to assess the likelihood of cancer, to enhance fine needle aspiration biopsy and cytology (FNA), to facilitate other thyroid diagnoses, and to teach thyroidology.

 

Previously, imaging of the thyroid required scintiscanning to provide a map of those areas of the thyroid that accumulate and process radioactive iodine or other nucleotides. The major premise of thyroid scanning was that thyroid cancers concentrate less radioactive iodine than healthy tissue and therefore provided triage in the selection for thyroid surgery. Unfortunately, however, since benign nodules also concentrate radioactive iodine poorly, the selection process is too inefficient to be cost-effective. Although, scintiscanning remains of primary importance in patients who are hyperthyroid or for detection of iodine-avid tissue after thyroidectomy for thyroid cancer, US has largely replaced nuclear scanning for the majority of patients because of its higher resolution, superior correlation of true thyroid dimensions with the image, smaller expense, greater simplicity, and lack of need for radioisotope administration. The other imaging methods, computerized tomography (CT), magnetic resonance imaging (MRI), and 18F-FDG positron emission tomography (PET) are more costly than US, are not as efficient in detecting small lesions, and are best used selectively when US is inadequate to elucidate a clinical problem (2,3).

 

 

Medical testing must be cost-effective. There is documentation that in a hospital or emergency department setting, the expense of thyroid ultrasound is quite low (5).

 

Although sonography can supply very important and clinically useful clues about the nature of a thyroid lesion, it does not reliably differentiate benign lesions and cancer. However, it can help significantly. US can:

 

  1. Depict accurately the anatomy of the neck in the thyroid region,
  2. Help the student and clinician to learn thyroid palpation,
  3. Elucidate cryptic findings on physical examination,
  4. Assess the comparative size of nodules, lymph nodes, or goiters in patients who are under observation or therapy,
  5. Detect a non-palpable thyroid lesion in a patient who was exposed to therapeutic irradiation,
  6. Give very important and clinically useful clues about the likelihood of malignancy,
  7. Identify the solid component of a complex nodule,
  8. Guide and facilitate fine needle aspiration biopsy of a nodule,
  9. Evaluate for recurrence of a thyroid mass after surgery,
  10. Monitor thyroid cancer patients for residual disease or early evidence of reappearance of malignancy in the thyroid bed or lymphadenopathy,
  11. Identify patients who have ultrasonic thyroid patterns that suggest diagnoses such as thyroiditis.
  12. Perhaps refine the management of patients on therapy such as antithyroid drugs,
  13. Facilitate delivery of medication or physical high-energy therapy precisely into a lesion and spare the surrounding tissue,
  14. Monitor in utero the fetal thyroid for size, ultrasonic texture, and vascularity,
  15. Scrutinize the neonatal thyroid for size and location,
  16. Screen the thyroid during epidemiologic investigation in the field.

 

TECHNICAL ASPECTS

 

Sonography depicts the internal structure of the thyroid gland and the regional anatomy and pathology without using ionizing radiation or iodine containing contrast medium (6,7). Rather, high frequency sound waves in the megahertz range (ultrasound), are used to produce an image. The procedure is safe, does not cause damage to tissue and is less costly than any other imaging procedure. The patient remains comfortable during the test, which typically takes only a few minutes unless there is a need to evaluate the lateral neck, does not require discontinuation of any medication, or preparation of the patient. The procedure is usually done with the patient reclining with the neck hyperextended but it can be done in the seated position. A probe that contains a piezoelectric crystal called a transducer is applied to the neck but since air does not transmit ultrasound, it must be coupled to the skin with a liquid medium or a gel. This instrument rapidly alternates as the generator of the ultrasound and the receiver of the signal that has been reflected by internal tissues. The signal is organized electronically into numerous shades of gray and is processed electronically to produce an image instantaneously (real-time). Although each image is a static picture, rapid sequential frames are processed electronically to depict motion. Two-dimensional images have been standard and 3-dimentional images are an improvement in certain circumstances (8). There is considerable potential for improving ultrasound images of the thyroid by using ultrasound contrast agents. These experimental materials include gas-filled micro-bubbles with a mean diameter less than that of a red blood corpuscle and Levovist, an agent consisting of granules that are composed of 99.9% galactose and 0.1% palmitic acid. They are injected intravenously, enhance the echogenicity of the blood, and increase the signal to noise ratio (9,10). Contrast-enhanced thyroid US features such as heterogeneous enhancement, slow ”wash in”, ill-defined enhancement of the border a the nodule, and fast “wash out” seem to be associated with increased association with malignancy (11).

 

Dynamic information such as blood flow can be added to the standard US signal by employing a physics principle called the Doppler effect. The frequency of a sound wave increases when it approaches a listener (the ear or, in the case of ultrasonography, a transducer) and decreases as it departs. The Doppler signals, which are superimposed on real time gray scale images, are extremely bright in black and white images and may be color coded to reveal the velocity (frequency shift) and direction of blood flow (phase shift) as well as the degree of vascularity of an organ (12,13). Flow in one direction is made red and in the opposite direction, blue. The shade and intensity of color can correlate with the velocity of flow. Thus, in general terms, venous and arterial flow can be depicted by assuming that flow in these two kinds of blood vessels is parallel, but in opposite directions. Since portions of blood vessels may be tortuous, modifying orientation to the probe, different colors are displayed within the same blood vessel even if the true direction of blood flow has not changed. Thus, an analysis of flow characteristics requires careful observations and cautious interpretations. The absence of flow in a fluid-filled structure can differentiate a cystic structure and a blood vessel.

 

Blood flow within anatomic structures can also be depicted by non-Doppler technology. This technique is called B-flow ultrasonic imaging (BFI). It is accomplished by transmitting precisely separated adjacent ultrasound beams and analyzing with a computer, the reflected echo pairs (14).

 

Various anatomic features and tissues result in different ultrasound characteristics (2,6). The air-filled trachea does not transmit the ultrasound. Calcified tissues such as bone and sometimes cartilage and calcific deposits in other anatomic structures block the passage of ultrasound resulting in a very bright signal and a linear echo-free shadow distally. Most tissues transmit the ultrasound to varying degrees and interfaces between tissues reflect portions of the sound waves. Fluid-filled structures have a uniform echo-free appearance whereas fleshy structures and organs have a ground glass appearance that may be uniform or heterogeneous depending on the characteristics of the structure.

 

The depth penetration and resolving power of ultrasound depends greatly on frequency (7). Depth penetration is inversely related and spatial resolution is directly related to the frequency of the ultrasound. For thyroid, a frequency of 7.5 to 10 - 15 megahertz is generally optimal for all but the largest goiters. Using these frequencies, nodules as small as two to three millimeters can be identified.

 

Performance and interpretation of thyroid sonograms are quite subjective and reflect probabilities, not certainty. Both overaggressive and excessively timid interpretation can be misleading. Routine protocols for sonography are not always optimal. Although some technologists become extremely proficient after specific training and experience, supervision and participation by a knowledgeable and interested physician-sonographer is usually required to obtain a precise and pertinent answer to a specific problem that has been posed by the clinician. For instance, one group has reported accurate, surgically proven preoperative identification of non-recurrent inferior laryngeal nerves (15). It is not that the ultrasound images depict an inferior laryngeal nerve. Rather, the diagnosis is suggested when, while performing the sonogram, the surgeon asks a specific, direct question about the anatomic region where the nerve should be located. Thereafter, a series of images are obtained with and without Doppler interrogation that reveal the presence of a small, linear structure that, on Doppler interrogation, is associated with blood vessels, allowing a probable answer to the inquiry. The surgeon is then in a position to operate, minimizing the risk of adverse consequences.

 

Standard sonographic reports may provide considerable information about major anatomic features, but are suboptimal unless the specific clinical concern is explored and answered. Indeed, because some radiologists may not address the clinical issue adequately, and for convenience, numerous thyroidologists and surgeons perform their own ultrasound examinations, in their office or clinic (point of service). However, it is essential that non-dedicated ultrasonographers have state-of-the-art equipment (might not be cost-effective) and that they are willing to expend a considerable amount of time for a complete study, in particular if there is a need to evaluate the lateral neck compartments. Technical ingenuity, electronic enhancements such as Doppler capability, and even artistry are frequently required. Special maneuvers, various degrees of hyperextension of the neck, swallowing to facilitate elevation of the lower portions of the thyroid gland above the clavicles, swallowing water to identify the esophagus, and a Valsalva maneuver to distend the jugular veins may enhance the value of the images. Nevertheless, sonography is rather difficult to interpret in the upper portion of the jugular region and in the areas adjacent to the trachea. Aiming the transducer obliquely may permit exploration of the region behind the trachea. Sonography is generally not useful below the clavicles.

 

To orient the imager, it can be useful to survey the entire neck and thyroid gland with a low-energy transducer before proceeding to 10-15 megahertz equipment. Protocols have been devised to assemble a montage of images to encompass an unusually large lobe or goiter. For an overview, panoramic ultrasound, which is a variation of conventional ultrasound allows one to produce images with a large anatomic field of view, displaying both lobes of the thyroid gland on a single image (16).

 

There may be considerable differences between sonographers in estimating the size of large goiters or nodules (17). One investigation has reported that curved-array transducers may avoid significant inter-observer variation that may occur when linear-array equipment is employed, especially when the gland is very large (18). The inter-observer variation may be almost 50% even among experienced ultrasonographers, because it is difficult to reproduce a two-dimensional image plane for multiple studies (19). Accuracy in volume estimation becomes most important when one uses ultrasound measurements to calculate an isotope dose or to compare changes over time in the size of a nodule or a goiter. Indeed, it has been suggested even for well-defined nodules, a change of less than 1 cm in size should not be accepted as a real change (17). The important aspect is that the clinician must be guided by the constellation of risk factors, local anatomy, and intervening events, when making a management decision. Stability of size is one factor, but not a major one.

 

Using planimetry from three-dimensional images reportedly has lower intra-observer variability (3.4%) and higher repeatability (96.5%) than the standard ellipsoid model for nodules and lobes, with 14.4% variability and 84.8% repeatability (p < 0.001) (20).

 

Importantly, for autonomous nodules, US-evidence of growth does not indicate a likelihood of malignancy. Rather, it usually reflects cystic or hemorrhagic degeneration, which correlates well with prior experience by pathologists and the literature. In contrast, growth of a non-functional follicular adenoma can be of concern and the lesion needs to be carefully evaluated for other suspicious signs (21). There may be imperfect concordance between the ultrasonic dimensions of large thyroid nodules compared with intraoperative findings (22).

 

SONOGRAPHY OF THE NORMAL THYROID AND ITS REGION

 

The anterior neck is depicted rather well with standard gray scale sonography (Figure 1). The thyroid gland is slightly more echo-dense than the adjacent structures because of its high iodine content. It has a homogenous ground glass appearance. Each lobe has a smooth globular-shaped contour and is no more than 3 - 4 centimeters in height, 1 - 1.5 cm in width, and 1 centimeter in depth. The isthmus is identified, anterior to the trachea as a uniform structure that is approximately 0.5 cm in height and 2 - 3 mm in depth. The pyramidal lobe is not seen unless it is significantly enlarged. In the female, the upper pole of each thyroid lobe may be seen at the level of the thyroid cartilage, whereas it is lower in the male. The surrounding muscles are of lower echogenicity than the thyroid and tissue planes between muscles are usually identifiable. The air-filled trachea does not transmit the ultrasound. Only the anterior portions of the cartilaginous rings are represented by dense, bright echoes. The carotid artery and other blood vessels are echo-free unless they are calcified. The jugular vein is usually in a collapsed condition and it distends with a Valsalva maneuver. There are frequently 1-2 mm echo-free zones on the surface and within the thyroid gland that represent blood vessels. The vascular nature of all of these echo-less areas can be demonstrated by color Doppler imaging to differentiate them from cystic structures (12,13). Lymph nodes may be observed. Nerves are generally not seen. However, a keen understanding of the local anatomy may permit critical interpretation of a series of gray scale US and Doppler images to gain useful insights into the probable presence or absence of an expected neurovascular bundle. Meticulous preoperative analysis that may include lymph node mapping can benefit surgical management (15). The parathyroid glands are observed only when they are enlarged and are less dense ultrasonically than thyroid tissue because of the absence of iodine. The esophagus may be demonstrated behind the medial part of the left thyroid lobe, especially if a sip of water distends it (Figure 2).

Figure 1. Sonogram of the neck in the transverse plane showing a normal right thyroid lobe and isthmus. L = small thyroid lobe in a patient who is taking suppressive amounts of L-thyroxine, I = isthmus, T = tracheal ring (the dense white arc represents calcification, distal to it reflects artifact), C = carotid artery (note the enhanced echoes deep to the fluid-filled blood vessel), J = jugular vein, S = sternocleidomastoid muscle, m = strap muscle.

Figure 2. Sonogram of the left lobe of the thyroid gland in the transverse plane showing a rounded lobe of a goiter. L = enlarged lobe, I = widened isthmus, T = trachea, C = carotid artery (note the enhanced echoes deep to the fluid-filled blood vessel), J = jugular vein, S = sternocleidomastoid muscle, m = strap muscles, E = esophagus.

GENERAL THOUGHTS ABOUT SONOGRAPHY

 

Thyroid US may play a useful role in the management of patients even when the thyroid examination is normal but it is debatable if the procedure is cost-effective as a screening test (1). Many thyroidologists/endocrinologists advocate routine use of US at the time of physical examination to discover subclinical, non-palpable thyroid abnormalities, which will be discussed presently, and to enhance the sensitivity and accuracy of palpation. This practice is called “point of service” US.

 

Whether US is performed at the point of service or in an US laboratory by ultrasonographers/radiologists, it is important to employ thyroid sonography selectively to supplement or confirm a physical examination especially when clinical perception is confused by obesity, great muscularity, distortion by abnormal adjacent structures, tortuous regional blood vessels, a prominent thyroid cartilage, metastatic tumor, lymphadenopathy, or prior surgery.

 

In practice, US may be used to supplement an examination when there is uncertainty about the palpation. It is important, however, to comprehend that the optimal clinical value of US depends on the quality of the examination, including the experience of the examiner and the characteristics of the equipment. Grossly misleading results may occur with quick, incomplete studies, insensitive US machines or substandard interpretations. “Routine” sonography in a medical office, clinic or in a radiology facility by an incompletely trained clinician or general radiologist can be misleading. Without study, training and practice, there are likely to be unacceptable results and adverse outcomes. Furthermore, the efficacy of US when performed in sub-optimal conditions has yet to be critically examined.

 

In the academic situation, sonography is useful to teach palpation of the thyroid gland.

There are claims that US can offer insights into thyroid function. For instance, among 4649 randomly selected adult subjects one investigation found that there was correlation between thyroid hypoechogenicity and higher than average levels of serum TSH, even in subjects without overt thyroid disease (23). One group reported TSH elevations in 26 patients with autoimmune thyroiditis when there was a well-defined, approximately 10 mm triangular area of low echogenicity, between the lateral margin of one or both thyroid lobes, the medial wall of the carotid artery, and, posteriorly, the pre-vertebral muscles. Euthyroid patients (71) with thyroiditis and controls (154) did not demonstrate a hypoechoic “triangle” (24). In contrast, the question arises how accurately a normal thyroid sonogram will predict normal thyroid function? In one study of normal-appearing US, TSH was normal in 41/48 (85.4%) subjects but was elevated in 7 individuals (14.6%) (p<0.001) and anti-thyroid antibodies were detected in 5 patients (10.4%) (25). Therefore, a normal sonogram does not preclude hypothyroidism or Hashimoto’s thyroiditis.

 

SONOGRAPHY USED TO FACILITATE AN UNCONVENTIONAL SURGICAL APPROACH TO THYROIDECTOMY OR REMOVAL OF METASTASES

 

US maybe used to guide a surgeon who performs a trans-axillary or sub-mammary approach to thyroidectomy, thereby avoiding a neck scar. Retro-pharyngeal thyroid metastases can be managed via trans-oral robotic and surgeon-performed US to localize and excise lymphadenopathy (26).

 

SONOGRAPHY IN THE PATIENT WITH AN ENLARGED THYROID GLAND (GOITER)

 

Thyroid sonography probably is not cost-effective in evaluating the average patient with thyroid enlargement. Since thyroid goiters are common and rarely associated with malignancy, there is little useful purpose to sonographic documentation of the size, shape, or uniformity of a goiter. However, US may be used in a goiter to identify non-palpable thyroid nodules for biopsy, or those that have enlarged or become harder. Importantly, US permits one to characterize nodules and estimate the risk of malignancy. The value of aspirating a selected nodule in a goiter is under current scrutiny. At this time, the data seems persuasive that the incidence of cancer in a particular nodule in a goiter is independent of the number of sonographically identified nodules, in distinction to prior belief. Therefore, this practice seems worthwhile (27,28).

 

At times, it will be useful to know the ultrasonic appearance of a dominant nodule in a goiter, a tender spot, a region of focal hardness because it might give a clue about pathology (Figure 2) (1). For example, sonography can identify one region in a goiter whose echo pattern is distinct from the rest of the goiter suggesting a second type of pathology, especially if the region is surrounded by an incomplete and irregular sonolucent rim, has punctate microcalcifications, or Doppler examination reveals internal vascularity. The significance of these ultrasonic features will be discussed below. Among the lesions that have been demonstrated in goiters using US are neoplasms and lymphoma. Other uses of sonography in goitrous patients include: differentiation of thyroid enlargement from adipose tissue or muscle, identifying a large unilateral mass in distinction to an asymmetric goiter, confirming substernal extension, providing the correct interpretation to varying clinical impressions among several examiners, and objectively documenting volume changes in response to suppressive therapy with thyroid hormone, which may be particularly useful information when patients change physicians.

 

An interesting public health use of US in underdeveloped countries has been to objectively identify goiter as a screen for iodine deficiency. Furthermore, in the epidemiological setting, with proper ultrasound equipment, assessment of thyroid volume and prevalence of thyroid nodules, but not echogenicity or echographic pattern, are comparable among different observers (29).

Sonography is useful to monitor patients undergoing long-term treatment with lithium for bipolar, major depressive, and schizoaffective disease. Their total thyroid volume in one investigation was significantly greater (23.7 ml vs. 13.6 ml) in the lithium-treated group (30) than among controls (96 sex- and age-matched control subjects). Furthermore, US detects thyroid enlargement with greater accuracy and sensitivity than palpation (31).

 

SONOGRAPHY WITH THYROIDITIS AUTONOMOUS NODULES, AND GRAVES' DISEASE

 

Routine sonography can be useful in distinguishing thyroiditis or Graves' disease, but it is uncertain whether this is cost-effective. Several publications have shown that the ultrasound pattern correlates with the presence of autoimmune thyroid disease and can predict thyroid dysfunction as will be discussed below (32). In subacute thyroiditis, the severely inflamed thyroid reflects very low intensity echoes, which is generally not seen with any other thyroid disorder (33). In the inflamed portions of the thyroid gland there is no increased vascular flow pattern on Doppler examination. The non-involved regions demonstrate normal vascularity and hemodynamics. In the recovery phase of subacute thyroiditis, the thyroid regains isoechogenicity and a Doppler study may show slightly increased vascularity (33-37). Hashimoto's thyroiditis and Graves' disease show moderately heterogeneous, reduced echogenicity (38-43). The diagnostic precision of this US pattern was compared to that of anti-thyroid peroxidase antibody (TPOAb) concentrations in 451 ambulatory patients with unknown thyroid status, excluding those with suspected hyperthyroidism or on drugs known to cause hypothyroidism. There was high intra-observer and inter-observer agreement on the abnormal thyroid ultrasound patterns, which were judged highly indicative of autoimmune thyroiditis and allowed the detection of thyroid dysfunction by other means with 96% probability (44). It has been reported that among 55 patients with hyperthyroidism (29 Graves' disease and 26 toxic nodules), color flow Doppler examination was useful to differentiate the etiology. Increased blood flow was successful in differentiating untreated Graves' disease from Hashimoto’s thyroiditis, both of which had similar gray scale findings (p < 0.001), and from controls (p < 0.001). “Hot”, autonomous nodules could also be differentiated from “cold” nodules because of more prominent vascular patterns and significantly higher peak systolic velocity values (p < 0.001) (45). The gray-scale US features of nontoxic autonomous nodules are similar to those of toxic autonomous nodules (46).

 

Investigation of patients with postpartum thyroiditis who had both high levels of antithyroid peroxidase antibody and a hypoechogenic thyroid gland also had a high risk of long-term thyroid dysfunction (47). In 119 patients with postpartum thyroiditis and 97 normal postpartum women as the control group, thyroid hypoechogenecity was present in 98.5% of patients and 7% of the control group (p <0.001). Initially, mean thyroid volume in the patients with thyroiditis was 77% greater than in the control group. After remission, mean thyroid volume decreased by 25% in the thyroiditis patients. Twelve months after delivery, hypoechogenicity persisted in 4 patients (48).

 

It has been reported that in children US findings of Hashimoto’s thyroiditis are present in only a third at the time of diagnosis and half of the Hashimoto’s children with normal initial thyroid sonography develop changes within 7 months. In some cases, characteristic Hashimoto’s findings may not develop for over 4 years (49).

 

Especially in Graves' disease, color Doppler imaging of the thyroid can demonstrate diffuse hyperemia of the thyroid gland (50) that has been called a "thyroid inferno" (51). In patients with amiodarone-induced thyrotoxicosis, Doppler flow sonography has been reported to differentiate two types of disorder with implications for therapy (52-56). Patients with moderate to high vascular flow had underlying thyroid disease, such as latent Graves' disease or nodular goiter. The latter are at risk of amiodarone-induced thyrotoxicosis type I (AIT I), which is caused by the organification of the high amounts of iodine in amiodarone. In contrast, AIT II is caused by a destructive thyroiditis caused by the drug and there is typically no demonstrable vascular flow. The clinical value of this observation is that the Type II patients seem to respond to treatment with glucocorticoids. In contrast, AIT I patients tend to respond to a combined regimen with methimazole and potassium perchlorate (52). Although this conclusion was originally based on a small number of patients, the observations were confirmed in a retrospective case-note audit of 37 patients (53). Interestingly, in that study, euthyroid amiodarone-treated patients failed to show hyperactive flow (52). In another investigation, looking at the data from the perspective of patients who had been treated for amiodarone-induced thyrotoxicosis, in a retrospective study of 24 patients, responsiveness to prednisolone correlated poorly with the absence of enhanced blood flow in the thyroid glands, but the presence of enhanced flow appeared to correlate with non-response to prednisolone (55). Interleukin 6 (IL-6) levels correlated with the ultrasound classification in one study (52), but not in another (53).

 

A report has successfully validated excessive mean peak systolic velocity of the superior thyroidal artery in Graves’ disease but not in patients with destructive thyroiditis (57).

An important application of standard US in patients with thyroiditis or Graves' disease is to assess those thyroid glands that have focal firm consistency or are enlarged or painful for coincidental tumor or lymphoma (1). In one report, patients with Hashimoto's thyroiditis had sonography to detect nodules and then had ultrasound-guided aspiration biopsy to elucidate the nature of the lesion. Two of 24 patients (8.3%) had papillary thyroid cancer (58).

 

In patients with thyrotoxicosis, US can assess the size of the thyroid gland to facilitate I-131 dosimetry. The size of each lobe is measured in the sagittal and transverse planes to provide the length (L), anterior-posterior depth (D), and transverse width (W), respectively. The volume of each lobe is calculated using the formula for a prolate ellipse: (volume = 0.5 {L x D x W}). 3D echography may improve the accuracy of assessment of thyroid volume (20).

 

Doppler sonography may become a useful tool for the clinical endocrinologist in the management of patients with Graves’ disease if observations are confirmed in large populations. It has been suggested that color-flow mapping of the thyroid gland may have a role in the selection of an optimal dose of methimazole needed to maintain a euthyroid state in patients with Graves' disease (59). Another study has characterized Doppler ultrasound data from patients with Graves' disease, Hashimoto's disease, and goiter to obtain a "hemodynamic index" to ascertain when antithyroid drugs should be withdrawn or ablative therapy given in patients with Graves' disease. The hemodynamics in the thyroid was significantly different between untreated thyrotoxic and medically well-controlled patients but there were no significant differences between untreated or medically poorly controlled patients. It would be interesting to ascertain whether the hemodynamics permit an identification of a subset of well-controlled patients who will relapse after a course of therapy (60). Furthermore, Doppler sonography has provided data from 40 patients with Graves' disease showing significantly increased thyroid blood flow in euthyroid patients who presented early in relapse after withdrawal of antithyroid drug therapy when compared with 16 age-matched normal control subjects. Conversely, there were no significant differences in euthyroid patients who remained in remission when compared with normal controls (61). The value of quantifying thyroid blood flow at the time of diagnosis has been assessed in 24 patients with Graves' disease, using percutaneous spectral Doppler recordings from the thyroid arteries, in an attempt to predict the likelihood of remission following withdrawal of antithyroid drug therapy. The mean duration of treatment was 14 months and follow-up in 13 women was at least 18 months (range: 18 - 39 months) after antithyroid drug withdrawal. Mean peak systolic velocity and volume flow rate values as well as thyroid volume measured at the time of diagnosis were significantly higher (139 cm/s, SD 46; 195 ml/min, SD 170; 52 ml, SD 18) in patients who relapsed after drug treatment compared with patients in remission (71 cm/s, SD 27; 67 ml/min, SD 61; 25 ml, SD 13) (62). Thyroid hypoechogenicity at onset of Graves’ disease is probably not a reliable prognostic index of relapse after medical treatment. However, the absence of thyroid hypoechogenicity after methimazole treatment seems to be a favorable prognosticator of remission (63). In another investigation, Doppler ultrasound determined increased peak systolic velocity in the inferior thyroid artery in untreated hyperthyroid patients with Graves' disease was significantly and positively associated with the maintenance dose of methimazole needed to keep TSH normal (64). Normoechoic Graves' hyperthyroid glands seem to be more resistant to therapy with I-131 than hypoechoic thyroids (65).

 

Another example of the value of Doppler ultrasound relates to the administration of iodide solutions that have been used traditionally prior to thyroid surgery for Graves' disease because it was thought that they reduce the vascularity of the thyroid gland. Doppler echography has demonstrated a significant decrease in thyroid vascularity in patients with Graves' disease after seven days of Lugol's solution, confirming the rationale of this form of treatment (66). Preoperative treatment with Lugol’s solution decreased the rate of thyroid blood flow and vascularity, as assessed by Doppler evaluation. Lugol’s solution also decreased intraoperative blood loss during thyroidectomy in another investigation (67). In contrast, US has also shown that preoperative iodide may increase the size of the thyroid gland, which could complicate surgery when a Graves’ thyroid is very large before the Lugol’s solution is administered (68).

 

Doppler examination has been used trans-vaginally in pregnant women with Graves’ disease to depict and assess the size of the fetal thyroid gland. Clinical benefits might include facilitating adjustment of the mother’s dose of antithyroid drug and anticipating or preventing fetal and neonatal hypothyroidism. The authors suggested that when reduction of the medication does not result in decrease in the size of the fetal goiter, trans-placental passage of thyroid stimulating immunoglobulin should be suspected (69).

 

SONOGRAPHY OF LYMPHOMA

 

In the author’s experience, the value of US to predict lymphoma is very limited. However, the sonographic patterns of thyroid lymphoma have been classified into three types based on internal echoes within the suspected lesion, the border of the lesion, and the intensity of the echoes behind (deep to) the lesion. The echoes behind the lesion in each type of lymphoma are increased, presumably because of enhanced transmission of the ultrasound through the lesion. In the nodular type of lymphoma, the internal echoes within a nodule are uniform and hypoechoic (may be sufficiently hypoechoic to be pseudocystic). The border between lymphoma and non-lymphomatous tissue is well-defined and the borderline is described as “broccoli-like or coastline-like” irregularity. In the diffuse type of lymphoma internal echoes are also exceedingly hypoechoic but the border between lymphoma and non-lymphomatous tissues is not distinct. It is difficult to differentiate the diffuse type lymphoma from chronic thyroiditis. The mixed type lymphoma shows multiple, patchy hypoechoic lesions, each with enhanced posterior echoes (70).

 

SONOGRAPHY OF THE THYROID NODULE

 

The most frequent use of US is to refine the diagnosis of a thyroid nodule. US can identify thyroid nodules, even when they are too small to palpate. Sonography can demonstrate nodules that have an enhanced risk of malignancy with the best sensitivity of any non-invasive technique, but with only fair specificity. In addition, FNA of thyroid nodules should be performed under US guidance whenever possible.

 

Thyroid nodules can be identified by sonography because they distort the uniform shape or echo pattern of the thyroid gland. Thyroid nodules may be large or small. They may distort the surrounding thyroid architecture or may dwell within a lobe and be unobtrusive. They may be solid tissue or consist of solid areas interspersed with echo-free zones that represent fluid-filled hemorrhagic or straw-colored degenerative zones (Figure 3). A smooth, globular area without echoes generally represents an epithelial-lined cyst, which is a rare benign lesion (Figure 4) (71). Most thyroid nodules have a less dense ultrasound appearance than normal thyroid tissue and some are more echo-dense (6). A sonolucent rim, which is called a halo, may be present around a nodule. A halo represents a capsule or another interface, such as inflammation or edema, segregating the nodule and the rest of the gland. Doppler technique may demonstrate increased vascularity within a nodule or in a halo (Figure 5) (12). “Nodules” are not a single disease but are a manifestation of different diseases including adenomas, carcinomas, inflammations, cysts, fibrotic areas, vascular regions, and accumulations of colloid.

Figure 3. Sonograms showing longitudinal (left panel) and transverse (right panel) images of the left lobe containing a degenerated thyroid nodule. Note the thick wall and irregularity. N = nodule, H = hemorrhagic degenerated region.

Figure 4. The left panel shows an anterior scintiscan of a euthyroid patient who had a firm nodule in the left thyroid lobe. The nodule is "cold". * * * = nodule. The right panel shows a sonogram of the neck in the longitudinal plane revealing that the nodule is a smooth-walled cystic structure without internal echoes. between the + + symbols. Note the dark dense echoes distal to the cyst. C = cyst, L = thyroid lobe.

Figure 5. Sonogram of the neck in the longitudinal plane showing a hypoechogenic nodule that was surrounded by an echo free rim, called a halo. Doppler examination demonstrates great vascularity in the halo, identified as bright spots. Small blood vessels are also seen elsewhere. N = nodule, L = heterogeneous thyroid lobe, m = muscle.

The ultrasonic appearance of a thyroid nodule does not reliably differentiate a benign thyroid lesion and cancer (1,6)but it does offer strong clues that help the clinician in the process of triage. Nevertheless, sonography cannot identify a specific kind of tumor such as a Hürthle cell lesion (72). The most reliable sonographic indicator that a nodule is malignant is observing vascular invasion by tumor, which is rarely seen. However, there are distinctions in echo-density, calcifications, distortions of the rim, and vascularity that favor a benign or malignant diagnosis (73,74). These characteristics are summarized in Tables 1 & 2. But it is important to understand that the features described reflect statistical probabilities and not dependable criteria.

 

Table 1. Ultrasound Characteristics Associated with an Increased Thyroid Cancer Risk

1. Hypoechoic

2. Microcalcifications

3. Central vascularity

4. Irregular margins

5. Incomplete halo

6. Taller-than-wide

7. Significant enlargement of a nodule

 

Table 2. Ultrasound Characteristics Associated with a Low Thyroid Cancer Risk

1. Hyperechoic

2. Large, coarse calcifications (except medullary thyroid cancer)

3. Peripheral vascularity

4. No hyper-vascular center

5. Spongiform appearance (puff pastry appearance)

6. Comet-tail shadowing

 

An important aspect is that single or even a few US features may be inadequate to select some nodules for FNA or to reliably assess the risk of thyroid cancer. In contrast, selections based on multiple characteristics that are associated with elevated cancer risk are more dependable indicators of probable malignancy. Nevertheless, certain single features should prompt for FNA including microcalcifications, a taller-than-wide shape, or irregular margins. Absence of elasticity-will probably identify nodules with a clinically meaningful increased, perhaps even high risk for malignancy (75).

 

  • ECHOGENICITY: Thyroid malignancies tend to be hypoechoic when compared with the rest of the thyroid (71,76-79). Since many benign thyroid nodules, which are far more common than malignancies, are also hypoechoic, this finding is not particularly useful except that it is reasonably safe to conclude that hyper-dense nodules are probably not cancerous. One group of investigators has concluded that hyperechogenic lesions occurring in thyroiditis-affected thyroid glands bear no-clinical relevance. Therefore, they advocate that aspiration biopsy of these nodules is not advisable (80) and many clinicians follow that practice.
  • CALCIFICATIONS: The presence of calcification is also not a straightforward diagnostic aid. Microcalcifications are relatively more common in malignant than in benign lesions and may represent psammoma bodies. Microcalcifications have been reported as demonstrating a 95.2% specificity for thyroid cancer, but a low sensitivity of 59.3 % and a diagnostic accuracy of 83.8% (77). B-flow ultrasonic imaging may be particularly sensitive in detecting microcalcifications by demonstrating “twinkling” in some nodules (14). However, large coarse calcifications and calcifications along the rim of nodule are common in all types of nodules and reflect previous hemorrhage and degenerative changes. Thus, since some cancers may have been chronic and have undergone degenerative change, they may demonstrate peripheral or coarse internal calcification. Therefore, diagnostic FNA biopsy may be appropriate even when there are large, coarse, or eggshell calcifications to avoid missing a cancer (79). Indeed, in one investigation, among 64 thyroid nodules with peripheral calcifications 19 (30%) were benign, and 45 (70%) were malignant. Interruption and thickening of peripheral calcifications and decreased internal echogenicity of a thyroid nodule with peripheral calcifications were associated with malignancy in this study (81). In our estimation, considerably more than 30% of such nodules are benign; thyroid calcifications that are greater than pin-point size provides little practical help in identifying cancer in the individual patient. In one study, the highest incidence of calcification was found in thyroid cancer (54%), followed by multinodular goiter (40%), solitary nodular goiter (14%), and follicular adenomas (12%). The authors reported that calcifications in a "solitary" nodule in a person younger than 40 years should raise a strong suspicion of malignancy: relative cancer risk of 3.8 versus 2.5 in patients older than 40 years (82). In contrast to the prior statements, it is important to note that large calcifications are seen with increased frequency in medullary thyroid carcinoma (83).
  • HALO: A halo around the nodule may be seen with benign or malignant conditions. It suggests that there is an acoustic interface around the nodule that does not reflect the ultrasound. It implies that there are two different types of histology in the region: the nodule and the surrounding thyroid (6,71,84). Some observers have suggested that cancer should be suspected when the periphery of a halo has a blurred appearance. We have not found that characteristic reliable. Since adenomas are more common than carcinomas, the finding of a halo is, in our opinion, more often seen with adenomas than carcinomas.
  • NODULE BORDER: There have been investigations of a possible correlation between the degree of definition of the border of a nodule and the likelihood of malignancy and even of the predictability of aggressive characteristics of a papillary cancer. In one series of 155 cases, poor definition of a nodule’s edge was observed in 21.5% of patients, all of whom showed worse disease-free survival (p = 0.0477) than those with a well-defined edge. Furthermore, this finding was directly linked to US-diagnosed lateral node metastasis (p = 0.0001) (85). Ultra-high frequency thyroid ultrasound (12–18 MHz) may reveal jagged edges and lobulated borders in 1 to 3 cm thyroid nodules. These findings have been reported to correlate with papillary thyroid cancer with a sensitivity of about 60 to 70% (86).
  • HEMODYNAMIC CHARACTERISTICS: Increased blood flow in the central part of a nodule is more likely associated with cancer than when the vascularity is along the periphery. An analysis of the hemodynamic characteristics of a nodule by high resolution pulsed and power Doppler ultrasonography also may offer valuable preoperative diagnostic insights. For example, one study of 25 follicular adenomas and 10 follicular carcinomas compared the vascular pattern and the velocimetric parameters (such as peak systolic velocity), end-diastolic velocity, pulsatility index or resistance index. Eight of 10 patients with follicular carcinomas showed moderate increase of intra-nodular vascularity using “Power Doppler”. In contrast, the 21 out of 25 follicular adenomas showed only a peripheral rim of color flow. Furthermore, the velocimetric analyses were significantly higher in the patients with cancer than those with adenomas (80).

 

Bayes' mathematical theorem has been used to evaluate the cancer diagnostic value of enhanced intranodular blood flow by Doppler analysis in determining the probability of cancer in thyroid nodules that demonstrate follicular cytology. The sensitivity of enhanced intranodular flow by Doppler analysis for detection of thyroid carcinoma was 80%-86% and the specificity of indicating cancer ranged from 85% to 89%. In contrast, the probability that a nodule is thyroid cancer before a Doppler test was estimated at 12%-14%. After Doppler examination, the probability of thyroid cancer increased to nearly 50% in the presence of intranodular flow but declined to 3% when there was no central intranodular flow (87). In one investigation of 230 patients, 203 of whom were treated surgically, the addition of color flow Doppler imaging to conventional sonography increased the screening sensitivity and accuracy in identifying 36 malignant thyroid nodules from 71.9% to 83.3% (88). Thus, Doppler ultrasound may be a particularly useful predictor of the risk of malignancy in thyroid nodules (89).

 

Doppler sonography employing ultrasound contrast medium may further enhance the diagnosis of thyroid cancer. In one investigation, carcinomas showed a significantly earlier arrival time of Levovist in the nodule than nodular hyperplastic benign nodules or adenomas (90).

  • SHAPE: There have been observations that some cancers tend to have a non-globular, “tall” shape, as if growing in one plane (i.e. the depth of the nodule is larger than the width in the horizontal plane). Nodules that are tall rather than wide should be viewed with considerable suspicion. This observation has recently been validated in 500 patients with thyroid microcarcinomas (91).
  • CYSTIC SPACES: Especially large benign or malignant thyroid nodules tend to undergo hemorrhagic or cystic degenerative changes. It has been reported that features associated with cancer in a cystic thyroid nodule include more than 50% solid tissue, eccentricity of the cystic space, and micro-calcifications (92).
  • MISCELLANEOUS CHARACTERISTICS: Ultrasonographers have observed that colloid nodules, which are benign with high probability, have a more or less characteristic appearance of a “stack of pancakes”, “puff pastry like a Napoleon”, or sponge. In one publication, among 201 nodules, no malignancies were found when the US appearance was greater than 75% “sponge-like” (spongiform) (93). There may be a small, echogenic, bright spot with “comet-tail shadowing” associated with colloid that must be differentiated from a pin-point bright spot. There seems to be triage-merit to these characteristics, which will require critical scrutiny. Furthermore, it is important to be aware that a cancer may co-occur in an otherwise nodular or colloid goiter. This issue will be further discussed below in the section on thyroid biopsy.

 

To summarize, the cancer-predictive value of ultrasonic characteristics varies considerably but is acceptable when multiple characteristics are considered together. The most supportive data we have found is that there was a 97.2% positive predictive value for cytologically diagnosed cancer and 96.1% predictive value for benign disease among 1,244 nodules in 900 patients who were stratified according to ultrasound characteristics on a scale of 1-5 assessing cancer-risk (85).

 

In contrast, the sensitivity for cancer, however, is lower as shown in the following studies. Retrospective examination of 849 nodules (360 malignant, 489 benign) revealed that statistically significant (P <0.05) sonographic characteristics of malignancy included: a taller-than-wide shape (sensitivity 40.0%; specificity 91.4%), a spiculated margin (sensitivity 48.3%; specificity 91.8%), marked hypoechogenicity (sensitivity 41.4%; specificity 92.2%), microcalcification (sensitivity 44.2%; specificity 90.8%), and macrocalcification (sensitivity 9.7%; specificity 96.1%). The US findings for benign nodules were isoechogenicity (sensitivity 56.6%; specificity 88.1%; P <0.001) and a spongiform appearance (sensitivity 10.4%; specificity 99.7%; P <0.001). The presence of at least one malignant US finding had a sensitivity of 83.3%, a specificity of 74.0%, and a diagnostic accuracy of 78.0% (74). In an iodine-deficient geographic region where there is endemic goiter and thyroid nodules are frequent, among 2,642 consecutive patients (3,645 nodules) a numeric score was assigned to nodules based on ultrasonic high-risk of cancer. Nodules with a score of over 5.5 out of 10 had a 66% sensitivity and a 76% specificity for cancer, both of which were much higher values than when the scores were below 5 (94).

 

It is noteworthy that the results of sonography may influence a management decision even when the results of needle biopsy are only “suspicious”. In one study, 303 patients who had thyroid nodules with an aspiration biopsy reading of merely suspicious for papillary thyroid cancer had surgery anyway. The pre-surgery ultrasound examination had a positive predictive value of 94.9%, and negative predictive value of 80.9% (95).

 

The use of a Bayesian classifier to differentiate benign and malignant thyroid nodules by using sonographic features is under investigation (96).

 

Preoperative US of a nodule that turns out to be thyroid carcinoma has a very limited ability to predict postoperative staging. In one study, the sensitivity of depicting metastases to lymph nodes was 36.7%, invasion of the muscles 77.8%, trachea involvement 42.9%, and esophagus 28.6% (97).

 

Postoperatively, sonographic features of nodules in a thyroid bed cannot reliably distinguish recurrent thyroid cancer and benign thyroid remnants (98). However, in remnants, increased vascularity, and microcalcifications of a lesion that is larger than 6 mm in size should be viewed with suspicion.

 

Perhaps more objective, computerized triage of ultrasound features of thyroid nodules will become possible. In one investigation an artificial neural network and binary logistic regression was significantly better than two experienced radiologists in distinguishing benign and malignant thyroid nodules based on 8 ultrasonographic parameters: size, shape, margin, echogenicity, cystic change, microcalcification, macrocalcification, and halo. The study included 109 pathologically proven thyroid lesions (49 malignant and 60 benign) in 96 patients (99).

It is important to note that there may be significant inter-observer variation in interpretation. The inter-observer variation in the interpretation of thyroid ultrasonograms among 4 experienced readers reviewing 144 patients, varied according to the characteristic examined. Echogenicity showed slight agreement (kappa = 0.34); composition, margin, calcification, and final assessment had fair agreement (kappa = 0.59, 0.42, 0.58, and 0.54, respectively); shape and vascularity showed substantial agreement (kappa = 0.61 and 0.64, respectively). Intra-observer variability showed better agreement (kappa > 0.61). For the four radiologists, the overall sensitivity was 88.2%, specificity 78.7%, positive predictive value 76.2%, negative predictive value 89.6%, and accuracy 82.8% (100).

 

There have been investigations into the differences in the biologic behavior of thyroid cancer based on preoperative US features. One study in patients with follicular variant of papillary thyroid cancer showed more aggressive cancer behavior when there were preoperative US characteristics that suggested malignancy when compared with those without such features (101).

 

In children, there is no consensus about the value of US characteristics as predictors of malignancy. One group is not enthusiastic (102). Another group of investigators who also did molecular genetics on aspirated thyroid nodules offered a more positive view (103).

 

SONOGRAPHY OF A PALPABLE DOMINANT NODULE IN AN ENLARGED OR NODULAR THYROID

 

We now know that a so-called “solitary nodule” in an otherwise normal thyroid gland often is a nodule in a gland that has sub-clinical nodules (see below). Even more frequently, clinicians encounter patients with a “dominant” nodule in an enlarged or nodular thyroid. It is generally agreed that for a dominant thyroid nodule FNA is the best test to assess malignancy. Furthermore, a diagnostic strategy using initial FNA was found to be more cost-effective than starting with ultrasonography or scintigraphy (104). Evidence is mounting in support of US for patients with palpable uninodular thyroid disease and goiter because non-palpable nodules are common and a few of these are cancerous. In many countries, US is being employed more often than previously especially when palpation is uncertain or skills are tentative. US has been reported to provide information to the clinician that importantly alters management in 63% (109/173) of patients who were referred to a tertiary endocrine group. Sonography showed an indication for needle aspiration or demonstrated that the procedure is not necessary. Among 114 patients who were referred because of a solitary thyroid nodule, US detected additional nonpalpable thyroid nodules that were at least 1 cm in diameter in 27 patients and no nodules in 23 subjects. Thus, among 50 patients US lead to an almost equal number of additional aspirations or no biopsy. Among 59 patients who were referred because of goiter, US showed no nodule in 20, thus avoiding biopsy, and revealed nodules at least 1 cm in diameter in 39 patients that required aspiration (27).

 

THE NON-PALPABLE THYROID NODULE OR INCIDENTALOMA

 

Sonography demonstrates micronodules (incidentalomas) of the thyroid that are less than 1 cm in diameter, non-palpable, common, and of questionable clinical significance (105) (Figure 6). Whereas palpable thyroid nodules occur in 1.5 - 6.4 % of the general population (106), the incidence of non-palpable nodules is at least ten fold greater when the population is screened by US (107). Non-palpable nodules increase with age to involve approximately 50% of older adults, especially women. The risk of malignancy among palpable nodules is approximately 10% and in micronodules had been thought to be considerably smaller (108). However, investigations reported a similar incidence of cancer in palpable and non-palpable thyroid nodules (109-111). One study actually reported a higher incidence of malignancy among incidentally discovered nodules than among clinically detected lesions (112). However, most microcarcinomas are clinically indolent. Yet, among 317 incidentalomas that were aspirated from 267 patients the rate of malignancy was 12% in a retrospective analysis. In addition, in this subgroup, 69% (25/36) of patients had either extra-thyroidal extension or regional node involvement and 39% had multifocal tumors at surgery, suggesting that the small size alone does not guarantee a low risk in incidentally found thyroid cancers (113). Therefore, the clinical impact of incidentalomas is quite small but they cannot be ignored. Rather, they should be monitored at intervals with US for suspicious characteristics, size, adenopathy, other clinical features, and - perhaps – a thyroglobulin measurement.

 

How useful are the sonographic characteristics of non-palpable nodules as an index of malignancy? Some insight to this question has been gained from a study performed on 16,352 self-referred patients in a health care center. Among 1,325 non-palpable thyroid nodules in 1,009 patients, marked hypoechogenicity, an irregular shape, a taller-than-wide shape, a well-defined spiculated margin, microcalcification, and an entirely solid nature were significant predictors for malignancy (P < .05) (114).

Figure 6. Sonograms of the right thyroid lobe in the longitudinal plane showing a 2.7 x 3.2 mm hypoechoic nodule that is delineated in the lower panel by the xx and ++ symbols. Note the linear hypoechoic structure below that (arrow). In the upper panel the bright structure is a Doppler signal and indicates a blood vessel below the nodule. The nodule is not vascular.

Non-palpable nodules or those that have escaped detection on examination are often discovered incidental to imaging of the neck for vascular or neurological reasons. They may be discovered during upper GI endoscopy (115). These thyroid lesions should be managed like other “Incidentalomas”, with observation, dedicated thyroid US, aspiration biopsy, or even surgery, as indicated by the data and mature judgment. This opinion is supported by an investigation in which thyroid nodules were found in 9.4% (116) of 2,004 consecutive patients undergoing carotid duplex ultrasonography. There was high correlation of the nodules with standard thyroid ultrasonography (presence of nodules, 97% (64 of 66) and size, r = 0.95, P<.001). Twenty-one (32%) of the nodules were smaller than 1 cm. Only two patients with unilateral masses noted on carotid duplex had a normal thyroid sonogram. Twenty-nine of the 66 (44%) were selected for fine-needle aspiration biopsy due to cancer-risk criteria. These results lead to surgery in 13 of the 66 (19.7%); pathology included 5 patients with cancer (3 with papillary cancer, 2 with follicular cancer), 4 patients with a follicular adenoma, and 2 with lymphocytic thyroiditis (117).

 

How successful is ultrasound-guided cytological diagnosis of non-palpable nodules? Intuitively, it is generally believed that success varies inversely with nodule size but the data are not conclusive. The diagnostic yield with nodules as small as 10 mm has been reported as comparable to that of aspirating larger nodules (110). Adequate material for cytological analysis reportedly was obtained in 64% of 0.7-cm lesions and 86.7% of 1.1 cm nodules. For nodules ≥1 cm, the sensitivity was 35.8% and false-negative results were seen in 49.3% (118). In contrast, a study of aspirates from 317 nodules in 267 patients reported that the size of impalpable nodules (0.9 +/- 0.3 cm, a range of 0.2 cm to 1.5 cm) was not related to the probability of getting an adequate specimen for cytological diagnosis (105). Of 201 thyroid nodules that were 5 mm or smaller in size, in 180 patients, investigators reported that were 162 adequate specimens (81%) (115). Personally, we generally do not routinely aspirate nodules smaller than 8 mm but have had limited diagnostic success in sampling incidentalomas as small as 5 mm. Based on a review of the literature, Mazzaferri et al. have concluded that thyroid nodules 5 mm or smaller have a high rate of false positive ultrasound findings and often yield inadequate cytology on fine needle aspiration biopsy. Therefore, they advise that nodules of this size with no other suspicious clinical findings should not undergo routine needle biopsy, even if they appear ultrasonographically suspicious (119). In contrast, more optimistic results have been reported. When ultrasound-guided FNA was done on 5 mm or smaller nodules, surgical confirmation was obtained in 62 nodules and there were 34 (55%) true positives, 0 (0%) false positives, 23 (37%) true negatives, and five (8%) false negative results for malignancy (sensitivity 87%, specificity 100%, positive predictive value 100%, negative predictive value 82%, accuracy 92%, false positive rate 0%, and false negative rate 8%) (120). However, considering the minimal clinical impact of thyroid microcarcinomas, the clinical value of aspirating nodules this small is uncertain. Importantly, the American Thyroid Association guidelines recommend avoiding cytological evaluation of nodules less than 1 cm in size (1). A selected, population base study of 485 thyroid nodules suggested that this advice would not miss any thyroid cancers with high risk features (121).

 

US has changed our clinical perception of what is a normal thyroid gland and has advanced medical practice. Current high-resolution ultrasonography of the thyroid has permitted the clinical detection of nodules that are as small as 2 mm. It frequently demonstrates that what appears to be a normal gland, actually contains a non-palpable nodule or is a subclinical nodular goiter (78,108). It may show that a solitary nodule on palpation really is a clinically palpable nodule in a gland that is subclinically multinodular. Pathologists have long known about the ubiquitous nature of thyroid micronodules and the relative frequency of occult thyroid carcinomas, which are rarely of clinical consequence. Now the clinician is often confronted with the challenge that micronodules are discovered as a consequence of investigations for orthopedic, neurological, vascular pathology or other pathologies, or together with a palpable thyroid nodule. As a rule, their discovery often results in needless expense, concern, and therapy because it is not known which of the myriad nodules that have been revealed is, or will progress to become a cancer with clinical impact.

 

It remains for future investigation to determine the appropriate management for micronodules. Because it is rare for one of these lesions to represent an occult thyroid cancer and rarer still for one to become a clinically significant malignancy, non-selective surgery, which has an exceedingly small yield of cancer and is not risk-free, seems ill advised. Also inappropriate is dismissal of the problem as unimportant. Rather, to this author, periodic sonographic reassessment for possible growth of the nodule or change in characteristics appears preferable. The role of ultrasound guided needle biopsy in the management of these patients, especially when there is a history of exposure to therapeutic x-ray will be discussed below.

 

Not all "incidentalomas" in the neck are thyroid in origin. Parathyroid adenomas have been observed within the thyroid gland or in the usual parathyroid anatomic location when ultrasonography was performed to evaluate thyroid nodules (122,123). An example of a misidentified lesion that demonstrates the extent of the lack of specificity of a “sonographic nodule” is an esophageal tumor that was erroneously characterized as thyroid (124).

 

SONOGRAPHY OF LYMPHADENOPATHY

 

Even in a patient with thyroid cancer, enlarged benign thyroid lymph nodes are more common than malignant ones. Nevertheless, US may be useful to diagnose and if appropriate, periodically reassess lymphadenopathy in the patient with a history of thyroid cancer, or if there is a history of exposure to therapeutic radiation in childhood or adolescence. A high-resolution ultrasound system equipped with a high-energy linear probe, a 12 -15 MHz transducer, B-Mode and Doppler capability, experience, and diligence are required to detect lymphadenopathy.

 

NORMAL LYMPH NODES: Normal lymph nodes are depicted by sonography as approximately 1 X 3 mm, well-defined, elliptical, uniform structures that are slightly less echo-dense than normal thyroid tissue and that have an echo-dense central hilum. Lymphadenopathy that is reactive to infection may be larger but the lymph nodes tend to maintain an oval shape; in contrast, malignant nodes more often have a "plump" rounded shape (125) (Figures 7, 8).

Figure 7. Sonogram in the longitudinal plane of the left side of the neck after thyroidectomy showing a small, elliptical benign appearing lymph node in the jugular region. It is delineated by the xx and ++ symbols.

Figure 8. Sonogram in the transverse plane after thyroidectomy for cancer, from a muscular man. There was no palpable mass. The image shows a rounded lymph node that was cancer. C=carotid artery, m=muscle, ++ marks the node.

Especially in children, inflammatory lymphadenopathy is common, which may complicate a search for cancerous nodes. Tuberculous cervical lymphadenitis can mimic metastatic lymph nodes from papillary thyroid carcinoma (126). Indeed, especially in a region where tuberculosis is endemic, even when a patient is known to have papillary thyroid cancer, adenopathy reportedly is more commonly due to tuberculosis than to thyroid cancer (127).

 

A source of confusion in diagnosing lymphadenopathy especially in the elderly and obese subjects is fatty change in a node that may mimic a macro-metastasis at palpation. US can offer useful insight. In one study, of 110 selected patients with a total of 247 nodes, the central “fatty”, hyperechoic hilum was quite large, extending more than one third of the transverse diameter. The ratio of the long to short axes of the node and the parenchyma to fat (P:F) were obtained. Differences between mean P:F ratio in diabetic and nondiabetic patients were significant (p=0.045). The mean P:F ratio was negatively related to body mass index (BMI) (r=0.62, p=0.015) and age (r=0.54, p=0.024). All of the nodes examined with a mean P:F ratio ≤ 1.2 (58) were found in patients older than 72 years and with a BMI higher than 27.8 (30).

 

CHARACTERISTICS OF MALIGNANT LYMPHADENOPATHY: There are ultrasonic characteristics of lymphadenopathy that correlate in a clinically useful fashion with metastases from thyroid cancer. The features that correlate most highly include microcalcifications, a spherical shape, a large cystic space, loss of the hilum, and neo-vascularization that is characterized by blood vessels penetrating the node from its periphery rather than its hilum.

The results of investigations are reasonably confirmatory. In one study of 19 patients who were referred for lymph node dissection, 578 nodes were removed, 103 of which were ultrasonically detected. The authors analyzed only the 56 nodes (28 benign and 28 malignant) that were unequivocally matched for US and pathology. The authors reported that the major criteria of malignancy were: cystic appearance, hyperechoic punctations, loss of hilum, and peripheral vascularization. If there was only cystic appearance or hyperechoic punctations, the risk of malignancy was lower but still suspicious of malignancy. They were of the opinion that nodes with “a hyperechoic hilum should be considered as benign, that peripheral vascularization has the best sensitivity-specificity compromise, and that round shape, hypoechogenicity, and the loss of hilum taken as single criterion are not specific enough to suspect malignancy”. The reported sensitivity and specificity of these criteria were 46 and 64% for round shape (long to short axis ratio < 2), 100 and 29% for the loss of fatty hyperechoic hilum, 39 and 18% for hypoechogenicity, 11 and 100% for cystic appearance, 46 and 100% for hyperechoic punctations, and 86 and 82% for peripheral vascularization (128). In several other investigations, the two most useful diagnostic characteristics are the ratio of the longitudinal to the transverse diameter of a lymph node ( L/T ratio) and the absence of a central echogenic hilum (125,129-131). In one study, the L/T ratio was less than 1.5 in 62% of metastatic nodes and greater than two 2 in 79% of reactive nodes (132). A wide cortex or narrow hilum was observed in 90% of malignant lesions, but only 45% of benign nodes. The absence of a hilum was observed in 44% of malignant lesions, but in only 8% of benign nodes. In this study the size and uniformity of a lymph node was not helpful in differentiating benign or malignant nodes.

The location of adenopathy in proximity to the thyroid in the central compartment of the neck may also be indicative of thyroid cancer. Multivariate analysis in an investigation addressing this question showed that only central location (odds ratio, 4.07; 95% confidence interval (CI), 1.64 to 10.10) and size (odds ratio, 5.14; 95% CI, 1.64 to 16.06) remained as significant corollaries of cancer (57).

 

It is not clear whether additional information about the nature of lymphadenopathy may be offered by color and spectral Doppler investigation. Although one group of investigators found that malignant nodes (29/32) more often than benign ones (6/16) demonstrate enhanced color flow signals (133), another group observed abundant color flow signals in all enlarged lymph nodes (134). There may be some diagnostic value to examining the ratio of systolic and a diastolic blood flow in a lymph node, which is called the resistive index. It has been reported that cancerous lymph nodes have a high resistive index (mean 0.92) while reactive nodes have a considerably lower value (<0.6) (134). Another investigator reported that metastatic nodes from papillary carcinoma frequently demonstrate prominent hilar vascularity similar to reactive nodes (135).

 

Among abnormal nodes that had cystic spaces, one study showed a high likelihood of papillary thyroid cancer as assessed by FNA. Cystic changes were not seen in 43 of 63 pathologic nodes that were either metastatic from other malignancies (22 patients) or benign reactive lymphadenopathy (21 patients) (136). Since cystic spaces due to necrotic material may be seen in tuberculous nodes, caution is warranted when one interprets the clinical meaning of this finding. An important diagnostic aspect of cystic masses that are lateral to the thyroid is demonstrated by one report that showed that among 37 adults (age 16-59 years), 10.8% of cervical cysts were lymphatic metastases from occult thyroid carcinoma (137). Others have reported similar observations and the point has been made that in younger patients, the lymph nodes might appear purely cystic, thereby mimicking branchial cysts (138).

 

In some studies, the addition of CT of the neck to US was found to be slightly superior to sonography alone for the detection of metastatic papillary thyroid cancer lymph nodes in the lateral compartment of the neck but not in the central compartment (139). Another investigation suggested that high-resolution ultrasound is accurate in preoperative evaluation for extra-thyroidal tumor extension and lateral lymph node metastasis. In contrast however, in this study, CT had greater sensitivity than ultrasound alone in the detection of central lymph node metastases (140).

 

In patients with suspected recurrent thyroid cancer, however, a combination of diagnostic techniques maybe necessary to differentiate a true recurrence and noncancerous images, called cryptic findings (141). Fused I-131 whole body scan SPECT when coupled with CT or PET can elucidate the nature of such images. Many of these findings prove to be inflammatory in nature, thereby avoiding unnecessary treatment with I-131 (142). Precisely directed US has been reported to enhance the specificity while maintaining sensitivity, especially in the neck and superior mediastinum (143).

 

Cytological, immunocytological, and biochemical (thyroglobulin) analysis of enlarged cervical lymph nodes, using the ultrasound-guided aspiration biopsy technique described below, can differentiate thyroid cancer metastases and inflammatory lymphadenopathy (144). It is important to add that is not necessary to require a classical cytological diagnosis of thyroid cancer in a lymph node aspirate. Any evidence of thyroid cells or the detection of thyroglobulin in the node is adequate proof of cancer; thyroid cells or thyroglobulin do not belong in non-cancerous nodes.

 

WHAT A THYROID ULTRASOUND REPORT SHOULD INCLUDE

 

The thyroid ultrasound report must answer the question that has been posed by the clinician and not be just a routine recitation. The ultrasonographer or the thyroidologist who interprets the images should note and record in the report the features listed in Table 3 and call specific attention to the features that reveal a higher than average risk of malignancy.

 

TABLE 3. ESSENTIAL ELEMENTS OF A THYROID ULTRASOUND REPORT

1. Each lobe and isthmus

A. Dimensions of Lobes (cm)

B. Shape of Lobes, (conventional shape or indentations and where they are)

C. Echogenicity of Lobes

·       Hyperechoic

·       Hypoechoic

·       Isoechoic

·       Heterogeneous

D. Vascularity of Lobes

·       Physiologic

·       Increased

·       Decreased

·       Avascular

E. Nodule(s) in Each Lobe or Isthmus

·       Location

·       Number of Nodules (1 or 2, a few, multinodular)

·       Do all nodules have uniform characteristics?

·       Does one nodule have noteworthy characteristics? *

·       Margins

o   Distinct

o   Ill-defined

·       Halo

o   Continuous

o   Discontinuous

·       Echogenicity of nodule

o   Hyperechoic

o   Hypoechoic *

o   Isoechoic *

·       Composition

o   Solid

o   Cystic

o   Complex (solid with cystic component)

·       Shape

o   Globular

o   Irregular

o   Taller-than-wide *

·       Vascularity

o   Physiologic

o   Decreased

o   Avascular

o   Increased

o   Peripheral

o   Central *

·       Calcifications

o   Punctate *

o   Coarse

o   Egg-shell

·       Other features

o   Puff-pastry “Napoleon-like” layers that are alternatingly echo-dense and echo-poor

o   Spongiform

o   Bright spot with “comet tail shadowing”

2. Lymph nodes *

·       Location

o   Ipsilateral to nodule

o   Contralateral to nodule

o   Standard levels or relation to another anatomic structure

·       Shape

o   Oval, elliptical

o   Globular *

·       Hilum

o   Fatty

o   Vascular

o   Absent *

·       Margin

o   Well-defined

o   Ill-defined *

·       Vascularity

o   Increased

o   Physiologic

o   Blood-flow from periphery rather than hilum *

·       Calcifications

o   Punctate *

o   Coarse

o   Egg-shell

·       Composition

o   Solid

o   Complex with cystic component *

·       Impact on surrounding structures

o   Deforming or infiltrating *

o   No impact

3. Extra-thyroid bed mass

·       Anatomic site (thyroglossal? sub-lingual?)

·       Ultrasonic characteristics

4. Comparison with prior examination, prior date, _____________

Comparison based on _____report or _____images?

·       Technically comparable? _____Yes  _____ No

·       Compare characteristics of lobes

·       Compare characteristics of nodules

·       Compare characteristics of nodes

*Enhanced risk of thyroid cancer

 

It is both logical and useful to separate a report into: 1) a brief statement of the reason for the US in the context of the history including pathology if any, 2) an objective narration of the findings, which represents the anatomy as defined by ultrasound, and 3) a brief, subjective, summary and conclusion or opinion. Mixing concepts 2 and 3 can be confusing to the clinician by mistaking what the interpreter sees in distinction to what he/she thinks, which may lead to variance in management.

 

There have been several attempts to codify thyroid ultrasound reports and stratify cancer-risk. An example is the Thyroid Imaging Reporting and Data Systems (TIRADS). One of them has, for example, been correlated with needle-biopsy results in 1959 thyroid nodules. The classifications were expressed as 1-5 with the following percentages of malignancy: TIRADS 2 (0% malignancy), TIRADS 3 (<5% malignancy), TIRADS 4 (5-80% malignancy), and TIRADS 5 (>80% malignancy). In a sample of 1097 nodules (benign: 703; follicular lesions: 238; and carcinoma: 156), the sensitivity specificity positive predictive value, negative predictive value, and accuracy were 88, 49, 49, 88, and 94%, respectively. The major problems of this approach are that the classifications are subjective and, as we shall see below, environmental and other factors may influence ultrasound appearance of nodules. Nevertheless, uniform, reproducible, and relevant reporting should facilitate clinical management and help the clinician to select nodules for aspiration biopsy, surgery or observation (145).

 

In one investigation, the American College of Radiology TIRADS structured reporting improved the “quality” of thyroid ultrasound reports. The authors reported an improved description of features that were predictors of malignancy. In addition, there was an increased number of definitive management recommendations that resulted in reducing the number of biopsies. In this author’s opinion, the value of management advice and reduced biopsies based on image appearance alone needs to be established on more firm evidence than is currently available (146,147). Attempts are in progress to unify thyroid ultrasound reporting features and recommendations, as well as the various TIRADS systems, across the various medical specialty societies and also internationally (148).

 

Several novel computer-based approaches taking advantage of developments in artificial intelligence for malignancy risk assessment of thyroid nodules in ultrasound images have been suggested. Local echogenic variance and boundary features are utilized to incorporate information associated with local echo distribution. Analysis of variance is performed utilizing feature vectors derived from all combinations of the characteristics under study. The classification results are evaluated with the use of receiver operating characteristics that are capable of discriminating between medium-risk and high-risk nodules (149). This promising field Is it in its infancy.

 

SONOGRAPHY IN THE PATIENT WITH A HISTORY OF HEAD AND NECK THERAPEUTIC IRRADIATION IN YOUTH

 

In the patient with a history of therapeutic irradiation to the head and neck in youth, the thyroid cancer risk may be as high as 30%. Since thyroid nodules may be detected with ultrasound before they become large enough to be palpable, sonography has been employed to screen irradiated people for tiny nodules. This selection process is quite inefficient because in the process, many more benign nodules are found than malignant ones. Furthermore, as has been observed after the nuclear event in Fukushima, Japan, the detected papillary microcarcinomas tend to be indolent (150). Consequently, some clinicians prefer not to detect micronodules contending that they are clinically irrelevant. In contrast, the author prefers to obtain a potentially useful baseline sonogram, but not to act on the presence of a micronodule unless a repeat sonogram after an interval of time demonstrates its growth or other circumstances that heighten the suspicion of malignancy. It is this author’s practice to obtain a thyroglobulin level when a micronodule is detected and again a year later in order to assess whether it has risen significantly. However, this conjecture and its validity have not been studied rigorously.

 

SONOGRAPHY TO MONITOR CHANGES IN THYROID OR NODULE SIZE

 

Changes in the size of a nodule may be clinically important, but difficult to perceive clinically. However, sonography can accurately and objectively assess changes in the volume of thyroid nodule(s) and the thyroid gland over a period of time. This is especially important during the course of therapy with thyroid hormone, in patients with a history of exposure to therapeutic irradiation, and when there is a history of thyroid cancer. Interval studies on such patients may be performed without discontinuing thyroid suppressive therapy, administering recombinant human TSH, or any preparation of the patient. Consequently, it is a simple matter to compare serial records, which may lead to changes in thyroid management earlier than palpation alone would warrant. Furthermore, since most patients tend to change doctors and residence over a period of years, an objective assessment of the size and volume of the thyroid gland or nodules will greatly facilitate the continuity of care.

 

Caution is warranted in interpreting the meaning of changes in the volume of thyroid nodules shortly after fine-needle aspiration has been performed. Bi-directional volume changes after the biopsy have been reported (151). Therefore, it is appropriate to assess nodule size at least weeks after FNA. For the same reason, to assess nodule size after a period of observation or suppressive therapy, the US should be done before another FNA is performed.

 

A Downside to Serial Sonography to Monitor Changes in Thyroid or Nodule Size/Volume               

 

Although it makes intuitive sense to repeat sonograms at some interval to detect early evidence of growth or malignant change, there is a downside to this practice too. There is indication that frequent screening with serial neck US is more likely to identify false positive abnormalities rather than significant disease. Therefore, coupling repeat US with clinically suspicious events or examination is warranted (152).

 

SONOGRAPHY IN THE PATIENT WHO HAD THYROID CANCER

 

Sonography has become a most useful imaging procedure in patients who have had either partial or complete thyroidectomy (Figure 8) (153). Sonography is done without interrupting the therapy with thyroid hormone, which is used universally in the thyroid cancer patient.

 

One study, in which 110 patients who had partial or total thyroidectomy for thyroid cancer were examined every 1-2 years, showed that ultrasonography is the most sensitive and important way to image postsurgical recurrences of thyroid carcinomas and lymphadenopathy in the neck (154). This observation is most important because recurrence in the neck is by far the most common location of reappearance of thyroid cancer. The authors suggest routine use of US in these patients.

 

Furthermore, a five-year observational study of 80 patients investigated the optimal initial follow-up strategy for patients who had near total thyroidectomy for papillary thyroid microcarcinomas (155). Sonography identified lymph node metastases not only in two thyroglobulin-positive patients but also in one thyroglobulin-negative patient. Importantly, after observation for 32 +/- 13 months after surgery, all US node-negative patients had undetectable thyroglobulin levels while on suppressive therapy and US remained negative. In contrast, whole body scanning showed no “pathological” uptake in any patient and was essentially useless, probably because differentiation of postoperative gland-remnants and tumor was not possible. Yet, radioiodine uptake in the region of the thyroid bed did correlate with recombinant human TSH (rhTSH)-stimulated thyroglobulin levels: 1 ng/ml or less in 45 patients without uptake and more than 1 ng/ml in 35 patients with uptake (r = 0.40, P < 0.0001). The authors concluded that in their population, the thyroglobulin probably derived mainly from small normal tissue remnants rather than cancer. Therefore, they contend that mild elevations of thyroglobulin are also of limited diagnostic value.

 

Sonography can detect post-operative thyroid remnants in the thyroid bed and thyroglossal region even when surgeons report a total thyroidectomy. One investigation found US remnants in 34 of 102 cases (156). This author believes that the frequency of remnants is highly experience- and surgeon-dependent.

 

It is important to appreciate that sonography may yield clinically erroneous or misleading results if it is performed during the initial several months following the surgery. During this time there may be abundant lymph nodes and heterogeneous, sono-dense regions that probably reflect postoperative changes such as edema and inflammation.

 

Sonography may serve to uncover unsuspected disease. After less than total thyroidectomy, sonography will detect nodules in the thyroid remnant, post-operative thyroid bed or in the contra-lateral thyroid lobe, which could be benign tissue or tumor. After total thyroidectomy but not following partial thyroidectomy, nodules and adenopathy are more likely to represent cancer when the concentration of thyroglobulin is elevated. Sonography may detect this disease even before it has grown sufficiently large to be palpable.

 

In patients in whom thyroid carcinoma has been diagnosed as the result of metastases to bone, lung or cervical nodes, sonography can detect an occult thyroid primary cancer even if the thyroid gland is normal to palpation.

 

One investigation has shown that rhTSH-stimulated serum thyroglobulin measurements combined with neck ultrasonography has the highest sensitivity in monitoring differentiated thyroid carcinoma in children, and many investigators believe in adults also (157). One group of investigators has reported that even when thyroglobulin levels remain low or undetectable after stimulation with rhTSH, sonography may identify lymph node metastases from thyroid cancer (158).

 

It may be difficult to differentiate a suture granuloma from recurrent thyroid cancer. A case report demonstrated a nodule that mimicked recurrent thyroid cancer on sonography and 2-{fluorine-18}-fluoro-2-deoxy-D-glucose positron emission tomography, but the diagnosis of a suture granuloma was confirmed by a US-guided fine needle aspiration biopsy (159). The ultrasonic appearance of suture granulomas includes echogenic foci larger than 1 mm in diameter (p<0.05) that are paired (p<0.05), and usually are clustered centrally or on near the middle of the nodule, unlike those in recurrent carcinomas (p<0.05) (160).

 

US is useful in the operating room during surgery. Intra-operative ultrasonography may enhance the ability to locate and resect recurrent thyroid cancer that does not accumulate radioactive iodine. Experience in seven patients suggests that sonography was particularly helpful after external beam radiotherapy to identify tumor nodules of 20 mm or less that were invasive or adherent to the airway (161). One investigation reported that intra-operative ultrasound performed by the surgeon influenced the management in 57 percent (41/72) of patients by identifying non-palpable adenopathy (162). However, one wonders if resection of non-palpable or even larger deposits of differentiated thyroid cancer will affect outcomes since historically even bilateral radical neck dissection was not associated with enhanced results when compared with thyroidectomy alone. Nevertheless, excision of non-palpable nodules that are in proximity to a vital structure could be palliative if the cancer is removed before it invades. In this author’s opinion intra-operative US could become standard to look for and remove undetected nodes after the surgeon has completed a thyroidectomy for cancer, even after a compartmental node dissection, before “closing”.

 

Preoperative sonography in the cancer patient may be associated with decreased recurrences. One group of investigators studied 275 patients who underwent pre-operative US and had a median follow-up of 41 months. They reported that patients who have had recurrence of papillary thyroid cancer were at an increased risk for subsequent recurrence of the tumor in the neck. US before the initial operation and followed by compartment-oriented surgery based on the US was related to decreased subsequent recurrence rates (162).

 

With respect to the lateral compartment of the neck, preoperative US is an excellent predictor of outcome for disease-free interval. Furthermore, a surgical approach based on preoperative US provides excellent long-term regional control (163).

 

SONOGRAPHY IN CONJUNCTION WITH NEEDLE BIOPSY

 

Fine needle aspiration biopsy of thyroid nodules and adenopathy in adults, children, and adolescents has become a major diagnostic tool that is safe and inexpensive (133,164-169). Major untoward effects are very uncommon and include bleeding (especially in patients who use anticoagulants or antiplatelet agents or those who have a bleeding diathesis), hoarseness, and infection. Many authorities, however, contend that it is reasonably safe to continue anticoagulants, including the newer novel agents, in patients who been taking these medications when performing an FNA (170). Having seen a few patients who have experienced excessive bleeding as a result of FNA while using aspirin or anticoagulants, it is this author’s practice to discontinue any agent that interferes with coagulation of blood prior to performing and FNA. Actually, there have been very occasional reports of fatal cervical hemorrhage related to FNA (171). Furthermore, even if there is no increased risk of significant hemorrhage due to FNA, the specimen maybe diluted by unnecessarily abundant red blood cells, complicating cytological interpretation. Seeding the needle track with thyroid cancer is a remote consideration (172,173).

 

Indications

 

The major indications for ultrasound-guided FNA are summarized in Tables 4 and 5. Ultrasound has made placement of the needle more accurate especially for small or complex nodules or nodes. Cytopathological interpretation is usually clinically satisfactory and promises to improve with tissue marker analysis of specimens (174). However, the accuracy of the puncture varies considerably depending on factors that are related not only to the operator and the cytologist, but also to the patient. The latter conditions include the size, homogeneity and vascularity of the nodule or node, its location in the neck, sampling errors, and the habitus of the patient. These issues affect biopsy technique.

 

Table 4. Needle Biopsy with Ultrasound Guidance is Generally Reserved For:

1. A small nodule in an obese, muscular, or large framed patient.

2. Nodules that are barely palpable or non-palpable

3. Nodule size less than one centimeter.

4. A nodule that is located in the posterior portions of the thyroid gland.

5. A dominant or suspicious nodule within a goiter.

6. All nodules that yielded non-diagnostic results on a free-hand biopsy.

7. Complex degenerated nodules if a prior biopsy without ultrasound guidance has not been diagnostic.

8. Incidentalomas that have been detected ultrasonically in patients with high risk factors for thyroid cancer such as exposure to therapeutic x-ray.

9. Small lymphadenopathy.

 

Table 5. Features That Warrant Percutaneous Fine-Needle Aspiration Biopsy of a “Solitary” Nodule or a “Special” Nodule in A Goiter

1. Clinical Features

 a. History of head and neck irradiation in youth

 b. Family history of medullary (or signs & symptoms) or less so papillary thyroid cancer

 c. Unusual firmness without calcification

 d. Growth of nodule especially during suppressive therapy

 e. Lymphadenopathy

2. Ultrasonic Features (at least two “suspicious” ultrasound features)

 a. Hypoechoic nodules with one or more of the following

  i. Irregular margins

  ii. Enhanced intranodular vascular spots (central vascularity)

  iii. Microcalcifications (punctate calcifications)

  iv. Blurred margins

  v. Taller-than-wide nodule shape

  vi. Enlargement of a nodule when compared to prior examination

 b. Lymphadenopathy (palpable or ultrasonographic)

3. In a goiter, biopsy the nodule that has “suspicious” ultrasonographic features rather than the largest nodule.

4. The size or number of nodules in a gland does not correlate with risk factors

 

Methods

 

Thyroid nodules or lymph nodes that are palpable are often biopsied directly. In some cases, correlation of the palpable anatomy with a sonographic film or screen image may be useful. In such cases, for small, complex, or deep nodules, or when a palpation-guided biopsy has resulted in an insufficient specimen, ultrasound-guided fine needle biopsy is employed (27,175), but with added cost ($289 by one estimate (176) and some inconvenience. Direct, real-time ultrasound guidance improves accuracy in puncturing the nodule. Ultrasound-guided biopsy is always required for impalpable incidentalomas and even then, it is difficult to reliably sample lesions smaller than 10 mm, as discussed previously.

 

Two methods for ultrasound-guided needle biopsy have been suggested: 1) A sonographer manipulates the transducer to locate the nodule and a second physician inserts the needle under direct vision. With practice, the assistance of a second operator is usually not required. 2) A special clamp is used to hold the transducer and fix the direction of insertion of the needle. Both require hand-eye coordination and experience is necessary to identify the spot on the skin over the target nodule to insert the needle. In our practice a dimple is produced on the skin with a blunt 1 mm wooden dowel directly over the nodule as the transducer identifies it. We have not found it appropriate to employ a "permanent marker" for this purpose, as has been suggested (177). Furthermore, this author finds the holder cumbersome and restrictive and prefers the free hand approach. With the free-hand method, the needle may be inserted parallel to, or at an angle to the ultrasound beam and at a short distance from the transducer, aiming at the nodule. The parallel approach may be technically challenging but is "comforting" to the operator because the image of the needle shaft may be viewed as it traverses the neck and into the nodule. Nevertheless, many experienced operators prefer an oblique to a perpendicular approach because of its simplicity and relatively fewer complications. The needle shaft is not imaged with this technique but its tip is seen as a very bright spot when it crosses the plane of the scan. The tip of the needle must be within the nodule during aspiration. However, even with ultrasound guidance, it is rather difficult to be certain that the tip of the needle is actually within a small nodule at the instant of aspiration, particularly if it is less than 7 or 8 mm in diameter (Figure 9).

Figure 9. Sonogram from an ultrasound guided fine needle aspiration biopsy showing a hypoechoic small nodule. The bright spot (above the arrows) is the tip of the needle within the nodule at the instant of aspiration. N=nodule.

Employing Doppler technique to identify and avoid puncturing blood vessels in the region of a nodule provides a distinct advantage of ultrasound-guided aspiration over palpation-guided biopsy. This precaution reduces the amount of blood in the aspirate and facilitates interpretation of the cytology (178). The same purpose is served by discontinuing antiplatelet and anticoagulant medication prior to a biopsy.

 

Samples of thyroid nodules and adenopathy may be obtained in either of two ways. One may aspirate the material with a syringe, employing a to and fro motion to produce a large quantity that frequently contains excessive blood, and complicates cytological examination. This author prefers the capillary technique that is done with a 25 or smaller gauge needle (without a syringe) using minimal trauma. The utility of a 27-gauge needle has been validated (179). Capillary action achieves a small, concentrated sample that remains in the needle shaft. The specimen is then expelled with an air-filled syringe quickly and gently on to a microscope slide (180). The diagnostic accuracy of the two methods is equivalent (181). One group has reported that the non-aspiration technique produces specimens of better quality and reduces inadequate results (182).

 

In this author’s experience, the capillary action aspiration method results in a superior cytological yield and the syringe/larger needle aspiration should be reserved for low-yield or fibrotic lesions.

 

Microscopic assessment of aspirates onsite for adequate cells by a cytologist at the time of the biopsy significantly reduces the number of non-diagnostic reports especially when the operator is not optimally experienced (183). It is likely that on-site assessment of cytopathologic adequacy of aspirates would help reduce the costs of needle biopsy, reportedly, by as much as 35.5%, by reducing unsatisfactory specimens that are sent to off-site cytologists (184). Furthermore, in some centers cytologists actually do the aspirations (185).

Ultrasound-guided FNA is an accurate method for identifying suspected recurrence of thyroid cancer in enlarged lymph nodes or in the thyroid bed.

 

Specimens

 

Obtaining material that is sufficient for a reliable cytological diagnosis involves competing realities. It is often necessary to do multiple punctures of a thyroid nodule to obtain enough cells even when ultrasound guided aspiration is employed. Yet, the first puncture is likely to be associated with less blood than subsequent samplings and may therefore be the best one for the cytologist to interpret. Especially for small nodules and those that are very vascular, gentle technique and point of service examination of the aspirate with a microscope to assess adequacy are important factors. In some cases when a hematoma has been produced it may be prudent to delay completing the aspiration until another day when the blood has been resorbed. Furthermore, especially when there are only a few benign-looking cells, the clinician should not be convinced that a nodule has been sampled adequately. Rather, a repeat biopsy after an interval of time may be prudent. In contrast, high suspicion is warranted when there are even a few cells that have features that are associated with malignancy. Sometimes cytology cannot suitably assess the pathological potential of a nodule. Such nodules are referred to as Atypia of Unknown Significance (AUS) or Follicular Lesions of Unknown Significance (FLUS), which will be discussed elsewhere. Caution is appropriate in accepting a report of negative cytology when the aspiration was done because a nodule grew during the course of suppressive therapy. Occasionally, when the specimen is inadequate, a better specimen maybe obtained with a needle with a larger lumen (186).

Effectiveness

 

One investigation retrospectively evaluated the effectiveness of ultrasound-guided fine-needle aspiration, in 37 patients previously treated for thyroid cancer, in identifying as cancer those cervical nodules that were suspicious of recurrence. There were 29 true positives, 6 true negatives, 1 false negative, and 1 inadequate biopsy. Therefore, US-guided biopsy had a sensitivity of 96.7%, a specificity of 100%, and an overall accuracy of 97.2% in detecting recurrence (187).

 

Caution with Respect to Negative Cytology in Children and Adults When the US is Suspicious

 

In a retrospective investigation of 35 children and adolescents, the global accuracy of FNA was 83%, with a sensitivity of 75%, and a specificity of 94%. Fourteen FNAs suggested malignancy (40%), only 1 of which was a false positive (7%). In significant contrast, 5 of the 21 FNAs suggesting benign lesions were false negatives (24%). These 5 cases had US findings suggestive of malignancy (188). Thus, a cautious approach is warranted especially in children when US findings suggest malignancy even if the cytology is benign.

 

In the postoperative thyroid bed, ultrasound-guided FNA may be particularly useful. In one series, among 21 cases there were 15 recurrent cancers, 5 benign nodules such as a parathyroid gland or regenerated normal thyroid, and 1 false positive (189).

 

There is limited ability to reliably aspirate and accurately diagnose a non-palpable nodule or node even with ultrasound-guidance (190). Ultrasound-guided cytological diagnosis of non-palpable nodules depends on the size of the lesion. One study suggested that the diagnostic yield of aspirating incidentally discovered, non-palpable 10 mm or larger thyroid nodules was high (99). Another study found that sampling of material that is adequate for cytological analysis was 64% for a 0.7-cm lesion and it increased to 86.7% when a nodule was 1.1 cm. For nodules that are 1 cm or smaller, the sensitivity was 35.8% and false-negative results were seen in 49.3% (108). In contrast, similar success has been reported in aspirating nodules that were 4 to 10 mm in size when compared with larger ones (191).

 

We have had mixed diagnostic success in sampling nodules or nodes as small as 5 mm. A few micro-cancers have been discovered in this way. The cost-effectiveness of aspirating nodules this small is uncertain considering the small (if any) clinical significance of thyroid microcarcinomas. We biopsy small lymph nodes that are “plump”. Generally, the width/depth must be almost 1 cm to yield adequate cells.

 

The cancer-predictive value of measuring thyroglobulin in the wash-out obtained from a cell-poor aspirate of nodes has been mentioned. Assaying thyroglobulin in aspirates from a thyroid nodule is not useful as an index of malignancy.

 

Suspicious Nodules in Goiters

 

It has been reported from a goiter zone in Italy that as many as 52% of histological malignant nodules in goiters were found only with the aid of ultrasound-guided FNAB. Therefore, the authors concluded that ultrasound-guided aspiration should be used in areas where multinodular goiter is endemic to assess nodules that are deemed suspicious by virtue of a hypoechoic pattern, a "blurred halo", micro-calcifications, or an intranodular color Doppler signal (192). In another report of patients with endemic goiter, 44 were selected for surgery based on suspicious ultrasonography and among 24 of them who had a “cold” nodule, aspiration biopsy revealed 2 with papillary cancer and surgery disclosed 2 more cases of papillary cancer and one case of insular cancer (193).

 

Predictors

 

One group has investigated the predictors and optimal follow-up strategy for initial non-diagnostic ultrasound-guided FNAs of thyroid nodules. Among 1,128 patients with 1,458 nodules that were biopsied over a 6-yr period, 1,269 aspirations (950 patients) were diagnostic, and 189 (178 patients) were non-diagnostic. The authors reported that the only significant independent predictor of non-diagnostic cytology (P < 0.001) was a cystic content of each nodule and the fraction of specimens with initial non-diagnostic cytology increased with greater cystic space. As emphasized above, for pathologic lymph nodes, in distinction to thyroid nodules, cystic degeneration is typical of thyroid cancer metastases. For example, diagnostic ultrasound-guided FNA was obtained on the first repeat biopsy in 63% of nodules and was inversely related to increasing cystic content of each nodule (P = 0.03). One hundred and nineteen patients with 127 nodules returned for follow-up as advised, and malignancy was documented in 5% (194).

 

For non-palpable thyroid nodules, the relative importance of sonographic features as risk factors of malignancy and the use of ultrasound-guided aspiration cytology was studied in 494 consecutive patients with nodules between 8-15 mm. It is noteworthy that 92 patients (19%) had inadequate cytology and were excluded from the study. Cancers were observed in 18 of 195 (9.2%) solitary thyroid nodules and in 13 of 207 (6.3%) multinodular goiters. The prevalence of cancer was similar in nodules greater or smaller than 10 mm (9.1 vs. 7.0%). The authors recommended that ultrasound-guided FNA should be performed on all 8-15 mm hypoechoic nodules with irregular margins, intranodular vascular spots or microcalcifications (194). In another study, among 402 patients with 8 mm to 15 mm non-palpable nodules, the cancers were most likely to be hypoechoic and solid, and have microcalcifications, irregular borders, or central blood flow. Since 125 (31 %) of nodules met those criteria, biopsies could be avoided in 69 percent of nodules, incurring a risk of missing 13 percent of the cancers (111).

 

It would appear that that no single parameter satisfactorily identifies the subset of patients whose nodule should be subjected to biopsy. In one investigation of 6,136 nodules in 4,495 patients, the best compromise between missing cancers and cost-benefit was achieved with at least two “suspicious” ultrasound features. The most useful were nodule shape (taller-than-wide), microcalcifications, blurred margins, and a hypoechoic pattern (195). Enhanced intranodular blood flow on Doppler examination also was reported as a helpful criterion (81). Another investigation of 1,141 nodules reported that logistic regression analysis showed that the size of the nodule affected the utility of ultrasonic characteristics of nodules in assessing cancer risk and selection for needle biopsy. In nodules smaller than 15 mm in size, hypoechogenicity (odds ratios, OR: 3.18), microcalcifications (OR: 19.12), solitary occurrence (OR: 3.29) and height-to-width ratio ≥1 (OR: 8.57) were independent risk factors for malignancy. The authors concluded that all lesions presenting at least one of the above-mentioned features should be biopsied (sensitivity 98%, specificity 44%). With nodules larger than 1.5 cm, the mentioned selection criteria were less sensitive than for smaller nodules. Useful features included, hypoechogenicity, taller than wide or microcalcifications (sensitivity 84%, specificity 72%) (196).

 

It is difficult to decide which nodule in a multinodular goiter to biopsy. Guidelines include selection by size, the ultrasound characteristics mentioned above, and most importantly nodules that are clinically suspicious. Perhaps one may be reassured that the pathology is likely benign when there are very many nodules in a goiter rather than a few. In one investigation of thyroid nodules that underwent ultra-sound-guided FNA, the authors found that the cancer risk is similar for patients with one or two nodules (over 1 cm) and decreases with three or more thyroid nodules (197).

 

It is particularly difficult to effectively select nodules for biopsy in an endemic goiter zone where nodules are ubiquitous. In one investigation in an iodine deficient region, a numeric score was assigned to nodules based on ultrasonic high-risk of cancer. Among 2,642 consecutive patients (3,645 nodules), nodules with a score of over 5.5 out of 10 had a 66% sensitivity and a 76% specificity for cancer, which was much higher than for those with lower scores. The data strongly facilitated the decision of which nodules to biopsy (94).

 

Combining the results of cytology and the tumor marker thyroglobulin after a patient has had a total thyroidectomy may enhance the accuracy of either single predictor of residual/recurrent thyroid cancer. One investigation reported that among 340 consecutive patients with differentiated thyroid carcinoma, who had been treated with near-total thyroidectomy, 131-I thyroid ablation, and TSH suppressive doses of L-thyroxine, rhTSH-stimulated thyroglobulin alone had a diagnostic sensitivity of 85% for detecting active disease and a negative predictive value of 98.2%. After adding the results of neck ultrasound, the sensitivity increased to 96.3%, and the negative predictive value to 99.5% (198). However, in one study, US and FNA did not seem useful to detect recurrent papillary thyroid cancer when the serum thyroglobulin level was undetectable (199).

 

One should be somewhat more suspicious that an incidentaloma could be cancerous when the patient has another non-thyroid cancer. In one investigation of 41 patients who had another cancer and who had an incidentally discovered thyroid nodule, surgical pathology revealed 4 papillary thyroid cancers, 4 microscopic papillary thyroid cancers, 2 metastatic cancers, and 7 benign lesions (200).

 

Not Biopsying Nodules that are Not Likely Malignant by US Criteria

 

Several studies have recognized sonographic morphological patterns that correlate with benign thyroid disease. The authors advise not biopsying these nodules or goiters in the interest of cost-effectiveness (201-203). In one study, 650 patients were identified for whom both a pathology report and ultrasound images were available. From an alphabetized list, the first 500 nodules were reviewed retrospectively. Most of the diagnoses were based on cytological rather than histological findings. Four patterns associated with benign disease were identified and seemingly attributed to colloid: spongiform configuration, cyst (cystic), a “giraffe pattern” (light blocks separated by black bands), and diffuse hyperechogenicity (201). One characteristic has borne the test of time: thyroid cancer is rarely if ever hyperechogenic.

 

It is useful to know that one group has reported that not performing a biopsy on nodules less than 5 mm in size seemed safe because when they underwent later surgery because of a 2 or 3 mm enlargement, there was no evidence of distant metastases or fatalities (204).

 

The rationale supplied by the authors (201,204) for not biopsying these nodules is that fewer biopsies, will lead to less delay of “necessary” biopsies and less false-positives. This author completely agrees with not biopsying non-suspicious nodules unless there are other factors that indicate cancer-risk. Fortunately, small low risk nodules generally do not adversely affect quality of life or survival. However, the practical outcome of this “leave the nodule alone” philosophy may result in a mind-set that, in this author’s opinion, should be avoided. The difference between focusing clinical attention on biopsying suspicious nodules and confidently dismissing nodules that “can be left alone” may result in a difference in the risk of missing a cancer. The outcome of the difference is similar to misjudging that a dog may bite, and giving it wide berth to avoid getting bitten, and mistaking that a dog does not bite and getting mauled.

 

Thus, I feel that we should not use tentative data from limited investigation to make a pivotal decision not to biopsy certain thyroid nodules and selection against surgery. A simple, logical, safe, inexpensive, and more reliable clinical attitude is employing sonography to enhance the efficiency and accuracy of biopsying ultrasonically suspicious nodules and nodules that have clinical or historical features that are associated with higher than average cancer-risk, and paying reasonable but not invasive attention to the rest of the nodules and the gland as a whole.

 

If the pattern approach to selecting nodules not to biopsy is employed by ultrasonographers, they should be cognizant that cancerous thyroid nodules in a radiation-exposed population may often not exhibit the classic ultrasonic features of malignancy. Rather, benign characteristics are more often encountered. Therefore in this setting especially, benign-looking nodules should be biopsied and not “left alone” (205).

 

Repeat Aspiration Biopsy in Patients with a Previously Benign Result

 

There is no consensus about how often FNA should be repeated after previous aspirations have indicated benign disease. Considerations that enter the decision include periodic US, historical risk factors, changes in physical examination, and even the patient’s or the physician’s level of anxiety. It seems reasonable that growth of the volume of the nodule, the emergence of adenopathy, or symptoms that suggest pressure on cervical structures such as hoarseness or dysphasia should be viewed with suspicion. Sometimes the observations of an ENT consultant may influence management. In this author’s experience routine re-aspiration rarely results in a discovery of malignancy. In a retrospective review of records of patients seen at the Mayo Clinic between January 2003 and December 2013 of 334 nodules with benign FNA, 85.3% were benign, 7.2% suspicious, 5.7% non-diagnostic, and 1.8% malignant. Importantly, the repeat FNA altered clinical management in only 9.5% of cases (206).

 

Non-Cytologic Examination of Aspirates

 

Ultrasound-guided aspiration can facilitate biochemical analysis. Needle washings of adenopathy (not applicable to thyroid nodules) may contain Tg, revealing papillary thyroid cancer even when there are insufficient or inadequate cells. It is noteworthy that assay of Tg in tissue is reportedly not effected by serum anti-thyroglobulin antibodies (207). Furthermore, the aspirate of nodules or lymph nodes may contain calcitonin in medullary cancer, a tumor marker such as galectin-3 (208) in papillary thyroid cancer, or lead to a non-neoplastic diagnosis such as tuberculosis (209) or amyloidosis (210). One anticipates that one day aspirates may be studied routinely for biochemical products, sub-cellular components, and, bacteriologic, fungal, or viral material. Examination for molecular genetic tumor markers will be discussed elsewhere.

 

Core Biopsy

 

There is also interest in sonographically-guided core biopsy of thyroid nodules. One group has concluded that percutaneous acquisition of tissue for histological rather than cytological evaluation is an accurate and safe alternative to aspiration biopsy in the assessment of thyroid nodules (211). However, one needs to be aware that there may be greater risk from core biopsy, including an occasional fatal case, in contrast to fine needle aspiration biopsy (170). Other investigators have reported on the use of an ultrasound-guided special compound needle that can accomplish both aspiration and core biopsy and suggest its use when prior aspiration has been unsuccessful (212).

 

SONOGRAPHY BY THE THYROID SURGEON

 

Although preoperative thyroid ultrasonography is not essential for successful surgery, many surgeons have come to recognize that it may be useful to identify pre-operatively suspicious lymph nodes in patients with biopsy-proven papillary thyroid cancer. Indeed, respected surgical authorities assert that ultrasound is an essential modality in the evaluation of thyroid malignancy and that surgeon-performed ultrasound has proved invaluable in the preoperative, intraoperative and postoperative setting (213-215). It has become increasingly popular for surgeons personally to perform a pre-operative sonogram since metastatic disease may not be clinically apparent to them intra-operatively. Preoperative identification of metastatic disease by cervical ultrasound may result in altering the surgical approach in as many as 40 percent of patients (27,28,97,192,216,217). Furthermore, pre-operative thyroid ultrasonography followed by compartment-oriented surgery may decrease recurrence rates in patients if performed before their primary operation (162). It is noteworthy that ultrasound guided FNA for thyroid nodules has been incorporated into some general surgery residency programs. (218).

 

Preoperative Labeling Lymph Nodes or Intraoperative US may Facilitate Intraoperative Identification and Removal of Adenopathy

 

It may be difficult for a surgeon to identify at surgery a small node that was discovered by preoperative ultrasonography. Insertion of a hook 20-gauge needle into a US-suspicious lymph node pre-operatively facilitates identification and removal of the pathological lesion (219-221). Alternatively, pre-incision, ultrasound-guided injection of blue dye into abnormal lymph nodes was very useful in the re-operative neck to facilitate their safe and efficient removal in one study (222). Other investigations have employed ultrasound-guided, preoperative injection of charcoal suspensions to tattoo the lesion. The rate of success is reportedly as high as 84-96% in small studies. However, in 1 case the charcoal was found several centimeters away from the lesion, tattooing a lesion behind a large blood vessel has not been achieved, and in 2 of 55 patients a charcoal dot remained in the skin after the procedure. There were no reported serious adverse effects (223,224). In strong contrast to this approach, other surgeons eschew selective removal of nodes in favor of classical compartmental dissection.

 

Intraoperative sonography may be very useful (161,225,226). In 26 of 31 patients with papillary thyroid cancer who had preoperative sonographic identification of adenopathy, intraoperative palpation did not locate adenopathy but intraoperative ultrasonography located and facilitated removal of the lesions (smaller than 10 mm in diameter) in all patients (225).

 

A method that may help find a thyroid sentinel node preoperatively has been reported in a porcine experimental model. US contrast agent and methylene blue dye were injected trans-cutaneously into the thyroid glands of pigs and draining lymphatic channels and sentinel lymph nodes were identified ultrasonically. Subsequently, a sentinel node biopsy was conducted; bilateral neck and upper mediastinal dissection was performed. The lympho-sonography of the thyroid gland in this porcine model correlated well with blue dye-guided sentinel node surgical biopsy. If applied to humans, this technique might potentially enable a detailed analysis of thyroidal lymphatic drainage and enhance thyroid cancer surgery (227).

 

SONOGRAPHY IN CONJUNCTION WITH PERCUTANEOUS THERAPEUTIC INTERVENTION

 

After an aspiration and cytology have demonstrated that a nodule is benign, ultrasound-guided puncture of a nodule may have a role in therapy to deliver medication or other therapy precisely into the lesion and to spare the surrounding tissue.

 

Percutaneous injection of ethanol has been used to reduce the function of autonomous thyroid nodules (228). One investigation has observed 34 patients, for up to three years, who had percutaneous ethanol injection of autonomous thyroid nodules. The patients required 1-11 sessions of 3-14 ml of ethanol injection (total amount of ethanol per patient: 20-125 ml). The authors report recovery of extra-nodular uptake on isotope scan and normalization of TSH levels within 3 months from the end of the treatment in 30/34 patients and an average reduction in nodule volume of 62.9%. 4/34 patients were refractory to the treatment, 3 of whom had had nodule volumes > 60 ml. There were no recurrences during 6 to 36 months of observation (229). Another study examined 20 patients with autonomous thyroid nodules for 763 +/- 452 days after ethanol injection. A mean of 2.85 +/- 1.1 injections per patient, and a mean volume of 4.63 ml of ethanol were required (nodule volume-dependent). After a mean time of 50 +/- 23 days TSH normalized and was maintained in 16 patients (80%), whose nodular volume reduced 60.8%. Four patients (20%) did not completely respond to the treatment (230). Less impressive but “clinically acceptable” results have also been observed in a study that reported a "complete cure" in only 22 of 42 patients (52%), mainly in small nodules, and little or no hormonal response in 4 patients (9%). However, nodule volume decreased in all cases and there were no recurrences or serious adverse effects (231). In the reported series, "mild to moderate" local pain often occurred after the injections and lasted a day or two. Local hematomas were seen. Major complications like permanent dysphonia or vascular thrombosis seem to be very uncommon. However, transient paralysis of the recurrent laryngeal nerve may occur. Thus this technique may be an option for large, but not very large autonomous nodules that cannot or should not be treated surgically or with I-131 (232).

 

Percutaneous injection of ethanol has also been used to treat toxic nodular goiter (231,233) and thyroid masses that are recurrent after non-toxic nodular goiters have been treated surgically (233), with results that are similar to those described above.

 

Recurrent cysts, and cystic spaces in a degenerated solid lesion have been obliterated in this fashion (234,235). Perhaps the procedure will have use in cosmetically unacceptable or very large structures. Prospective studies will be required to ascertain if ultrasound-guided placement of medication will reduce the intensity or duration of pain after the injection and improve success over palpation-directed injection.

 

Sonographically guided percutaneous ethanol injection is a treatment option for patients with cervical nodal metastases from papillary thyroid cancer that are not amenable to further surgical or radioiodine therapy. In a study of 21 metastatic nodes in 14 patients, all treated lymph nodes decreased in volume, some impressively. No major complications occurred in this series (116). Yet, in other studies severe untoward effects have been reported including necrosis of the larynx and adjacent skin due to ethyl alcohol (236). It seems that this option may be palliative when there are large nodes that threaten to impact on surrounding structures. However, since ethanol-treated nodes may increase in size due to inflammation, caution is warranted especially when there are bulky nodes in the thoracic inlet or adjacent to vital structures.

 

Greater use of percutaneous administration of ethanol for a variety of benign and malignant conditions seems likely. However, prudence dictates that the injection should only be used when essential and not as an optional therapy to reduce the size of routine cysts, euthyroid nodules and goiters, or even non-threatening malignant nodules.

 

Thermal ablation techniques for benign thyroid nodules, toxic adenomas and even for papillary microcarcinomas are increasingly used, particularly in Korea and Europe (237). Both radiofrequency ablation (RFA) and laser ablation are used and have been shown to be efficient, cost-effective, and to have a low rate of complications (238,239). For example, one investigation evaluated the efficacy of ultrasound -guided laser thermal ablation in reducing the volume of hypofunctioning benign thyroid lesions that caused local compression symptoms or patient-concern in 20 patients, when the patients refused or were ineligible for surgical treatment. A 75-mm, 21-gauge spinal needle was inserted into the thyroid gland under ultrasound-guidance, and a flat-tipped 300-micron quartz fiberoptic guide was placed into the tissue that was to be destroyed with a 1.064-micron continuous-wave neodymium yttrium-aluminum-garnet laser for 10 minutes. Ultrasonograms were used to assess the decrease in nodule volume at 1 month and 6 months after therapy. The mean nodule volume decreased from a baseline value of 24.1 +/- 15.0 mL to 13.3 +/- 7.7 mL at 1 month (43.8 +/- 8.1%) and to 9.6 +/- 6.6 mL at 6 months (63.8 +/- 8.9%). Untoward effects included burning cervical pain, which rapidly decreased after the laser energy was turned off and treatment with betamethasone for 48 hours in 3 patients. No patient had local bruising, cutaneous burning, or dysphonia (240).

 

In a multicenter study on 44 patients with toxic adenomas or autonomous nodules and a follow-up of 19.9±12.6 months showed that the mean  nodule volume decreased from an initial volume of 18.5±30.1 ml to 11.8±26.9 ml at 1 month and to 4.5±9.8 ml at the last month (241). The thyroid function tests improved significantly and 35 of the hot nodules became cold or normal when followed by scintigraphy, and 9 had a decreased uptake. There were no major complications.

 

Several studies have shown that radiofrequency ablation may be an alternative to active surveillance for papillary thyroid microcarcinomas (PTMC). For example, in a study involving 107 patients, the mean volume reduction ratio at 18 months was 0.999 ± 0.002 (range: 0.992-1) at 12 months (242). Thyroid function tests remained normal, and there was no tumor regrowth, local recurrence, or distant metastases during follow-up visits. In an Korean study with 152 biopsy-proven PTMCs from 133 patients complete disappearance was found in 91.4% (139/152) of ablated tumors (mean follow-up 39 months) (243). All patients were either of high surgical risk or refused to undergo surgery. In the 13 tumors that did not show complete disappearance, none of the PTMC showed regrowth of the residual ablated lesion during the follow-up period, and there were no local recurrences, lymph node or distant metastases in any of the patients. The complication rate was 3% (4/133), including one voice change.

 

SONOGRAPHY TO DISCOVER PELVIC THYROID TISSUE

 

Trans-vaginal and trans-abdominal pelvic sonography has been employed to identify a 16 cm mass in the right adnexa that was a cystic teratoma, a struma ovarii, containing a 5 mm focus of papillary cancer within the thyroid tissue (244).

 

SONOGRAPHY OF THE FETAL THYROID

 

Ultrasonography in pregnancy can be become a helpful tool to assess thyroid status in utero. Gestational age-dependent and age-independent nomograms for fetal thyroid size have been developed by performing ultrasonograms in 200 fetuses between 16 and 37 weeks of gestation (245). Fetal hyperthyroidism can be detected by the presence of increased blood flow within a goiter in contrast to peripheral vascularity when goiters are associated with hypothyroidism (246). Fetal goiters and hypothyroidism have been studied, and successful treatment has been reported (247). It is thought that intrauterine recognition and treatment of congenital goitrous hypothyroidism may reduce obstetric complications and improve the prognosis for normal growth and mental development of affected fetuses. One report cited a fetal goiter diagnosed at 29 weeks of gestation during routine ultrasound examination. Fetal blood sampling performed at this time documented fetal hypothyroidism and treatment was given using a series of intra-amniotic injections of tri-iodothyronine and subsequently, thyroxine. Following birth, neonatal serum TSH levels were within the reference range (248). A case of fetal goitrous hypothyroidism associated with high-output cardiac failure was diagnosed at 32 weeks of gestation based on ultrasound examination. The fetus' thyroid function was examined by amniocentesis and cordocentesis. The fetus was treated by injection of L-thyroxine into the amniotic fluid at 33 weeks of gestation. Thereafter, the goiter decreased in size, and the high-output cardiac failure improved (249). Similarly, fetal goiter and hypothyroidism that resulted from the treatment of maternal Graves’ disease with propylthiouracil was diagnosed with trans-vaginal US and treated by levothyroxine injection into the amniotic fluid. Successful ultrasound-facilitated treatment of fetal goitrous hypothyroidism has been reported in 12 cases (250). Assessing the fetal thyroid size ultrasonically may also be beneficial in adjusting the dose of antithyroid drug in mothers with Graves’ disease and in preventing fetal and neonatal goiter and hypothyroidism, as discussed before (67). In addition, determining fetal thyroid size with ultrasonography in mothers with a history of Graves' disease has been reported to facilitate achieving normal fetal thyroid function (251).

 

SONOGRAPHY OF THE THYROID IN THE NEWBORN

 

There are several uses of ultrasonography in newborn infants. Normative data in 100 (49 male) healthy term Scottish neonates showed a mean thyroid length of 1.94 cm (SD 0.24, range 0.9-2.5), width of 0.88 cm (SD 0.16, range 0.5-1.4), depth of 0.96 cm (SD 0.17, range 0.6-2.0), and volume of 0.81 ml (SD 0.24, range 0.3-1.7) (252). There was considerable variation (-0.8 to + 0.7 ml) between the two lobes in individual babies. Another investigation revealed that the ratio of thyroid width to tracheal width is a simple, practical parameter for estimating the size of the thyroid gland in neonates and small children (253).

 

In permanent primary congenital hypothyroidism, ultrasonography has been reported to identify 66 instances where the thyroid gland was not located in the usual anatomical position and hemiagenesis in one case. The diagnosis was confirmed by scintigraphy. The authors concluded that sonography might be used as the first imaging tool in patients with congenital hypothyroidism, but scintigraphy should be used to distinguish agenesis from ectopia (254).

 

EPIDEMIOLOGICAL USE OF ULTRASONOGRAPHY

 

Ultrasonography has been used effectively even in the field in undeveloped areas to evaluate thyroid anatomy and size in iodine-deficient regions or to search for cancer in radiation-exposed populations. Inter-observer agreement on estimates of thyroid volume has been good in epidemiologic studies but agreement on echogenicity has been poor (29). One study has revealed that in the Chernobyl population thyroid cancers often exhibit benign ultrasound characteristics, that malignant features are uncommonly encountered, and as many nodules as is feasible should be biopsied (205). Correlation of age, body size and thyroid volume in endemic goiter areas have been reported (255). Data for thyroid volumes that are specific to a geographic region, iodine status, sex, and pubertal stage may be more appropriate than a single age-specific international reference (256,257). Systematic ultrasound screening has been found useful in Belarus for the early detection of thyroid carcinoma in children 4-14 years of age who were exposed to radioactive fallout due to the Chernobyl accident (258).

 

After the nuclear accident in the Fukushima Daiichi Nuclear Power Plant, large-scale ultrasound screening has been implemented (for review see (259)). This led to a high rate of detection of thyroid cancer in younger individuals within the studied cohort of approximately 300,000 subjects in Fukushima prefecture. This observation resulted in significant concerns in the population because it was felt that these cancers might have been caused by radiation. The current evidence indicates, however, that these findings are largely explained by the effect of screening.

 

Ultrasonography has also shown that the prevalence of thyroid cancer has not increased in a population exposed to the accidental release of I-131 in Hanford, Washington during 1944-1957 (260). Ultrasonography has also been used to monitor thyroid nodule development among workers in nuclear power plants (261).

 

The value of ultrasonographic mass screening to uncover thyroid carcinoma depends on the cancer-risk status of the population. In a population with average cancer risk the value of screening is controversial because of the presumed low benefit/cost of the screening as contrasted with subsequent discovery of the small number of tumors that will progress to palpable, clinical, but low-virulence tumor. One group studied 1401 women who were scheduled to undergo a breast examination. Thyroid nodules were detected in 25.2% and thyroid cancer in 2.6% of all subjects. The size of the tumors was significantly smaller in the ultrasound-studied group than that of a clinically detected cancer group (P < 0.05) (262). Another group studied thyroid sonography in 5549 patients who were undergoing breast sonography. Forty-two (0.76%) thyroid cancers were found; all were papillary carcinomas. The incidence of thyroid cancer was significantly higher in the group with breast cancer than in the group who did not have breast cancer (37). In contrast, epidemiologic investigation of the long-term risk of developing thyroid cancer has been useful in a population with a higher risk of cancer such as irradiated people. In a prospective ultrasound examination of 2637 atomic-bomb survivors the hazard ratio for cancer development was significantly high at 23.6 (95% confidence interval, 7.6-72.8) and even higher, 40.2 (95%, confidence interval, 9.4-173.0) in 31 people who initially had cytologically benign solid nodules. The hazard ratio was only 2.7 (95% confidence interval, 0.3-22.2) in 121 subjects who had thyroid “cysts”. Importantly, sex, age, TSH level, thyroglobulin level, radiation dose, nodule volume, and increase in nodule volume did not predict cancer development in the solid nodule group but sonography did reveal the risk of cancer (263).

 

ELASTOGRAPHY

 

Ultrasonography can estimate the rigidity or stiffness of tissue, which is called elastography. The deformability of a tissue may be assessed from a change in Doppler signal in response to externally applied pressure or vibrations, or by tracking shear wave propagation. This phenomenon may correlate with palpable consistency and cytology of a nodule or goiter. The technique may enhance the cancer-predictive value of sonography of non-cystic non-calcified thyroid nodules (75,264-267). However, it is premature to judge the clinical value of the test and the literature contains controversial data. One retrospective investigation revealed that among 16 malignant and 20 benign thyroid nodules elastography correlated with FNA in a sensitivity of 100% and specificity of 75.6% in detecting malignant thyroidnodules (267). Other investigators reported that elastography is not able to select cancers among follicular lesions of indeterminate significance (268). Elastography was not very useful in detecting thyroid cancer in patients affected by Hashimoto’s thyroiditis (269). Elastography may be useful in the diagnosis of inflammatory conditions of the thyroid like sub-acute thyroiditis (270).

 

OTHER USES OF ULTRASONOGRAPHY

 

There have been other novel and inventive applications of ultrasound to thyroidology and the list grows. Just as medical practices have evolved as a result of sonography, surgical techniques may change as well, as was discussed previously. Intra-operative diagnostic sonography is already used in the patient with thyroid cancer and one suspects that it will impact favorably on surgical methods, complications and outcome. Another example of the potential is a recent report that used ultrasonography to demonstrate that routine insertion of drains into the thyroid bed to prevent formation of hematoma or seroma following thyroid surgery may not be necessary. The authors contended that not draining the wound did not adversely influence the volume of the sequestered fluid (p = 0.313) and actually was beneficial by reducing morbidity and decreasing hospital stay (p = 0.007) (271).

 

Thyroid sonography may also be useful before neck surgery for non-thyroid disease. In a retrospective study of 1200 consecutive patients who were treated surgically for primary head and neck tumors and who had routine preoperative neck ultrasound by the surgeon, 47%, (477/1195) of the patients had coexisting thyroid disease. Preoperative fine-needle biopsy of sonographically detected thyroid nodules was performed in 20%, which was cost-effective in limiting concomitant thyroid surgery to fewer patients (6%; 21/350) (272).

 

Ultrasonic energy can be used therapeutically to destroy tissue, as discussed previously, and also to activate mechanical equipment. An example of the latter is ultrasonically activated shears for thyroidectomy that have been reported not to increase complications, shorten operative time, improve cosmetic results, and reduce the patient’s pain, without greater expense than conventional methods (273). An ultrasonically activated scalpel significantly improved bleeding control during thyroid resections and may also be beneficial with respect to cost containment by reducing operative time (274). Ultrasound-guided percutaneous interventional procedures to deliver medications, enzymes, recombinant materials such as RNAS, monoclonal antibodies, or energetic forces to the thyroid gland, nodules, or nodes also challenge the imagination. US-guided high-intensity, focused ultrasound maybe use to ablate benign thyroid nodules (275).

 

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Adipose Tissue: Physiology to Metabolic Dysfunction

ABSTRACT

 

Like the obesity epidemic, our understanding of adipocytes and adipose tissue is expanding. Just in the past decade, substantial advances have led to new insights into the contributions of adipose tissue to normal physiology and obesity-related complications, which places adipocyte biology at the epicenter of a global pandemic of metabolic diseases. In addition to detailing the types, locations, and functions of different adipose tissue depots, this chapter will review the secretory capacities of adipose tissue. Arguably one of the most significant discoveries in the last two decades of adipocyte research is that not only do adipocytes release endocrine hormones, but fat cells and adipose tissue secrete a variety of effectors, including exosomes, miRNA, lipids, inflammatory cytokines, and peptide hormones that act in both paracrine and endocrine capacities to impact local and systemic metabolic responses. The origins of adipocytes via progenitor cells and the process of adipocyte development are discussed. Inflammation, metabolically healthy fat, and adipose tissue expansion are also considered. Finally, several emerging research areas in fat cell biology with therapeutic potential in the management patients who are overweight and have obesity are summarized.

 

INTRODUCTION: AN HISTORICAL PERSPECTIVE ON ADIPOSE TISSUE BIOLOGY

 

The first published citation referencing adipose tissue (AT) dates to 1837. Subsequent sporadic single AT citations appeared in in the literature until the 1940’s, including a 1933 publication in Biochemical Journal examining the degree of fatty acid unsaturation in human AT in relation to its depth from the skin surface (1). The first year in which two AT-related citations were recorded was in 1942. In 1947, nearly ten AT citations appeared. Adipose tissue remained understudied for decades due to the misconception that it was simply an inert energy storage depot, but recent discoveries of AT’s wider role in cell and whole-body signaling have created a scientific renaissance in this field. As of early 2019, over 139,000 citations involving adipocytes or AT are now discoverable.

 

The earliest recognized function of adipocytes was the storage of energy in the form of triacylglycerols (TAGs). It was not until the mid-1980s that the secretory functions of AT and the production of adipocyte-specific proteins were revealed. At that time, a serine protease named adipsin was shown to be secreted from cultured adipocytes and reported to be reduced in mouse models of obesity compared to lean littermates (2). Acylation stimulating protein, a member of the alternative complement family, was also revealed to be produced by AT (3) and implicated in lipid storage (4). Although the functions of these AT secretory products remain poorly understood, their discovery revealed adipocytes and AT to be significant sources of a variety of protein products, including many endocrine hormones. Arguably one of the most important of these discoveries was leptin (5), a bona-fide adipocyte-derived hormone that clearly acts not only as an afferent “adipostat” signal of fat mass to central brain centers in the regulation of body weight (5) but also has peripheral actions that impact glucose metabolism (6) and immune function (7).

 

In addition, adipocytes are also highly sensitive to insulin and involved in the regulation of blood glucose levels. Insulin action on fat cells stimulates glucose uptake and modulates lipid metabolism by increasing the accumulation and decreasing the breakdown of TAGs (and subsequent release of free fatty acids into the circulation) within the adipocyte. The importance of each of these 3 fat cell functions (Figure 1) – lipid storage, secretory function, and insulin sensitivity – is underscored by the demonstration that disruption of any one role has profound systemic ramifications in mice and man that can contribute to a variety of obesity-related metabolic disease states (8).

 

The first CDC statistics reporting obesity rates over 20% in many US states also appeared in the late 1990’s, as did literature from a variety of disciplines showing that obesity, or excess adipose tissue, enhanced the risk of metabolic diseases, particularly type 2 diabetes (T2D). This was a substantial shift in thinking from the previous two decades when AT was not considered to have much importance or relevance to T2D. In addition to metabolic diseases, obesity is associated with increased risk of 13 types of cancer that account for ~40% of all cancers diagnosed in the United States (9).

 

Today, obesity and accompanying epidemics of co-morbidities have become global problems.  While in 2015–2016 the prevalence of obesity was 39.8% in adults and 18.5% in youth in the USA (10), the World Health Organization (WHO) reports that obesity has nearly tripled across the world since 1975, and in 2016 more than 1.9 billion adults were overweight and over 650 million were obese. Today, with most of the world's population living in countries where overweight and obesity account for more deaths than malnutrition (underweight), excess AT presents a major challenge to chronic disease prevention and health across the planet. This global epidemic can be attributed to advancing economies and the adoption of mechanized transport, urbanization, commercial growth, industrialization, a progressively more sedentary lifestyle, and a nutritional transition to processed foods and high calorie diets over the last 30 years (11). Besides preventing obesity by promoting a healthy lifestyle through diet and exercise, one of the best ways for modern-day physicians and scientists to combat the global menace of obesity is to better understand AT.

Figure 1. Physiological characteristics of adipocytes. Disruption of any one of these fat cell functions may lead to the development of systemic metabolic dysfunction.

ADIPOCYTE PHYSIOLOGY

 

Adipocyte Hues – White, Brown, Beige and Pink

 

Adipose tissue has historically been classified into two types, white adipose tissue (WAT) and brown adipose tissue (BAT), which are visibly distinguishable based on tissue color. The white and brown adipocytes comprising these depots exhibit physiological differences, which give rise to specialized tissue functions. White adipose tissue, which is critical for energy storage, endocrine communication, and insulin sensitivity, comprises the largest AT volume in most mammals including humans. In contrast, BAT is largely present in mammals postnatally and during hibernation. Brown adipose tissue uses energy for non-shivering heat production, which is critical for body temperature maintenance. While BAT was originally thought to only be present in infant humans, imaging studies have revealed metabolically active BAT in the supraclavicular and thoracic regions of adults (12–14). Although women have increased BAT mass and activity over men (14,15), the chance of detecting BAT activity in either sex has been shown to be inversely correlated with age and body mass index (BMI) (14). Seasonal correlations have also been observed with BAT activity being higher in the winter and lower in the summer, possibly due to either the temperature or, more likely, the photoperiod (14,15). In healthy humans, BAT activity contributes to whole-body fat oxidation and diet-induced thermogenesis (16), supporting a physiological role for this AT depot in adults.

 

Brown and white adipocytes differ in shape, size, and the intracellular structure of their organelles (Figure 2). White adipocytes are generally spherical in shape and each contains a large, single lipid droplet that pushes all other organelles, including the nucleus, to the cell’s periphery. Brown adipocytes contain multiple lipid droplets dispersed throughout a more ellipsoidal-shaped cell that is enriched with iron-containing mitochondria, giving the cell (and the BAT as a whole) a brownish hue. The thermogenic activity of brown adipocytes is conferred by the presence of its numerous mitochondria containing uncoupling protein 1 (UCP-1), a proton transporter that short-circuits the ATP (energy)-generating proton gradient and allows for concurrent heat production as protons flow back into the mitochondrial matrix (17). Brown fat cells typically grow to 15 to 50 µm, while white fat cells have a larger capacity for lipid storage and can expand to nearly 100 µm in diameter (18). The capacity of white adipocytes to expand in number and size is depot-dependent and is discussed in more detail in the Adipose Tissue Expandability and Metabolic Health section.

Figure 2. Adipocyte types are described by color hues. The primary characteristic of an adipocyte is its ability to store lipid; white, brown, beige, and pink adipocytes all share this property. However, each type of fat cell is somewhat specialized and has a distinct intracellular distribution of organelles and gene expression profile. All fat cells have Golgi and endoplasmic reticulum, but these organelles make up a more significant portion of pink adipocytes than other adipocyte types.

Recently, two additional adipocyte hues – beige and pink – have been described. Beige adipocytes display characteristics of both brown and white fat cells (Figure 2) and typically develop within subcutaneous WAT from a distinct subset of preadipocytes (19) or via the  

transdifferentiation of existing white adipocytes (20,21). However, gene expression analyses indicate that beige fat cells represent a distinct type of thermogenic fat cell (19). Beige adipocytes were originally observed to arise in response to cold exposure in rodents (22,23); however, many studies have since identified that diet (24), exercise (25), pre- and post-biotics (26), pharmaceutical agents, numerous plant-based bioactives, and even adipokines (27)can also induce “beiging” or “browning” of WAT, which may protect against obesity and associated metabolic dysfunction. The “beiging” of WAT is inducible in both mice and humans (28), but this process is more highly observed in mice.

 

Pink adipocytes were first described in 2014, arising in the subcutaneous WAT of female mice during days 17-18 of pregnancy and persisting throughout lactation. These fat cells appear to derive from white adipocytes that take on epithelial-like features to form milk-secreting alveoli, giving the tissue a pink hue (29). Pink adipocytes are characterized by compartmentalized lipid droplets, cytoplasmic projections, and abundant organelles including mitochondria, peroxisomes, and rough endoplasmic reticulum, that show a structure more typical of epithelial cells. While reversible transdifferentiation appears to be responsible for the development and disappearance of pink adipocytes during pregnancy, lactation, and post-lactation in rodents (30), it remains uncertain whether or not pink adipocytes form in humans. Notably, loss of a key adipogenic transcription factor within the mammary secretory epithelium creates a pro-breast tumorigenic environment and indicates that the reversible white-to-pink transition might reveal insights into breast cancer biology (29,31). Further investigations into adipocyte plasticity might therefore identify novel therapeutic targets to combat obesity and its pathological consequences, as well as cancer. However, since WAT makes up the largest AT volume in the human body and undergoes the most expansion during obesity, in this chapter we will focus on the roles that white adipocytes and WAT play in normal physiology and metabolic dysfunction.

 

Adipose Tissue in the Regulation of Lipid Metabolism

 

Adipose tissue stores body fat as neutral TAGs and represents the chief energy reservoir within mammals. Although many diverse cell types are found in whole AT, adipocytes constitute the largest cell volumes and are the defining AT cell type. White adipocytes are characterized by their large unilocular central lipid droplets (cLDs). However, the biogenesis of unilocular LDs in adipocytes is poorly understood due to the fragile nature of WAT.

 

Using live-cell imaging combined with fluorescent labeling techniques, the cytoarchitecture of unilocular adipocytes (Figure 3) and spatiotemporal dynamics of lipid droplet formation have been investigated (32). As shown in Figure 3, cytoplasmic nodules containing micro LDs (mLDs; small green fluorescent protein (GFP)-negative spheres within the cytoplasm) appear on the surface of fat cells, pushed to the edges by the large cLD. Surprisingly, the cytoplasm and organelles do not distribute uniformly around the edge of the cell, but instead form numerous, discrete cytoplasmic nodules connected via a thin layer of GFP-positive cytoplasm. The largest nodule also contains the nucleus, which is surrounded by a thicker layer of cytoplasm. The electron micrograph (Figure 3F) shows the close contacts between mLDs and mitochondria. Furthermore, additional nascent lipid droplets can be visualized budding off from the smooth ER (sER). Studies using a fluorescent-labeled free fatty acid (FFA) analog revealed that exogenously added lipids were rapidly taken up by the fat cell and concurrently esterified to TAG and absorbed by mLDs prior to packaging within the cLD. The lipid transfer followed a unidirectional path from mLD to cLD and provides insight into adipose tissue growth via fat cell hypertrophy (32).  

Figure 3. Architecture of primary unilocular adipocytes. Figure adapted from (32). The cytoplasm and nuclei of adipocytes and stromovascular cells were labeled by infecting visceral WAT explants from nonhuman primates with an adenoviral vector encoding enhanced green fluorescent protein (eGFP). Two days after infection, live explants were examined by for GFP expression using confocal microscopy. Cellular and subcellular features are labeled: cLD, central lipid droplet; Cyt, cytoplasm; LDM, lipid droplet membrane; mLD, micro-LD; N, nucleus; PM, plasma membrane; sER, smooth ER. (A) GFP-positive unilocular adipocytes (spheres) and stromovascular cells (asterisks) residing in WAT. The image represents the sum of all confocal slices. Bar, 10 um. (B) Single confocal section of the image in A. Enhanced magnification of adipocytes containing cytoplasmic nodules (C) and perinuclear cytoplasm (D). (E) Schematic representation a unilocular adipocyte demonstrates that the cLD is a sphere tightly fitted within the cell, whereas the cytoplasm collects in multiple organelle- and mLD-containing nodules. (F) Electron micrograph of a unilocular adipocyte from a visceral WAT explant that was fixed and processed for electron microscopy. Asterisks mark contact sites between mitochondria and mLDs, whereas arrowheads point towards vesicles budding off the ER tubules. Bar, 500 nm.

Adipocytes store TAG under conditions of energy surplus and release fatty acids to supply to other tissues during fasting or times of high energy demand. As such, AT is central to the regulation of systemic lipid metabolism, and nutritional and hormonal cues serve to balance lipid storage and breakdown within the fat cell (Figure 4).

 

Figure 4. A critical balance between lipogenesis and lipolysis within adipocytes must be established to maintain whole body insulin sensitivity and energy homeostasis. Lipogenesis is shown on the left (gray arrows mark the pathway), whereas lipolysis is shown on the right and is marked by black arrows. Nutritional and hormonal cues regulate both processes. Lipid droplet associated proteins, such as perilipin and comparative gene identification-58 (CGI-58) are not shown but play important roles in lipolysis. CD36 (cluster of differentiation 36) is a fatty acid transporter that facilitates entry of free fatty acids (FFAs) into the cell. Insulin stimulates glucose uptake into fat cells by increasing the localization of the insulin responsive glucose transporter, GLUT4, within the plasma membrane. Other abbreviations: VLDL-TG – triglyceride-containing very low density lipoprotein; LPL – lipoprotein lipase; ACC - acetyl-CoA carboxylase 1; FAS – fatty acid synthase; G3P – glycerol 3 phosphate; DGAT - diacylglycerol acyltransferase; β-AR – β-adrenergic receptor; NA – noradrenaline; AC – adenylyl cyclase; PKA – protein kinase A; ATGL - adipocyte triglyceride lipase; HSL - hormone sensitive lipase; MGL - monoacylglycerol lipase; TAG – triacylglycerol; DAG – diacylglycerol; MAG – monoacylglycerol.

LIPOGENESIS

 

Adipocytes accumulate lipid via one of two processes (Figure 4).  In the first process, under normal daily feeding conditions adipocytes take up dietary lipids from the circulation in the form of FFA’s liberated from circulating TAGs via the action of lipoprotein lipase (LPL) (33). Adipocytes secrete LPL, which is transported to the adjacent capillary lumen to catalyze the hydrolysis of FFA’s from circulating triglyceride-containing lipoproteins (34,35), such as chylomicrons produced in the small intestine and very low density lipoproteins (VLDLs) synthesized by the liver (36). Adipocytes also take up glucose, which is converted to glycerol and serves as the backbone for the sequential esterification of fatty acids for form TAG. The final step in TAG synthesis, re-esterification of circulating free fatty acids, mediated by diacylglycerol acyltransferase (DGAT) (37,38). The second process is by de novo lipogenesis (DNL) within the adipocytes themselves. Lipogenesis comprises both de novo synthesis of fatty acids from acetyl-coenzyme A (acetyl-CoA) and the esterification of these fatty acids to a glycerol backbone producing TAGs (Figure 4). De novolipogenesis can occur in the fasting and fed states (36). Following a meal, especially one high in carbohydrates, excess glucose oxidation yields elevated levels of acetyl-CoA that become substrate to generate fatty acids.  This occurs through actions of the DNL enzymes acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) to convert acetyl-CoA to palmitate, which can then be elongated and desaturated to form other fatty acid species (39).

 

Surprisingly, in rodents DNL is relatively low in WAT compared to BAT and liver, and it plays an even lesser role in WAT lipid storage in humans under physiological conditions (40,41). Typically, hepatic DNL activity exceeds that of AT and is a more substantial contributor DNL-generated circulating lipids. However, in humans fed high-carbohydrate diets, liver DNL contributes only a small portion of total de novo fat biosynthesis, suggesting that AT contributes significantly to whole body DNL when there is a carbohydrate surplus (39,42). Under this condition, adipocyte DNL is usually quite low but has been shown to be important for whole body substrate metabolism (43,44) as inhibition of WAT DNL is associated with insulin resistance (45).

 

A primary transcriptional regulator of adipocyte DNL is carbohydrate response element-binding protein (ChREBP) (39). Mice lacking AT ChREBP have decreased DNL and insulin resistance (46). The other major DNL regulator in AT is sterol regulatory element-binding protein 1 (SREBP1). Mice with whole body knockout of SREBP1 do not display decreased lipogenic gene expression in AT (45,47), thus supporting ChREBP as the primary lipogenic transcription factor driving AT DNL. However, a new mouse model of inducible, overexpression of insulin-induced gene 1 (Insig1), an inhibitor of SREBP1 activation and transcriptional activity, demonstrated that several acute and chronic white adipocyte-specific compensatory mechanisms are activated to restore adipocyte DNL in the absence of SREBP1 activity (44). Decreased SREBP1 activity prior to this compensation and during conditions where compensation was inactivated result in decreased lipogenic gene expression, impaired whole body glucose tolerance, and elevated lipid clearance (44) suggesting that both SREBP1 and ChREBP play important roles in adipocyte DNL.

 

Enhanced AT DNL can produce favorable lipid species that may be therapeutically advantageous in the context of obesity and insulin resistance (48). Adipocytes synthesize and secrete a novel family of bioactive lipids, known as the branched fatty acid esters of hydroxyl fatty acids (FAHFAs). Although FAHFAs are found in many tissues, the highest levels are in white and brown AT, and their production is likely dependent on AT lipogenesis as disruption of adipocyte DNL impairs their synthesis (39,49). Over 1000 structurally distinct FAHFAs have been predicted based on in silicoanalyses and at least 20 FAHFA families have already been identified in mammalian tissues (50). The serum and subcutaneous AT levels of one FAHFA family, palmitic acid esters of hydroxyl steric acids (PAHSAs) have been shown to be higher in insulin-sensitive compared to insulin-resistant individuals (51). In animal models, PAHSAs have been shown to decrease inflammation and enhance whole body insulin sensitivity (39,49). Recent evidence from a mouse model of high-fat diet (HFD)-induced insulin resistance demonstrates that PAHSAs act via both direct and indirect mechanisms to improve insulin sensitivity in multiple metabolic tissues, such glycolytic skeletal muscle, heart, liver, and AT. In WAT explants, PAHSAs directly inhibit lipolysis and enhance insulin’s ability to suppress lipolysis. While PASHAs can also directly inhibit endogenous glucose production (EGP) in isolated hepatocytes, the decreased AT lipolysis indirectly attenuates EGP because of reduced glycerol (gluconeogenic substrate) delivery to the liver (50).

 

Additional evidence from humans support a role for increased DNL and ChREBP activity in maintaining metabolic health. These include restoration of DNL in WAT following as bariatric surgery-induced weight loss (52) and reported observations of elevated WAT DNL in other metabolically favorable states including caloric restriction and adaptive thermogenesis (53,54). Collectively, these studies in mice and man support a potential role of WAT DNL in metabolic health.

 

LIPOLYSIS

 

Under physiological conditions when metabolic fuels are low and/or energy demand is high, such as fasting, exercise, and cold exposure, adipocytes mobilize their TAG stores via the catabolic process of lipolysis to supply fuel to peripheral tissues (55). Lipolysis is a highly regulated biochemical process that generates glycerol and FFAs from the enzymatic cleavage of TAGs by lipases (36) and can occur in all tissues, although it is most prevalent in AT where the bulk of TAG is stored. As shown in Figure 4, TAGs are broken down into diacylglycerols (DAGs) and monoacylglycerols (MAGs) by the sequential action of adipocyte triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoacylglycerol lipase (MGL). At each step a single FFA is released, and in the final step MGL releases the glycerol backbone from the last FFA. These breakdown products can be re-esterified within the adipocyte or released into circulation to be used by other tissues (36,55), including by the liver for gluconeogenesis (glycerol) and for oxidative phosphorylation by muscle or other oxidative tissues (56).

 

Lipolysis is controlled by sympathetic nervous system (SNS) input as well as a variety of hormones (55). The best understood of these regulators is the catecholamine, noradrenaline (NA), also known as norepinephrine. Noradrenaline stimulates β-adrenergic receptors (Figure 4), which, in turn, stimulate protein kinase A (PKA) via adenylyl cylase (AC)-mediated production of cyclic AMP (cAMP). PKA activates the lipolytic action of ATGL and HSL by different mechanisms. Several lipid droplet-associated proteins, such as perilipin 1 (PLIN1) and comparative gene identification-58 (CGI-58) are also important in regulating lipolysis (57,58). Whereas PKA can directly phosphorylate and activate HSL (59–62), it primarily stimulates ATGL activity indirectly by phosphorylating PLIN1. This phosphorylation releases CGI-58 to potently activate ATGL (58,63,64). Non-adrenergic lipolytic stimuli include glucocorticoids, natriuretic peptides, growth hormone, and tumor necrosis factor alpha (TNFα) (58). These hormones are typically less potent lipolytic inducers than β-adrenergic stimulation and the molecular mechanisms responsible for their lipolytic abilities have not been clearly elucidated. However, some of these hormones clearly utilize different pathways than β-adrenergic signaling with additive or synergistic affects to increase lipolysis (55,58).

 

After a meal, the post-prandial increase in circulating insulin readily suppresses lipolysis (65) by increasing the activity of phosphodiesterase 3 (PDE3B) and decreasing cAMP levels (58). In the fasting state, insulin levels drop and NA is released, thus promoting lipolysis (66). Physiologically, exercise is another major pro-lipolytic stimulus in humans (58). Growth hormone, along with NA, adrenaline, and cortisol increase with exercise intensity, while insulin levels decrease. These changes culminate in an overall lipolytic response, the magnitude of which depends on exercise intensity and duration (58,67).

 

When AT becomes insulin resistant, as occurs in patients with diabetes and may also be present in patients with obesity, insulin’s ability to inhibit adipocyte lipolysis and reduce serum levels of FFA and glycerol are impaired. As a result, excessive lipolysis leads to increased FFA levels in both the fasted and fed state. Constant exposure of the liver and muscle to these high FFA levels is thought to promote the uptake and ectopic storage of lipids in these tissues (68). Ectopic lipids have been shown to impair insulin signaling, and thus insulin resistance at the level of adipocyte via increased lipolysis may be a major contributor to whole body insulin resistance (69). In addition to impaired insulin responsiveness in fat cells, elevated lipolysis in obesity may be mediated by decreased expression of adipocyte lipid droplet proteins such as PLIN1 and Fsp27/Cidec (fat-specific protein 27/cell death-inducing DFFA-like effector c) (70). These proteins coat the lipid droplet and promote TAG retention via the inhibition of lipolysis, and mice or humans deficient for PLIN1 (71) or Fsp27 (72,73) exhibit lipodystrophy and insulin resistance (70). Interestingly, adipocyte-selective gene deletions or transgenic overexpression mouse models of proteins involved in insulin signaling, glucose and lipid metabolism demonstrate parallel modulation of adipocyte insulin action and systemic insulin sensitivity or glucose tolerance.

 

In addition to potent anti-lipolytic action (58), insulin also stimulates lipogenesis (74) by activating LPL (activation) and increasing the transcription of lipogenic enzymes (74). Growth hormone antagonizes insulin by promoting lipolysis and inhibiting lipogenesis (36,58). The insulin-sensitizing, anti-inflammatory lipids (PAHSAs) generated during AT DNL and the excess basal lipolysis associated with ectopic lipid deposition and insulin resistance make both AT lipogenesis and lipolysis attractive targets for pharmaceutical intervention. On the other hand, the balance between lipid storage, mobilization, and utilization is homeostatically regulated through a complex interaction of often redundant hormonal signaling, neurological input, and nutrient flow. These intricacies complicate attempts to develop therapies targeting one aspect of lipid metabolism since disrupting the balance between lipolysis and lipogenesis may, in turn, have unanticipated effects on insulin sensitivity and whole-body energy homeostasis.

 

Figure 5. The Adipocyte Secretome. Fat cells express and release numerous protein, lipid, and nucleic acid factors that can act on other nearby or distant tissues within the body in a paracrine or endocrine manner. Leptin, adiponectin, and resistin are highlighted here because they are exclusively secreted from mouse adipocytes, while the other factors can also be secreted from other cell types. The arrow-headed line representing secretion of resistin is dashed since in humans, macrophages, and not adipocytes, primarily produce this adipokine. Abbreviations are RBP4 – retinol binding protein 4, BMPs – bone morphogenetic proteins, PAI-1 – plasminogen activator inhibitor 1, miRNA – microRNA, FFA – free fatty acid, FAHFA - fatty acid esters of hydroxyl fatty acids, PAHSA – palmitic-acid-hydroxy-steric-acid, FGF21 – fibroblast growth factor 21.

 

Endocrine Properties of Adipose Tissue

 

Adipocytes and other AT cells secrete a variety of mediators, including exosomes, miRNA, lipids, inflammatory cytokines, and peptide hormones that act in both paracrine and endocrine modes (Figure 5) (75). Although adipocytes secrete a large variety of bioactive molecules with widespread systemic effects contributing to numerous physiological and pathological processes, the autocrine and paracrine actions of these molecules are highly complex, and our understanding of these processes is likely rudimentary. However, substantial progress has been made studying three endocrine hormones that are almost exclusively produced in adipocytes and function to regulate food intake, the reproductive axis, insulin sensitivity, and immune responses. These hormones are leptin, adiponectin, and resistin, and we review their expression in obesity, their receptors, and effects in target tissues including metabolic actions (Figure 6). While not produced in human adipocytes directly but secreted instead by AT macrophages, resistin has similar functions in mouse and man. The dysregulation of any one of these hormones can contribute to systemic metabolic dysfunction, as well as to the pathogenesis of chronic metabolic diseases and some types of cancer.

Figure 6. Summary of adipocyte-specific adipokines, and their actions on other tissues. Abbreviations: TLR4 - Toll-like receptor 4; CAP1 - adenylyl cyclase-associated protein 1; AdipoR1 & R2 - Adiponectin receptors 1 and 2; CNS - Central nervous system; FAO – fatty acid oxidation; EE – energy expenditure.

LEPTIN

 

The first discovered endocrine hormone of adipocyte origin was leptin (5). In 1949 spontaneously occurring obese offspring in a Jackson Laboratories’ non-obese mouse colony were determined to be homozygous for a recessive mutation, termed “obese” (ob) (76). These ob/ob mice appear normal at birth, but soon begin gaining excess fat mass and displaying hyperglycemia and hyperinsulinemia (77). In the 1950s, ob/ob mice and their non-obese littermates underwent parabiosis experiments, where two animals are surgically joined (usually by peritoneum or a long bone of the leg) to allow for the exchange of whole blood between them (78). Weight gain was inhibited in ob/ob mice parabiosed with non-obese littermates, providing evidence that the ob/ob gene product was a circulating factor transferred from the blood of the lean littermate. In 1972, a similar study demonstrated that parabiosis with lean animals not only reduced weight gain in ob/ob mice, but also improved hyperglycemia, hyperinsulinemia, and insulin sensitivity (6). Finally, in the 1990s, positional cloning studies identified the product of the ob gene, dubbed leptin, which was derived from the Greek word “leptos” meaning to be thin. Further characterization of leptin revealed that adipocytes were its predominant source (5). Following this discovery, the first directly observed function of leptin was its effect on food intake (79) followed shortly thereafter by demonstration that leptin levels in mice and men strongly correlate with fat mass and play a key role in body-weight (energy) homeostasis as described below. 

 

Leptin Receptor and Signaling

 

Another spontaneously arising mutant mouse that developed obesity and type 2 diabetes is the diabetes or db/dbmouse (80). In contrast to ob/ob mice, parabiosis of db/db mice to wild type littermates did not improve body weight or diabetes, but instead resulted in unhealthy weight loss in the lean littermates, leading investigators to deduce that mice with the db mutation lacked a functioning receptor for the ob gene but still manufactured a circulating protein that crossed over to the lean littermates to induce anorexia (81). Further confirmation of these hypotheses came when parabiosis of ob/ob with db/db mice induced weight loss in the ob/ob mouse while the obese state was preserved in the db/db mouse (81–83). Although the db gene was cloned in 1990 (84), it was not until almost 5 years later (85)following the identification of leptin that the db gene was identified to encode the leptin receptor (LR).

 

The LR is a class 1 cytokine receptor with substantial homology to glycoprotein 130, a plasma membrane receptor that mediates the actions of many cytokines. Unlike other plasma membrane receptors, such as the insulin receptor, the LR lacks intrinsic kinase activity and signals via Janus kinases (JAKs). Six LR isoforms exist, designated LRa-LRf, with LRb being the best characterized. It is the longest LR isoform that is capable of full signaling via the JAK/STAT pathway (86).

 

Leptin-regulated circuits involved in energy homeostasis have been mapped to distinct yet diverse brain regions (87)expressing the long form of the LR (88). Increased central leptin signaling inhibits food intake and elevates energy expenditure, while leptin deficiency (such as during fasting or starvation) has opposite effects.  Expression of the LR has also been detected in peripheral tissues, but the exclusivity of the central leptin circuits to modulate energy intake and expenditure is supported by studies showing that deletion of the long form LR in peripheral tissues had no effects on these processes (89). Leptin levels strongly correlate with fat mass in mice and men (90,91), and as such leptin acts as a sensor of energy stores signaling the availability of body fat to the brain and regulating adipose reserves. However, during obesity the negative feedback loop between increasing leptin levels that signal high energy availability and inhibit food intake becomes disrupted due to the development of leptin resistance (92) — the inability to respond to leptin despite having sufficient or excess levels in circulation during accumulation of excess adipose stores. Although the physiological causes of leptin resistance are not well understood, it has been shown that hyperleptinemia is required for the development of leptin resistance during obesity. When leptin levels of mice are clamped to low levels (similar to lean mice), these clamped mice still develop obesity on HFD, but they do not become leptin resistant (93). The inability to overcome leptin resistance by giving supplemental doses has precluded leptin’s use as an anti-obesity therapeutic. Interestingly, leptin resistance that accompanies obesity appears to result from selective impairment of leptin’s ability to reduce food intake, while preserving its other capacity to raise energy expenditure (94). The molecular basis for this phenomenon has not yet been elucidated and remains under active investigation. 

 

Leptin also has central nervous system effects not directly related to energy balance, including modulation of reproduction and thermoregulation. Additionally, research into the role of leptin to mediate anxiety and depression is currently ongoing (95). Leptin can also act peripherally on hepatocytes and pancreatic β-cells to regulate glucose and lipid metabolism independently of its central effects (96). Leptin has also been shown to affect innate and adaptive immunity (7), bone formation (97), bone metabolism (98,99), angiogenesis, and wound healing (88). Skeletal muscle, liver, and intestines have been described as targets for leptin action (100), and some evidence suggests that leptin may also act in an autocrine manner on AT (101). How leptin mediates responses in peripheral tissues is poorly understood and complicated by the existence of its six receptor isoforms, their differential expression across tissues, the pleiotropic nature of leptin’s effects, the demonstration of “selective” or tissue-specific leptin resistance, and the complexity of the signaling pathways involved.  

 

Leptin and Cancer

 

Given the elevated risk for many cancers in patients with obesity in whom leptin levels are also high, it is not surprising that leptin has been implicated in tumorigenesis. Indeed, leptin levels or leptin signaling has been found to be dysregulated in breast, thyroid, endometrial, and gastrointestinal malignancies (102). Ectopic leptin expression in colorectal adenomas increases during the progression to colorectal cancer (103,104) yet associates with a favorable prognosis of the cancer (103). In papillary thyroid cancer, increased circulating leptin levels occur independently of body mass index (BMI), coincide with elevated LR expression on the tumor cells, and associate with aggressive carcinogenesis and poor prognosis (105,106). In contrast, reported associations between leptin levels and endometrial cancer are not maintained when adjusted for BMI, suggesting that leptin is not likely a causative factor in the development of this cancer (107–109).

 

Studies show that postmenopausal women with obesity have a 20-40% greater risk of developing breast cancer compared to normal weight women (110). In breast cancer, particularly in high-grade tumors, overexpression of both leptin and LR is associated with cancer progression and poor patient survival. Leptin’s ability to stimulate angiogenesis, regulate endothelial cell proliferation, and crosstalk with insulin and human epidermal growth factor receptor 2 (HER2) signaling pathways represent a few of the possible mechanisms by which leptin plays a role in breast cancer (111). As obesity rates continue to rise, it is likely that studies examining the relationship between leptin and cancer will become even more relevant. 

 

ADIPONECTIN

 

Adiponectin is a unique and extensively studied adipocyte-derived hormone with complex biology. Efforts to identify genes regulated during adipogenesis led to the discovery of adiponectin in 1995 (112) and 1996 by three separate research groups employing different approaches (113–115). Secreted by adipocytes, adiponectin is characterized by its remarkably high circulating levels reaching plasma concentrations in humans of 2-20 ug/ml (116), values that are more than 1000-fold higher than most other secreted factors (Figure 7). Unlike leptin, adiponectin levels decease as a function of increasing fat mass in both rodents and humans with obesity (115); thus, they are lower in patients with obesity than those who are lean. Adiponectin’s widely reported anti-hyperglycemic, anti-atherogenic, and anti-inflammatory effects have made it an attractive therapeutic target for the treatment of obesity and insulin resistance. However, efforts to develop therapies targeting adiponectin function have been impeded by its complex structure and regulation (117). 

 

Figure 7. Typical circulating concentrations of select adipokines and insulin for normal weight, healthy humans. Adipokine levels in the blood are several orders of magnitude higher than that of insulin.

Adipocytes secrete different forms of adiponectin: low-molecular weight (LMW) trimers (the most basic form), medium-molecular weight (MMW) hexamers, and high-molecular weight (HMW) oligomers (118), as well as globular adiponectin, a proteolytic fragment of the protein (119,120). In humans, the MMW and HMW oligomers make up most of the circulating adiponectin while the LMW trimer constitutes less than 30% of serum adiponectin. The HMW oligomer is most closely associated with enhanced insulin sensitivity and reduced glucose levels (121).

 

Adiponectin signaling is complex and incompletely understood. Three adiponectin receptors have been identified. Adiponectin receptor-1 and -2, referred to as AdipoR1 and AdipoR2, bind the LMW and globular forms (122). T-cadherin binds HMW adiponectin (123). Both AdipoR1 and AdipoR2 can modulate insulin sensitivity and metabolic gene expression in insulin-responsive tissues, and both receptors have demonstrated roles in the pathophysiology of insulin resistance and T2D (124,125). T-cadherin, which is expressed in a variety of tissues including the liver (126), belongs to a family of cell surface proteins involved in cell-cell interactions (127). Mice lacking T-cadherin accumulate adiponectin in circulation and have a similar cardiovascular phenotype to adiponectin knockout mice (128), suggesting that T-cadherin is the primary effector of cardioprotection by adiponectin (129–131).

 

Adiponectin enhances fatty acid oxidation through activation of AMP-activated kinase (AMPK) (132,133), a cellular energy sensor, which then inhibits acetyl CoA carboxylase, a rate limiting enzyme in DNL (134). This, in turn, reduces malonyl-CoA production and enhances fatty acid oxidation. Adiponectin can activate AMPK through two independent pathways, and can also modulate lipid metabolism by increasing mitochondrial density and mitochondrial DNA content (135,136). Adiponectin has diverse effects in many tissues, including bone and cartilage (137), and can act in an autocrine or paracrine manner in AT and other tissues (138). Adiponectin also appears to modulate a wide range of biological processes, including reproduction and embryonic development (139,140). The heart, liver, and skeletal muscle are considered the primary targets for adiponectin action, and adiponectin’s prominent insulin-sensitizing effects have been most fully characterized at the mechanistic level in liver and muscle (117) (Figure 6).

 

The liver performs a critical function in maintaining normal blood glucose levels by releasing glucose (i.e. hepatic glucose output) into circulation in conditions such as fasting, exercise, and pregnancy. Conversely, the ability of the liver to reduce its glucose output when demand is low, as in the fed state, is also crucial to preventing hyperglycemia, and this process is often impaired with obesity and insulin resistance. Adiponectin can robustly reduce plasma glucose levels predominantly by inhibition of hepatic glucose production as opposed to effects on whole-body glucose uptake into cells and glycolysis (141). The importance of adiponectin in regulating glucose output in the liver is underscored by studies showing that mouse models with genetic deletion (142) or overexpression (143) of adiponectin have impaired or enhanced hepatic insulin sensitivity, respectively. Adiponectin levels are increased bythiazolidinediones (TZDs), which is thought to be the predominant mechanism of action that improves insulin sensitivity and glucose tolerance with this class of medications (142–144). Adiponectin also promotes hepatocyte survival, inhibits hepatic fibrosis and inflammation, stimulates fatty acid oxidation (133,145) and modulates fatty acid uptake and metabolism (146). In patients with nonalcoholic fatty liver disease (NAFLD) who are insulin resistant, low plasma adiponectin levels are associated with the progression of NAFLD and non-alcoholic steatohepatitis (147,148). In summary, adiponectin has beneficial effects in the liver, where it protects against metabolic dysfunction and hepatic diseases (Figure 6).

 

Skeletal muscle is responsible for up to 80% of insulin-mediated glucose uptake in healthy individuals (149,150). Adiponectin can promote glucose uptake (151,152), enhance fatty acid oxidation (152,153), and enhance insulin sensitivity (154) in cultured muscle cell lines and mouse skeletal muscle. Adiponectin administration to obese, insulin-resistant adiponectin-knockout mice improves skeletal muscle insulin sensitivity (146,155,156). In human myotubes, adiponectin promotes fat oxidation via AMPK activation; this response is impaired in myotubes from patients with T2D and obesity (157). Thus, adiponectin has an important role in skeletal muscle metabolism in humans as well as rodents, and defective adiponectin signaling in skeletal muscle may contribute to insulin resistance.

 

Finally, in addition to its insulin-sensitizing and glucose-lowering effects in liver and skeletal muscle, adiponectin is also cardioprotective. Low circulating adiponectin levels correlate significantly and independently with coronary artery disease (158), and are considered a risk factor for cardiovascular diseases (CVD) such as hypertension, coronary artery disease, and restenosis (159). The vascular endothelium is believed to mediate some of the cardioprotective effects of adiponectin via AMPK activation and subsequent activation of eNOS (endothelial nitric oxide synthase) (160).

 

In light of these beneficial functions, adiponectin has significant therapeutic potential in the treatment of T2D, CVD, and NAFLD. Several years ago, small molecule screening efforts produced the first small molecule AdipoR agonist.  “AdipoRon”, as it was named, not only recapitulated adiponectin’s effects on AdipoR signaling pathways but also had profound anti-hyperglycemia effects in both diet-induced obese mice and a genetic mouse model for diabetes (161). A more recent study has now shown that AdipoRon can also decrease ceramides and lipotoxicity, and mitigate diabetic nephropathy (162). Hence, small molecule activators of adiponectin signaling show promise in the management of obesity-associated metabolic diseases like insulin resistance, NAFLD, and T2D.

 

RESISTIN

 

Resistin, the most recently discovered of the major adipocyte-derived hormones, was independently identified by two laboratories. In one case, the gene coding for this novel endocrine factor was identified in a screen for genes inhibited by TZD drugs and was named “resistin” because it induced insulin resistance (163). Another group identified the same gene in a screen for genes expressed exclusively in adipocytes and induced during adipogenesis; they named it ADSF, for adipose tissue-specific secretory factor. In this study, the product of the gene was shown to inhibit differentiation of adipocytes in vitro (164), later confirmed in a separate study (165).

 

Elucidating resistin’s role in physiology has been challenging. While resistin is expressed in both white and brown fat in mice, the various WAT depots (inguinal, gonadal, retroperitoneal, and mesenteric) and BAT exhibit distinct patterns of resistin expression (166). In addition, circulating levels of resistin are directly proportional to its gene expression in some conditions, but inversely proportional in others (167,168). Remarkably, while resistin produces similar metabolic and inflammatory effects in humans and mice, human resistin is predominantly secreted from macrophages, not adipocytes (169–171). The complex regulation of resistin expression and the fundamental differences in resistin biology between species are significant obstacles to fully understanding this hormone’s functions and mechanisms of action in humans.

 

Resistin interacts with two known receptors: the toll-like receptor 4 (TLR4) and adenylyl cyclase-associated protein 1 (CAP1) (172,173). Resistin signaling through TLR4 contributes to monocyte recruitment and chemokine expression, and is involved in inflammatory responses in atherosclerosis and acute lung injury (135,174). Both knockout and overexpression studies of CAP1 indicate that this receptor can also mediate proinflammatory effects of resistin (173).  Overall, the similarities, differences, and tissue specificity of resistin signaling through TLR4 versus CAP1 remains poorly understood.

 

Resistin has also been shown to regulate fasting blood glucose levels in mice (175). Elevated levels of circulating resistin are reported in genetic and diet-induced mouse models of obesity (163). Anti-resistin antibody administration improves insulin sensitivity in diet-induced obese mice, and conversely, resistin injection impairs glucose tolerance in normal mice; supporting a causative role of resistin in mediating insulin resistance in mouse models (176). Moreover, both human and mouse resistin have been shown to impair insulin-stimulated glucose uptake in cultured murine myocytes in vitro (177). Other studies have shown similar insulin desensitizing effects of resistin in liver and brain(178,179).

 

The finding that human resistin originates not in adipocytes but in mononuclear lymphocytes raised the possibility that the hormone may have distinct roles in the two species. An elegant mouse model was generated to address this issue, the so-called humanized resistin mouse.  In these mice, the endogenous resistin gene (normally expressed in adipocytes) was deleted, and the macrophage-expressed human resistin gene was inserted (180). Data from this study revealed that like murine adipocyte-derived resistin, the humanized resistin induced systemic insulin resistance, adipose tissue inflammation, and elevated circulating free fatty acids in high-fat diet (HFD)-fed mice.

 

In humans, epidemiological, genetic, and clinical data support a role for resistin in dysfunctional metabolism and related pathologies (181). As in mouse models, serum resistin levels are elevated during human obesity (182,183). Furthermore, high circulating resistin concentrations in humans have been associated with atherosclerosis, coronary heart disease, congestive heart failure, as well as inflammatory conditions including systemic lupus erythematosus, inflammatory bowel disease, and rheumatoid arthritis (184–188). Whether the relationship between resistin and insulin resistant states is merely correlative and whether interventions to antagonize resistin action will be of therapeutic value in the treatment of metabolic or cardiovascular disease in humans remains undetermined.

 

Cell Types in Adipose Tissue

 

Besides adipocytes, AT is comprised of endothelial cells, blood cells, fibroblasts, pericytes, preadipocytes, macrophages, and several types of immune cells (189). These non-adipocyte cell types are commonly referred to as the AT stromal vascular fraction (SVF) (Figure 8). Our understanding of the complexity of the cell types present in the SVF and how this milieu is altered by metabolic disease states is an area of active investigation. Cells in the SVF produce hormones and cytokines that can act in a paracrine manner on adjacent adipocytes. In the early 1990s, it was shown that TNF alpha production was increased in AT during metabolic disease states, in particular, T2D (190).Yet, it wasn’t for another ten years that adipose tissue macrophages (ATMs) were identified as the primary cellular source of AT TNF alpha (191). It is now largely accepted that in conditions of obesity and T2D, TNF alpha is produced in ATMs and acts on adjacent adipocytes within AT to promote insulin resistance. Hence, it is important to consider the presence and dynamic interactions of the SVF cells, especially when determining the cellular sources of AT-derived paracrine and endocrine hormones.

 

Figure 8. Constituents of adipose tissue (AT). Left: Along with mature, functional adipocytes and precursor cells, many cell types related to vasculature and immune function reside within AT. They perform both physiological and pathophysiological functions by communicating with the adipocytes via secreted factors and scavenging lipid from dying fat cells. The number and diversity of these cell types increases with developing obesity and metabolic dysfunction. Right: The non-adipocyte cells are collectively referred to as the stromal vascular fraction (SVF), and the SVF can be separated from lipid-containing adipocytes by digesting the extracellular matrix (ECM) and centrifuging the cellular mixture. The SVF will form a pellet at the bottom of the tube, while the adipocytes will float and form a visible lipid layer at the top of the aqueous medium. This separation technique is critical to studying the cellular composition of adipose tissue and gaining insight regarding the individual functions of these diverse and distinct cell types under physiological and pathophysiological conditions.

Adipogenesis

 

To understand how adipocytes contribute to systemic metabolic regulation, it is important to understand their development. Adipogenesis refers to the process by which precursor cells differentiate and become committed to storing lipid and maintaining energy homeostasis as adipocytes. Adipogenesis is regulated by hundreds of factors, including nutrients, cellular signaling pathways, miRNAs, cytoskeletal proteins, and endocrine hormones such as growth hormone, insulin-like growth factor 1, insulin, and several steroid hormones, as well as cytokines. Generally, pro-inflammatory cytokines inhibit adipogenesis (192), although some cytokines within the same family exert opposing effects (192). Cytoskeletal proteins (193), ECM proteins and their regulators (194), microRNAs (miRNAs) (195), and long noncoding RNAs (lncRNAs) (196) differentially modulate adipogenesis. Dozens of different transcription factors, briefly described below, also regulate adipogenesis (Figure 9).

Figure 9. Transcriptional regulation of adipogenesis as determined in vitro in a fibroblast-like preadipocyte clonal cell line. Preadipocytes are grown to confluence and become growth arrested. Following induction of differentiation, they re-enter the cell cycle and undergo several rounds of proliferation, a process known as mitotic clonal expansion. At the end of this short proliferative phase, preadipocytes terminally differentiate into adipocytes as they begin synthesizing lipid and assume characteristics of mature fat cells. Numerous transcription factors have been determined to promote (green arrows) or inhibit (orange horizontal ended line) adipogenesis either during clonal expansion or at later stages of terminal differentiation. The timing of activation (i.e. when each transcription factor is turned on and off) is critical to the progression of adipocyte differentiation.

 

PROMOTORS OF ADIPOGENESIS

 

The transcription factor peroxisome proliferator activated receptor gamma (PPARg) is considered the principal adipogenesis regulator (197). Its discovery substantially enhanced our understanding of the adipocyte and its role in metabolic disease. For example, mice with adipocyte-specific PPARg deletion have decreased AT mass and are insulin resistant (198). In humans, PPARg gene mutations can also cause lipodystrophy (partial or generalized loss of fat in the body) and severe insulin resistance (199–201). The discovery of PPARg as the functional receptor for the insulin-sensitizing TZDs resulted in a significant effort to understand PPARg action and identify additional agonists.Synthetic TZDs induce weight gain in humans and rodents by increasing fat mass, more so in the subcutaneous adipose depot, which is associated with improved metabolic outcomes. However, this weight gain is also considered as a negative side effect of TZD treatment, especially in the typical patient who has pre-existing obesity. Other adverse side effects of TZDs, such as bone fractures and heart failure, have spawned the search for structurally distinct PPARg ligands capable of inducing unique receptor-ligand conformations with signature affinities for diverse co-regulators (202). Several selective PPARg modulators (SPPARMs) with fewer side effects have been identified. These act as partial PPARg agonists, alter specific post-translational modifications of PPARg, and preserve anti-hyperglycemia effects while minimizing or eliminating the adipogenic effect that leads to increased fat mass via activation of distinct gene profiles that may be cell and tissue specific (203,204). Interestingly, the TZD, rosiglitazone, is capable of improving glucose homeostasis even in the absence of PPARg in mature adipocytes (205), suggesting that its adipogenic effects (in addition to its non-adipogenic ones) may also be important for its anti-hyperglycemic action.

 

In addition to TZDs, PPARg binds endogenous lipophilic molecules, including: long chain fatty acids (LCFAs), oxidized or nitrated FAs, prostaglandins, and arachidonic acid derivatives (206). Interestingly, serotonin (5-hydroxytryptamine, 5-HT) has also been shown to be a high affinity agonist for PPARg (207). Many of the endogenous PPARg ligands enhance adipocyte differentiation and regulate fat cell functions such as lipolysis, glucose uptake, and lipogenesis through PPARg-dependent and independent methods (208–214). Overall, these endogenous ligands have low affinity and limited subtype selectivity for PPARg relative to other PPARs, suggesting that much remains to be understood regarding this critical adipogenesis regulator. While there is no question that PPARg is essential for adipogenesis and lipid accumulation within fat cells, a better mapping of its gene expression profiles in discrete cell and tissue types and with endogenous and synthetic ligands will improve our understanding of AT development and function under both physiological and pathophysiological conditions.   

 

The CCAAT/enhancer-binding proteins (C/EBPs) are widely expressed transcription factors that regulate proliferation and differentiation of various cell types in mammals. Studies in vivo and in vitro have identified C/EBP isoforms α, β and δ as important regulators of adipogenesis (215). C/EBPs β and δ work together in early adipogenesis to promote fat cell differentiation by inducing expression of C/EBPα and PPARg (216). Additionally, the transcription factors Krox20 and ZNF638 can modulate adipogenesis by affecting C/EBPβ function (217,218).

 

The Signal Transducer and Activator of Transcription (STAT) family of transcription factors was first identified over 20 years ago (219). Both the protein expression of STATs and their ability to regulate gene expression are tissue-specific (220). In AT, STATs regulate gene expression during adipogenesis, and the expression of STATs 1, 3, 5A, and 5B is induced during differentiation of murine and human preadipocytes (221,222). Notably, the ability of STAT5 proteins to promote adipogenesis has been documented by over a dozen independent laboratories using both in vitro and in vivoapproaches (17).

 

Of the three isoforms of Sterol Response Element Binding Proteins (SREBP-1a, SREBP-1c, and SREBP-2), SREBP-1c is the predominant form expressed in white AT (223,224) and is an important regulator of lipogenesis genes, while SREBP-2 regulates the expression of cholesterol biosynthesis genes (225). Intriguingly, two miRNAs (miR-33a and miR-33b) located within the SREBP genes are highly induced during adipogenesis (226). Although SREBP-1 clearly plays a promoting role in adipogenesis in vitro, in vivo studies suggest that SREBP-1 is not critical for AT development and/or expansion, perhaps due to compensatory SREBP-2 overexpression (47,227).

 

Members of the early B-cell factor (EBF) family of transcription factors are characterized for their ability to modulate islet beta-cell maturation and neural development. Three primary members of this family (EBFs 1, 2, and 3) are expressed in fat cells. EBFs 1 and 2 can promote adipogenesis (228,229), and EBF2 can also play roles in determining brown versus white adipocyte identity in vivo (230) and the beiging process of adipose tissue in mice (231).  

 

INHIBITORS OF ADIPOGENESIS

 

The interferon-regulatory factor (IRF) family of transcription factors has functionally diverse roles in the immune system, but also plays a role in adipocyte development. All nine IRF family members are regulated to different degrees during adipogenesis in vitro, and some members can repress adipogenesis (232) and contribute to insulin resistance (233). For example, knockdown of IRF3, whose is expression is elevated in visceral and subcutaneous AT of obese mice as well as in subcutaneous AT from humans with obesity and diabetes decreases fat mass and prevents insulin resistance in high fat diet-fed mice (233).

 

Wingless-related integration site (Wnt) proteins regulate development and cell fate through both autocrine and paracrine signaling (234) by using three well-characterized pathways: the canonical Wnt signaling and the planar cell polarity and Wnt/calcium pathways, which are non-canonical. The canonical pathway is dependent upon the transcription factor, β-catenin (235). Wnt10b is the best studied member of the Wnt signaling family in terms of adipocyte development. In the presence of Wnt10b, β-catenin translocates to the nucleus where it inhibits PPARγ and C/EBPα activity, thereby impeding adipogenesis (236,237). On the other hand, extracellular antagonists of Wnt/β-catenin signaling have been reported to promote adipocyte differentiation (238,239). 

 

The GATA family of transcription factors were named based on their ability to bind the DNA sequence GATA (240). Only GATAs 2 and 3 are expressed in preadipocytes residing in white AT (241), and both are repressed during adipogenesis. In fact, GATA2 can directly bind to the PPARγ promoter to suppress its activity (241). In addition to inhibition of PPARγ expression, GATAs 2 and 3 can also associate with C/EBPs to disrupt their transcriptional activity (242). GATA3 expression is driven by the canonical Wnt signaling pathway (243,244). Collectively, these studies demonstrate that two GATA proteins can attenuate adipocyte development via multiple transcriptional and signaling pathways.

 

TRANSCRIPTION FACTOR FAMILIES THAT CAN EITHER PROMOTE OR INHIBIT ADIPOGENESIS

 

The Krüppel-like transcription factors (KLFs) include 17 members that can either activate or repress transcription. In relation to adipocyte development, KLFs 4, 5, 6, 9 and 15 can promote adipogenesis, while KLFs 2, 3 and 7 repress adipocyte development. Most studies on the roles of KLFs in adipogenesis have been performed in vitro using a variety of cell culture models, and have demonstrated that KLFs act in concert with other transcription factors modulate adipogenesis (245).

 

The transcription factor activator protein 1 (AP-1) consists of Jun proteins (c-Jun, JunB, and JunD), Fos proteins (c-Fos, FosB, Fra1, and Fra2), ATF and JDP family members, several of which are induced during adipogenesis (222). In humans, a mutation in the c-fos gene that is associated with lipodystrophy has been shown to reduce c-fos activity and adipocyte development (246). Many in vitro and in vivo studies demonstrate that, like KLFs, AP-1 transcription factors can positively and negatively regulate adipogenesis.

 

Many of the zinc finger proteins (ZFPs) function as transcription factors with several contributing to adipocyte determination and/or adipogenesis. Zfp423 and Zfp467 can promote adipocyte differentiation by enhancing PPARγ expression and activity (247,248). In addition to stimulating adipogenesis, Zfp423 can suppress ‘beige-like’ properties in white adipocytes that are typically associated with improved metabolic health (249). Zfp521 can inhibit Zfp423 to reduce adipocyte development and is also considered a critical regulator of the commitment to either osteogenic or adipogenic lineages (250,251).

 

The transforming growth factor beta (TGF-β) superfamily encompasses a large number of proteins, including bone morphogenetic proteins (BMPs) (252). BMPs and TGF-β have been reported to be involved in both adipocyte commitment and differentiation (253–255). Specifically, BMPs 2 and BMP4 can promote adipogenesis via the Smad signaling pathway (256) to regulate transcription of target genes such as PPARγ (257,258). While BMPs are known to promote adipogenesis, in vitro and in vivo studies demonstrated that TGF-β primarily inhibits fat cell differentiation.

 

HORMONAL REGULATION OF ADIPOGENESIS

 

Steroids are prominent regulators of AT development and distribution, and adipocytes express high levels of many steroid hormone receptors. These lipophilic hormones diffuse through plasma membranes, dimerize, and bind to their specific receptors to impart both genomic and non-genomic responses (259,260). Since steroid-bound receptors act as transcription factors, their capabilities should be fully considered in the transcriptional regulation of adipogenesis.

 

Two types of estrogen receptors, ERα and ERβ, are expressed in rat and human preadipocytes, mature adipocytes, and in other AT cells (261–263). Although many studies describing the role of estrogens in AT are contradictory, most investigations indicate that estrogen inhibits adipocyte differentiation (245) and the adipogenic action of PPARγ (264). Aromatase is an enzyme found in several tissues, including AT, that aromatizes androgens into estrogens. Both ERα-and aromatase-knockout mice have increased adiposity, suggesting that both estrogen and its receptor can reduce adipocyte development (265,266). Mice lacking ERα have enhanced visceral AT deposition and increased weight gain compared with wild-type mice (267).

 

Androgen receptors (AR) are also expressed in rodent (268,269) and human AT (270).  Similar to estrogen, many studies report contradictory actions of androgens on the differentiation and function of adipocytes. These inconsistent results highlight the importance of accounting for sex-, depot- and organism-specific effects. In studies of human AT, testosterone and the non-aromatizable androgen, dihydrotestosterone, inhibit differentiation of preadipocytes obtained from subcutaneous and omental depots of both men and women, although the magnitude of the inhibitory effect may differ between the sexes  (271,272). Overall, most studies indicate that androgens exert inhibitory effects on adipogenesis.  

 

Glucocorticoids (GCs) are well-known promoters of adipocyte development. GCs also promote adipocyte hypertrophy and differentiation of central fat depots that can lead to abdominal obesity and insulin resistance (273). In vitroadipogenesis studies include the wide use of the synthetic GC, dexamethasone. Although the mechanisms of action and target genes of GCs involved in adipocyte differentiation are not completely clear, it is known that GCs induce expression of C/EBPs beta  and δ and that GC-induced C/EBPδ coordinates with C/EBPβ to induce PPARg expression and adipogenesis (274).

 

To understand the actions of GCs via the glucocorticoid receptor (GR), it is important consider the enzyme that affects circulating levels of cortisol, the active form of GR’s endogenous ligand. 11β-hydroxysteroid dehydrogenase type 1 (11 beta HSD1) is an enzyme highly expressed in AT and liver that in AT converts inactive cortisone to the active hormone cortisol. Hence, it is not surprising that 11 beta HSD1 mRNA expression and activity is essential for the induction of human adipogenesis and that adipocyte development can be blocked with a 11 beta HSD1 specific inhibitor (275). In addition to inducing the expression of early adipogenic transcription factors, GCs promote adipocyte development by mechanisms that include suppression of anti-adipogenic factors (Pref-1 and Runx2); anti-proliferative effects on preadipocytes; and sensitizing or ‘priming’ of human preadipocytes to insulin action (276). Recent attention has focused on the potential contributions of environmental pollutants known as endocrine disrupting chemicals (EDCs) in the development of metabolic diseases. Studies reveal that EDCs can promote adipogenesis through GR activation (277), thereby implicating these compounds in the rising rates of obesity and diabetes.

 

In addition to regulating water and salt homeostasis, the mineralocorticoid aldosterone and its receptor (MR) have also been shown to play a role in the regulation of adipocyte development. This is important since MR is a high-affinity receptor for both mineralocorticoids and GCs. Aldosterone promotes adipogenesis in an MR-dependent manner (278)and a MR antagonist can inhibit adipogenesis (279). Although GRs and MRs are expressed in AT and thought to mediate cortisol’s actions on AT, the levels of GR are several hundred-fold higher than MR in both human preadipocytes and adipocytes (280). Loss of GR, but not MR, blocks the adipogenic capabilities of cortisol in human preadipocytes (280). However, MR expression is higher in omental than in subcutaneous AT, so there could potentially be depot differences in the relative importance of MR and GR in cortisol-induced adipogenesis (280). There could also be differences in the contribution of MR to adipogenesis during obesity when MR and 11 beta HSD1 expression levels are increased, while the GR and 11 beta HSD2 (the enzyme that deactivates cortisol) levels do not increase accordingly (280). Most of the current evidence suggests that the ability of aldosterone to modulate adipogenesis in vitro is largely dependent on MR. Additional studies are needed to determine if MR plays a role in adipocyte development in vivo.  

 

Vitamin D is another steroid hormone with strong experimental evidence that it can regulate adipogenesis. Unlike most of the water-soluble vitamins that are excreted via urine when in excess, Vitamin D, along with the other fat-soluble vitamins (A, E, and K), can be stored within fat-laden adipose tissue. The vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates vitamin D, are expressed in human AT, primary preadipocytes, and newly-differentiated adipocytes (281). The most active form of Vitamin D, 1, 25-Dihydroxyvitamin D, represses adipocyte differentiation (282,283) and the VDR can block adipogenesis by inhibiting C/EBPβ expression (284). Vitamin D-induced inhibition of adipogenesis also involves direct suppression of C/EBPα and PPARg (285). Vitamin D and VDR also play a role in the inhibition of adipogenesis of bone marrow stromal cells (286), in part by suppressing the expression of inhibitors of the canonical Wnt/β-catenin signaling pathway (287). Although vitamin D inhibits adipogenesis in the widely used murine and bone marrow-derived cells, both 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D3 can promote the differentiation of human subcutaneous preadipocytes (281). Overall, a case could be made that concentrations of vitamin D as well as the type of adipocyte precursor determine whether this hormone exerts pro- or anti-adipogenic actions via the VDR.

 

On the other hand, evidence regarding Vitamin D’s role in adipocyte development in humans is controversial and contradictory. According to a systematic review and meta-analysis of 23 studies between 2002 and 2014, overweight or obese subjects exhibit a higher prevalence of Vitamin D deficiency (288). In two double-blind, placebo-controlled randomized clinical trials, Vitamin D-supplemented individuals with healthy overweight or obesity lost significantly more fat mass than the placebo group when fed either a calorie-restriction (289) or weight-maintenance (290) diet for 12 weeks. While decreased fat mass may result from Vitamin-D induced inhibition of adipogenesis, this hypothesis was not directly tested in the studies, and two other longer term studies demonstrated no change in fat mass with Vitamin D supplementation between 14,000 and 20,000 IU per week (291,292).  

 

The relationship between adipocyte development and thyroid hormones has been recognized since 1888 when a report on myxedema proposed that obesity was a requirement for a diagnosis of hypothyroidism (293). The most biologically active form of thyroid hormone, T3, can induce brown adipocyte differentiation (294). Hyperthyroidism in rodents induces adipocyte hyperplasia, whereas hypothyroidism impedes AT development (295). Overall, studies on the involvement of thyroid hormones in AT development are controversial. While the induction of adipogenesis is differentially regulated by various thyroid hormone receptor (TR) isoforms, studies largely indicate that TRs promote adipogenesis in the majority of model systems (245).   

 

Adipocyte Progenitors

 

In AT, pools of adipose stem/progenitor cells (APs) exist that can differentiate into mature adipocytes (296,297). At least two distinct progenitor populations give rise to adipocytes:  developmental APs and adult APs (296,298). Our understanding of the molecular characteristics of APs has dramatically increased in recent years as discussed below.

 

APS IN ADIPOSE DEVELOPMENT

 

AT organogenesis in mice and humans begins during embryogenesis, and ends in the postnatal period for mice and just before birth in humans (296,297,299). AT is widely accepted to be of mesodermal origin (297). However, some of the spatiotemporal and molecular differences observed in formation of different AT depots suggest diverse developmental origins (297,300). Further, white and brown adipocytes, once considered to have common APs are now known to have different origins (297,301,302).

 

In the generation of white adipocytes, developmental APs express the master adipogenesis regulator PPARg but have distinct functional and molecular properties compared to adult APs (298,302). Developmental APs do not contain lipid but express the mature adipocyte markers perilipin and adiponectin, are able to replicate, and are located along the vasculature in developing white adipose tissue (298,302,303). Brown adipocytes can arise from myogenic Myf5-expressing precursors that also give rise to skeletal myocytes (297,302,304). Interestingly, brown-like adipocytes, known as beige adipocytes, emerge in white adipose from Myf5-negative precursors in response to cold or adrenergic stimuli, which suggests that the developmental origins of brown adipocytes and beige adipocytes are different (297,304). Collectively, these findings highlight the complex developmental heterogeneity of APs observed among adipose tissue depots in animals and humans.

 

APS IN ADULT ADIPOSE TISSUE

 

The notion that we are born with all the fat cells we will ever have is now considered archaic and inaccurate. Adipose tissue continues to generate new adipocytes throughout the lifespan, with a median adipocyte turnover rate of 8.3 years (302,305). Adult APs have been found in the SVF of AT depots in both rodents and humans (302,306–308) and are thought to represent an AP pool that contributes to this adipocyte renewal. Flow cytometry techniques that use a variety of cell surface and stem cell markers, have helped identify stromal cells that can undergo adipogenesis (302,306,307,309). These adult APs arise from tissue-resident mesenchymal stem cells, and are a major source of new adipocytes in AT (297,310). Bone marrow-derived APs from the myeloid lineage can also be recruited to AT where they become adipocytes (Figure 10). Bone marrow-derived adipocytes (BMDAs) are more abundant in female mice and are more frequent in visceral depots (297,311,312). Though BMDAs have been observed in human AT and are increased in patients with obesity, the processes and factors involved in BMDA recruitment to AT remain unclear. Compared to normal adipocytes, BMDAs have reduced expression of lipid metabolism genes and increased pro-inflammatory gene expression, suggesting that they may have negative metabolic effects (297,311,313).

 

Figure 10. Adipocytes are derived from both resident mesenchymal cells in the stromal-vascular fraction of adipose tissue and hematopoietic progenitors that reside in the bone marrow. In addition to adipocytes, mesenchymal progenitors can form other connective tissue cells, such as myocytes and osteocytes. Myeloid progenitors derived from hematopoietic progenitors in bone marrow give rise to adipocytes as well as neutrophils, macrophages, dendritic cells, and granulocytes.

Most of the information regarding AP proliferation in obesity comes from rodent models. In mice fed high-fat diets to induce obesity, APs form new adipocytes primarily in the visceral depot (299,302). Although limited data report decreased AP proliferation and differentiation capacity from humans with obesity compared to lean individuals (314), convincing evidence for depot-specific AP populations in humans has emerged. Subcutaneous APs were shown to have a higher growth rate and adipogenic potential than visceral APs, giving rise to more functional adipocytes (315,316). Increasingly sophisticated methods for assessing APs in mice will help facilitate the identification of the origins of all APs for each adipose depot as well as the niches in which they reside.

 

Adipose Extracellular Matrix: From Normal Development to Fibrosis

 

An underappreciated influence on AT physiology is the adipose extracellular matrix (ECM). The dynamics and composition of the ECM are critical for proper adipocyte development and function (317). During adipogenesis, there is increased synthesis of laminar ECM constituents and maintenance of peri-adipocyte fibrillar collagens that ultimately allows the adipocyte to embed itself in the basal lamina (317). In the growth phase of adipogenesis, adipocytes require ECM-mediated traction forces to properly accumulate lipids and increase in size. A number of inhibitors, enzymes, and modifiers contribute to adipocyte ECM maintenance and renewal; these reactions consume a large amount of energy in the mature fat cell (317). In obesity, the ECM expands to accommodate the adipocyte hypertrophy and hyperplasia, and subsequent tissue growth, induced by the increased demand for lipid storage (317–321). This process appears to occur in a similar fashion in both animal models and humans.

Figure 11. Differences in AT between lean and obese mammals. The AT extracellular matrix (ECM) is important for normal tissue function but can also contribute to its dysfunction. In obesity, accumulation of ECM components can restrict AT expansion, promote inflammation by recruiting immune cells, and impair adipogenesis. These combined effects can worsen insulin resistance.

Adipose tissue expansion during obesity, coupled with immune cell accumulation and hypoxia, can lead to AT fibrosis (Figure 11) (317,319,322). Fibrosis is the excessive accumulation of ECM components, such as collagens, that typically results from an imbalance of the synthesis and degradation of ECM components (319,323). Ultimately, adipocyte dysfunction will result from the decreased ECM flexibility conferred by the accumulation of fibrillar ECM components (317,320,323). Abnormal ECM collagen deposition is associated with immune cell infiltration, which can worsen fibrosis and contribute to AT dysfunction that often occurs in obesity (319,323). The removal of collagen VI, a major AT ECM component, improves adipocyte function and metabolism in obese mice by both decreasing AT immune cell infiltration and “weakening” the ECM, which allows uninhibited adipocyte hypertrophy (317,318,323). In humans, AT collagen VI expression is increased in obesity, and subjects with higher collagen VI have increased macrophage content and AT inflammation (324). Endotrophin, an adipocyte-derived cleavage product of collagen VI, directly stimulates AT fibrosis and macrophage accumulation, and can lead to systemic insulin resistance (325). Endotrophin can also cause fibrosis and endothelial cell migration in mammary tumors, leading to tumor expansion and the enhancement of metastatic growth (325,326).

 

Accumulation of ECM components and increased ECM-receptor signaling are associated with insulin resistance in obesity thought to be mediated by several possible mechanisms. In addition to physically restricting AT expansion, excess ECM components can also increase AT inflammation by interactions with their cell surface receptors (CD44, CD36, and integrins) (320). These ECM-receptor interactions can induce adipocyte death, inhibit angiogenesis, and promote macrophage infiltration and inflammation in adipose tissue, thereby driving insulin resistance (320). Interestingly, these downstream effector pathways of ECM-receptor signaling are similar to those involved in tumor growth and pulmonary fibrosis development. 

 

The ECM has clear roles in the normal development and function of adipocytes, but in excess can also play roles in obesity development and metabolic dysfunction. Our understanding of the adipose ECM has deepened in recent years, but more research is necessary to better delineate how ECM components and their interactions can directly influence AT physiology and pathophysiology. Since many AT cell types produce ECM components, studies to determine the specific contributions of adipocyte-derived ECM components to normal AT function as well as dysfunction will be required.

 

Rodent versus Human Adipose Depots

 

Much of the knowledge about the depot-specific characteristics and metabolic profiles of AT has been obtained from rodents. However, the validity of translating studies conducted in rodent fat to humans remains controversial. Relative to humans, rodents have substantially more BAT and rely heavily on this highly-inducible depot to stimulate thermogenesis (327). While BAT activation in rodents has been shown to elicit beneficial effects, including improvements in glucose and lipid metabolism (328,329), BAT function in humans is more controversial. Overall, the majority of studies have reported that the amount of active BAT in humans appears insufficient to induce meaningful changes in energy metabolism and, thus, is not thought to impact whole-body physiology and metabolic control in humans (330) as described in rodents.

 

With regard to white AT, notable differences exist with respect to fat depot structure and function between species (18). Humans have subcutaneous depots primarily in the abdominal and gluteal-femoral regions; whereas rodents have subcutaneous fat pads located anteriorly and posteriorly (Figure 12). With regards to location, the inguinal (posterior) fat pad in rodents is considered comparable to the gluteal and femoral depots in humans. Human subcutaneous

abdominal AT can be categorized as superficial SAT or deep SAT (331), which are  morphologically and metabolically different. Deep SAT has been reported to be closely related  to the pathophysiology of obesity-related metabolic complications, while superficial SAT is more closely related to the protective lower-body SAT (332–334). However, these subcutaneous layers are not present in rodents. In humans, intra-abdominal fat refers to visceral AT, which surrounds the inner organs, and includes omental, mesenteric, retroperitoneal, gonadal, and pericardial depots (335). For most purposes, however, when used in reference to human studies, visceral AT refers to omental and mesenteric depots that are quantified by abdominal computed tomography or MRI scans. On the other hand, visceral fat pads in rodents are classified as perigonadal (epididymal in males and periovarian in females), retroperitoneal, and mesenteric. While the mesenteric fat pad is most analogous to abdominal (visceral) AT in humans, it is not often studied in rodents due to surgical limitations. The perigonadal fat pads are the largest and most the readily assessable fat in rodents; hence, they are most frequently used in mouse studies and cited the most often in the literature as surrogates for human visceral AT. However, humans do not have an AT depot analogous to the rodent perigonadal fat pads. In addition, the omental depot is clearly defined in humans, but in mice it is difficult to detect. Overall, striking anatomical differences in AT distribution exist between rodents and humans, and these differences should be considered when interpreting rodent studies and potentially translating these observations to humans.

 

Figure 12. Rodent versus Human AT depots. Several differences exist between rodent and human subcutaneous (SubQ) and visceral AT depots. In the figure SubQ depots are colored as beige, while visceral depots are white, and BAT or BAT-like depots are brown.

It is well-established that the various adipose depots display metabolic heterogeneity and are intrinsically different within each species. In humans, fat deposition in the upper body, mainly the visceral but also the subcutaneous abdominal depot, is linked to a higher risk of metabolic dysfunction; while lower body adiposity in the subcutaneous gluteal and femoral regions is associated with lower risk and may even be protective (336). Rodent studies reveal that surgical removal of visceral fat pads improves insulin action, glucose tolerance, and longevity (337,338), while the removal of subcutaneous fat pads can cause metabolic syndrome (339). In addition, subcutaneous, but not visceral, donor AT transplanted into the visceral region of recipient mice improves glucose metabolism (340). In contrast, human studies have shown that the removal of small amounts of omental AT in individuals with obesity provided no metabolic health benefits (341). Likewise, liposuction (~10 kg) of subcutaneous AT in humans neither harmed nor improved the cardio-metabolic profile (342,343). Nevertheless, fat is redistributed from the subcutaneous to visceral depots during aging (344) in conjunction with increasing prevalence of chronic diseases such as hypertension, T2DM, and cardiovascular disease, suggesting that subcutaneous AT may be metabolically beneficial in humans as has been extensively reported in rodents.

 

Studies of depot-specific expression patterns have enhanced our understanding of the mechanisms underlying abdominal versus gluteal and femoral adiposity (345–347). Unique expression patterns in different adipose tissue depots in mice indicate substantial difference in the expression of homeobox (HOX) developmental genes (348). Not surprisingly, HOX genes exhibit differential expression patterns in human compared to mouse fat depots (346,347). In contrast, structural and hormonal regulators, including collagen VI (349,350) and glucocorticoids (351,352), respectively, that influence fat distribution are similarly associated with AT expansion in both rodents and humans.

 

Similar to humans (353), female rodents have a higher percentage fat mass relative to males, yet remain more insulin sensitive (354). However, there are many notable sex differences in rodent versus human depots. The inguinal depots of female mice contain mammary glands and the gonadal fat pad is near reproductive tissue, which is not the case in humans. In addition, high-fat diet-induced obesity affect men and women alike, but in many strains of mice females are resistant HFD obesity, unlike male mice (355,356). Furthermore, the periovarian (visceral) fat pad in female mice has been shown to be more insulin sensitive than the inguinal fat pad (354), which is contrary to human data that indicates in women the gluteal and femoral depots are more insulin sensitive relative to the visceral AT (357).

 

Current literature suggests that the secretion patterns of adipokines (including leptin, interleukin-6, and tumor necrosis factor α) in the visceral versus subcutaneous depots of humans are relatively similar to that of rodents. Interestingly, lower body AT has been shown to secrete more metabolically favorable adipokines such as adiponectin (358). These observations are similar in rodents studies (340).

 

While lipolysis can be stimulated in rodents and humans under similar physiological conditions, important biological differences in AT lipolysis among these species have been suggested. The β1 and β2 adrenergic receptors (AR) are ubiquitously expressed in rodents and humans, while β3-AR expression is confined to white AT in rodents and only marginally expressed in human adipocytes (359). The α2-ARs are highly expressed in the subcutaneous AT of humans and act to inhibit lipolysis (360), but are absent in rodent adipocytes. Though common factors, including catecholamines, growth hormone, and cortisol, are similar among species in regulating lipolysis, differences in the response to other lipolytic agents have also been reported. Natriuretic peptide induces lipolysis in humans, but not in rodents (361), while adrenocorticotropic hormone and alpha-melanocyte-stimulating hormone modulate lipolysis in rodent but not human adipocytes (362,363). Therefore, it is important to account for these differences and commonalities in AT lipolysis among species.

 

Rodent studies are essential to expand our understanding of pathways underlying the associations between fat distribution and metabolic health and disease. Fortunately, there are many shared traits among rodent fat pads and human fat depots. However, given the clear differences in adipose depot location and physiology between the species, interpretation of experimental data and the extrapolation of conclusions drawn from rodent data to humans should be conducted with appropriate caution and caveats.

 

Dermal Adipose Tissue

 

A thick layer of adipocytes, historically referred to as subcutaneous AT, underlies the reticular dermis in both rodents and humans (364). Recent studies have revealed major differences between the adipocytes from this dermal layer and more typical subcutaneous adipocytes found in other locations (364–366). Today, dermal adipose tissue (dWAT) is considered a separate adipose depot that is distinguishable from subcutaneous fat (364). Two unique features of dermal adipocytes in this regard are that they can alter their cellular characteristics and have high turnover rates (366). An additional distinguishing factor for dWAT is its organization. In rodents, dWAT forms several adipocyte layers between the dermis and muscle layer (panniculus carnosus) (367). Human dWAT is present as individual units referred to as dermal cones. These cones are concentrated around pilosebaceous units that functionally interact with each other to form the dWAT structure (366,368). Interestingly, only body regions prone to scarring contain dermal cones (368), indicating a potential role for dWAT in scarring and wound healing. Also, dWAT can regenerate after injury. Following injury, adipogenesis is activated in the proliferative phase of wound healing and dermal adipocytes repopulate the wound (366,367). This is a critical event, as mouse models lacking mature adipocytes cannot recruit the fibroblasts required for wound healing (369–371).

 

Other identified roles for dWAT include insulation (372), barrier protection from skin infection (373), and hair follicle cycling (374). It is well known that brown adipose tissue (BAT) rapidly responds to cold temperature challenges by mobilizing lipids for heat generation (adaptive thermogenesis), yet dWAT slowly responds to these challenges by thickening/expanding over days to provide an effective layer of insulation (367,372). Mouse models lacking adequate dWAT undergo chronic activation of BAT since the dWAT cannot provide adequate mitigation of body temperature (367). Conversely, obese mice with excess dWAT undergo minimal adaptive thermogenesis (367). The dWAT thickening observed with cold exposure also occurs with bacterial exposure. Adipocytes in dWAT differentiate and become hypertrophic and result in a thicker dWAT layer in response to epidermal Staphylococcus aureus. This dWAT adipocyte reaction is also critical for immune response to bacterial invasion (373). Hair follicles go through repeated rounds of death and regrowth, referred to as the hair follicle cycle (367). Robust dWAT expansion is characterized by increased adipogenesis and dermal adipocyte hypertrophy that accompanies the regrowth of hair follicles (374). Conversely, inhibiting adipogenesis impedes hair follicle regeneration. In several species of mammals, a thickening of the hair coat accompanies dWAT expansion in response to cold exposure (367). In summary, dWAT has distinct roles from subcutaneous AT. Thus far, unlike other AT depots, the contribution of dWAT to metabolic health has not been investigated. Nonetheless, there is clear evidence that dWAT has distinct structures and functions and plays a role in variety of physiological processes.

 

Epicardial AT

 

Epicardial AT (EAT) has recently emerged as an important player in the development of cardiovascular disorders (375,376). Notably, EAT is distinct from pericardial fat. While pericardial AT surrounds the pericardium, EAT lies between the visceral pericardium and the myocardium and shares a blood supply with the coronary arteries (375–378). The adipocytes in EAT are smaller than those in other visceral or subcutaneous depots and are outnumbered by preadipocytes; this is thought to be related to the high energy requirement of the heart, which normally favors oxidation of fatty acids over other substrates (376,379). Furthermore, the gene expression and adipokine secretion profiles of EAT are unique from those of other depots (376,380,381).

 

In normal physiological conditions, EAT behaves like BAT and serves to protect the coronary vessels and myocardium against hypothermia (376,382). In pathologies such as coronary artery disease and type 2 diabetes, EAT can display an extensive pro-inflammatory signature (383–385). Macrophages and mast cells have been shown to infiltrate EAT, undergo activation, and through a cascade of signaling events facilitate lipid accumulation in atherosclerotic plaques (376,384). Pro-inflammatory adipokine secretion from EAT has also been shown to induce atrial fibrosis (381). Further, insulin sensitivity and EAT thickness are inversely correlated, whereas fasting glucose and EAT size are positively correlated, with enlarged EAT depots often found in individuals with type 2 diabetes (376,386,387). These data suggest that EAT functions as a distinct fat depot with important physiological and pathological roles.

 

METABOLIC DYSFUNCTION ASSOCIATED WITH ADIPOSE TISSUE

 

Adipose Tissue Expandability and Metabolic Health

 

White AT retains the ability to expand during adult life to accommodate chronic excess caloric intake. AT expansion is characterized by adipocytes accumulating lipid and growing in size (hypertrophy) or number (hyperplasia or adipogenesis) or increasing in both size and number. Evidence suggests that the capacity of subcutaneous AT to expand as well as the manner of expansion (hypertrophy vs. hyperplasia) can influence cardiometabolic health. This mechanism is thought to underlie the benefits of thiazolidinedione (TZD) medications, which are approved for the treatment of type 2 diabetes (388,389). These PPARg agonists stimulate preadipocyte differentiation and the proliferation of adipocytes (390,391), especially in subcutaneous depots as compared to visceral adipose tissue (392), which leads to increased adiponectin levels and improved insulin sensitivity (393,394). Hence, there is a clear rationale to further characterize the mechanisms of AT expansion through adipocyte proliferation in humans that may inform future effective drug therapies.

 

On the other hand, the presence of enlarged, hypertrophic adipocytes, a lack of hyperplasia, and development of AT inflammation and fibrosis reflect impaired AT expansion and is associated with metabolic derangements (395–398). These observations support the “AT expandability hypothesis”, which postulates that a lack of adipogenesis (or hyperplasia) results in the limited capacity of AT to expand and store lipid, causing ectopic fat accretion and “lipotoxicity” in non-adipose tissues such as skeletal muscle and liver (399–401). The degree of ectopic lipid deposition in the liver and skeletal muscle is a significant determinant of metabolic syndrome (MetS) and the development of T2D and CVD (402).

 

Other findings do not support the AT expandability hypothesis and indicate that higher adipogenesis does not necessarily denote improvements in metabolic health. These studies report a higher population of small adipocytes (a measure of hyperplasia) in the AT of individuals with insulin resistance and T2D (403–406) and in those with more visceral AT and liver fat (406,407). Experimental overfeeding intervention studies have shown that individuals with smaller adipocytes at baseline have poorer metabolic health outcomes (i.e. impaired insulin sensitivity) in response to substantial weight gain than those with larger adipocytes (408,409). In addition, one in vivo analysis in humans demonstrated that increased hyperplastic expansion correlated with an increased number of metabolic syndrome components (410). Collectively, these data imply an alternative model of impaired AT expansion, as compared to the mechanisms proposed by the AT expandability hypothesis, and suggest that there is not a deficiency in hyperplasia but an abundance of adipocytes with a limited capacity to adequately expand and accommodate lipid, whether large or small. This inability to store excess lipid in AT is thought to be a key feature that leads to metabolic dysfunction.

 

Although the mechanisms of adipose expansion and its precise role in promoting glucolipid dysregulation remain a matter of debate, all of the aforementioned studies support the view that AT’s capacity to expand is intimately related to metabolic homeostasis, as the failure to store excess lipid appropriately in AT can contribute to many obesity-related complications.

 

AT Inflammation

 

A variety of cell types from both the innate and adaptive immune systems have been found in AT (411–413). Though resident AT immune cells are critical to normal adipocyte function in healthy individuals, AT inflammation, as mentioned in several preceding sections, is considered a major contributor to the metabolic dysfunction associated with obesity (413,414).

 

During nutrient excess as AT expandability reaches its limit, a strong association exists between adipocyte size and adipocyte death (415). In response to adipocyte death, pro-inflammatory macrophages surround dead and dying cells and remove debris from the damaged area. During this process, macrophages acutely produce inflammatory cytokines (413,416). In obesity, this cytokine production often fails to resolve, becomes chronic, and leads to impaired adipocyte insulin signaling, further inflammation, and a continued worsening of AT dysfunction (413,416,417). In a field that is rapidly changing, it is worth mentioning that some degree of inflammatory signaling might be required for normal AT function. The pro-inflammatory cytokines TNF alpha and oncostatin M have been shown to be required for proper AT expansion and maintenance of insulin sensitivity in mice (414,418–420). Although AT inflammation clearly has detrimental effects in obesity, evidence also indicates adaptive and homeostatic roles for pro-inflammatory signaling in AT expansion and function.

 

Metabolically Healthy (MHO) versus Metabolically Unhealthy (MUO) Obesity

 

An estimated 10-30% of individuals with obesity are considered to have “metabolically healthy obesity” (MHO) with favorable metabolic profiles (421). Although there is currently no consensus for parameters used to classify MHO,these individuals are characterized by normal insulin sensitivity, normal fasting glucose levels, low incidence of hypertension, and blood lipid profiles in the healthy range (422,423) (Figure 13). In contrast, individuals with “metabolically unhealthy obesity” (MUO) have comparable body mass indices (BMI) but develop metabolic aberrations. Factors that distinguish individuals with MHO from MUO (Figure 13) highlight the premise that metabolic health risk is not solely dependent on body weight and are described in more detail below. Understanding these characteristics and potential mechanisms underlying the MHO and the perceived healthy metabolic state of these individuals is an important area of ongoing research.

 

Figure 13. Clinical and biological factors thought to distinguish metabolically healthy obesity (MHO) from metabolically unhealthy obesity (MUO). Abbreviations: VAT – Visceral AT, SubQ AT – Subcutaneous AT, EMCL - extramyocellular lipid; IMCL – intramyocellular lipid; HDL – high density lipoprotein.

Evidence suggests that WAT plays a critical role in the development of MHO vs MUO, as its properties, location, and function are closely linked with cardiometabolic risk. Fat distribution (422), as well as changes associated with AT expansion, including the capacity for adipocyte differentiation (403) and parameters related to ECM remodeling (424), may also contribute to the MHO phenotype. In addition, adipose-derived circulating factors that impact whole-body metabolism have been implicated in MHO vs MUO differences (425).  However, studies have shown that the location of AT, rather than overall obesity, may be a stronger predictor of metabolic health risks (336). The accumulation of upper-body fat, namely visceral AT (VAT) but also subcutaneous abdominal (scABD) adipose tissue, confers a higher risk of obesity-related disorders (426), while lower-body fat (subcutaneous gluteal and femoral) may be metabolically protective (427). The preserved metabolic function of individuals with MHO may be attributed to significantly lower accumulation of VAT relative to MUO (422,428,429). As described in the previous section, enlarged adipocyte size, independent of adiposity, is positively correlated with the development of insulin resistance and impaired metabolic health (396). MUO individuals have been shown to have larger adipocytes than their MHO counterparts (430,431). Hypertrophic adipocytes may represent the failure of subcutaneous AT to expand and store excess fat, which can ultimately lead to ectopic lipid deposition in non-adipose tissues such as the liver and skeletal muscle (402).

 

Ectopic lipid accumulation in both the liver and skeletal muscle is of pathophysiological significance as part of the “lipotoxicity” hypothesis and may also impact the varying health risk of MHO vs MUO. Extramyocellular lipid (EMCL) and intramyocellular lipid (IMCL) are postulated to cause defects in insulin signaling and reduce insulin-stimulated skeletal muscle glucose uptake (432). These lipid stores are strong correlates of insulin resistance and are increased in individuals with T2D (402). Paradoxically, increased IMCL is also observed in ‘insulin sensitive’ athletes, which may be attributed to the oxidative capacity of skeletal muscle (433) and increased glucose transport in trained muscle (434). Intrahepatic lipid accumulation strongly associates with impaired insulin-induced suppression of hepatic glucose production, even independently of visceral AT amount, and the development of T2D (435). Ectopic fat in both the liver and skeletal muscle has been shown to be lower in MHO than MUO individuals (422,436,437) (refer to Figure 13).

 

The differential secretion of pro-inflammatory adipokines has also been proposed as a mechanism underlying the MHO phenotype (438–440) by some investigators, although others have reported conflicting results (441). Nevertheless, studies show reduced macrophage infiltration in MHO (442,443), supporting a reduced inflammatory state in these individuals. Intriguing data implicating potential genetic differences among MHO vs. MUO indicate that specific polymorphisms in genes, including the adiponectin receptor 1 and hepatic lipase, may be associated with the MHO phenotype (436). In addition, genes encoding some proinflammatory cytokines can be more highly expressed in the adipose tissue of MUO compared with MHO individuals (444,445).

 

A lingering question that remains unanswered is whether MHO subjects will sustain a healthy metabolic state throughout their lifespan or if they will eventually become MUO. An additional question is if a healthy lifestyle can help to maintain a favorable profile and prevent the transition to MUO. Indeed, longitudinal data clearly show that not all MHO individuals remain metabolically healthy, as up to 30% progress to MUO over a 5-10 year time frame (446–448). Of note, the length of time for follow-up assessments of MHO individuals is an important factor that may have considerable effects on the observed outcomes, because the total number of years as obese and aging can independently increase mortality risk. A major obstacle in advancing the understanding of the MHO phenotype is the manner by which metabolic health is described, including the parameters used to define insulin sensitivity and metabolic syndrome (449). Defining metabolic outcomes based on differing criteria can result in a broad range of reported prevalence, discrepancies regarding the observed characteristics, varied interpretations of health and mortality risks, and disagreement concerning the implications of therapeutic interventions. In addition, the use of the “healthy” descriptor may be misrepresentative of the true medical risks to these individuals, as long-term adverse health outcomes have been observed in individuals with MHO during follow-up years, thus no longer characterizing them as “metabolically healthy” (450,451). Additional long-term prospective studies are necessary to assess features of the MHO phenotype and to observe how the factors discussed above are altered over time. In addition, these studies may reveal if WAT function is a cause or consequence of the MHO and MUO phenotypes.

 

Lipodystrophy

 

While excessive adiposity, or obesity, can have adverse health consequences, deficiency of AT mass, as seen in lipodystrophy, can also lead to derangements in glucolipid metabolism. Lipodystrophy encompasses a group of rare, heterogeneous, genetic or acquired disorders characterized by varying degrees of severe reduction or absence of body fat (452) . Anatomically, this disease can present as a partial (i.e. localized to certain body areas) or generalized lack of AT. The combined overall prevalence of lipodystrophy is estimated to be 1 in 1,000,000 individuals, with ~1000 patients reported with genetic forms (453). Lipodystrophy associated with highly active antiretroviral therapy for HIV is one of the most common acquired forms worldwide (454). The diagnosis of this disorder mostly relies on clinical criteria. In most cases of generalized lipodystrophy, standard physical examination is sufficient to establish this diagnosis. In contrast, partial lipodystrophy may be represented by mild physical abnormalities and can sometimes be misdiagnosed as common forms of central (abdominal) obesity, suggesting that this form of lipodystrophy may be an underestimated condition (455). Although the pathological basis of most lipodystrophies remains unclear, it is well-accepted that AT dysfunction is a primary determinant of the resulting health consequences in these patients. Limited development and non-expandability of AT and failure of AT to accommodate excess lipid leads to the redistribution and storage of fat ectopically in the liver and skeletal muscle and the development of non-alcoholic fatty liver disease, often severe insulin resistance and type 2 diabetes, hypertriglyceridemia, and associated diseases (456–458).

 

Markedly reduced levels of leptin and adiponectin may also contribute to the pathology of lipodystrophy. As described above, leptin plays an important role in the regulation of body weight and energy metabolism, and leptin deficiency is common in lipodystrophic patients, due to the lack of AT (452). Low leptin levels can not only impact glucose metabolism (459) but also contribute to increased appetite and excessive caloric intake in these patients (460). Transgenic animal models shed light on the pathology of lipodystrophy and confirm the importance of AT in normal physiology. Fatless rodents, created via AT ablation, display hypertriglyceridemia and ectopic lipid accumulation, along with severe insulin resistance (457,461). In addition, several groups have successfully treated the metabolic derangements in these fatless mice by transplanting AT from wild-type animals (461–464). However, transplantation of AT from leptin-deficient mice did not improve the metabolic abnormalities in fatless mice (465), while leptin administration in the fatless mice ameliorated insulin resistance and hepatic steatosis (466). In humans with total lipodystrophy, leptin treatment also markedly improves the severe hypertriglyceridemia and insulin resistance that accompanies this disorder (467).  These studies confirm that both AT and leptin deficiency play a central role in lipodystrophy-associated pathologies.

 

Adiponectin has insulin-sensitizing and anti-inflammatory effects, and low levels of this adipokine have also been observed in patients with lipodystrophies (468). Recombinant adiponectin, adiponectin analogues (i.e. osmotin), and compounds that upregulate endogenous adiponectin (i.e. TZDs) have all been proposed as treatment approaches for lipodystrophy (469). In a fatless mouse model, treatment with the globular domain of adiponectin significantly improved the hyperglycemia and hyperinsulinemia characteristic of these lipoatrophic diabetic mice (146). Interestingly, the insulin resistance observed in these mice was completely ameliorated by treatment with both adiponectin and leptin, but only partially by either adiponectin or leptin alone (146), suggesting that both adiponectin and leptin deficiency may contribute to the insulin resistance in humans with lipodystrophy.

 

Studies to date support the premise that too little AT, as seen in lipodystrophy, appears to be just as deleterious as too much AT. Emerging data reveal that patients with lipodystrophy may have reduced survival and high mortality at an early age, predominantly due to cardiometabolic complications (470–472). Lipodystrophy has no cure; therefore, the primary treatment option is to improve metabolic outcomes via physical activity and dietary and pharmacological interventions. Conventional insulin-sensitizing agents, such as metformin and TZDs, are often used (453,473), and leptin replacement is also an approved therapy for total congenital lipodystrophy (467). Future investigations to better understand the pathogenesis and the clinical manifestation of lipodystrophy syndromes are essential for the development of improved therapeutics.

 

Adipose Tissue and Reproduction

 

While many studies have primarily examined the influence of white AT on the metabolic consequences associated with obesity, less frequently mentioned is the interplay between AT and reproductive health. Nevertheless, it is well established that AT is important for the normal function of the reproductive system, including the production and regulation of sex and reproductive hormones, pubertal development, and the maintenance of pregnancy and lactation (474).

 

Leptin and adiponectin are the most investigated adipokines as mediators of reproductive health and pathology. Receptors for both leptin and adiponectin have been identified in all major reproductive tissues, including the testes, placenta, ovaries, oviducts and endometrium (475). Obese mice that are deficient in leptin or the leptin receptor are unable to reproduce (476). Although rare, humans with leptin mutations have been identified, and studies in these individuals have validated the infertility findings in rodents (477).

 

Leptin administration in rodents was shown to increase luteinizing hormone (LH), follicle stimulating hormone (FSH), and ovarian and uterine weights in females, and testosterone, testicular weights and sperm counts in males (478,479). During human pubertal development, there is a steady increase in leptin, stimulating a rise in testosterone levels and fat-free mass in boys and in estradiol and fat mass in girls (480,481). Adiponectin administration was shown to inhibit gonadotrophin releasing hormone (GnRH), LH, and FSH (482,483) in pigs and increase estrogen and progesterone (484) in rats, while circulating levels in humans have been shown to be associated with serum levels of sex hormones (primarily estrogens), though this correlation was largely mediated by body weight (485).

 

In humans, leptin levels increase during pregnancy and rapidly fall in the post-partum period (486), and other reports have suggested that adiponectin may influence the amount of gestational weight gain and weight maintenance post-partum, even after adjusting for the sum of skinfold thickness and BMI (487,488). The effects of leptin on fetal development continue to be investigated, and have been suggested to correlate with fetal growth, birth weight, and organogenesis (489). Adiponectin plasma levels were shown to be significantly lower in overweight patients than normal weight women during pregnancy and negatively correlated with progressive gestational age and weight gain (490). In addition, women with low adiponectin concentrations experienced a significantly increased risk of gestational diabetes mellitus (491,492), and large reductions in adiponectin levels during pregnancy may also predict large-for-gestational-age offspring and increased birth weight (493). Interestingly, several studies have shown that higher adiponectin levels are associated with increased conception success in women undergoing assisted reproduction approaches (494). Overall, these studies are consistent with adipose-derived leptin and adiponectin having critical roles in reproductive function.

 

Many lines of evidence also demonstrate that either insufficient or excessive AT can have detrimental effects on reproductive health. Women with lipodystrophy disorders (see above), are characterized by AT and leptin deficiency and are frequently infertile (495,496). Anorexia nervosa is an eating disorder characterized by very low AT mass that is often accompanied by amenorrhea (absence of at least three menstrual periods in a row) (497). It is estimated that ~38% of women affected by anorexia experience infertility (498). Leptin deficiency is common in these patients (499)and may lead to disruptions in downstream neuroendocrine signaling (500).  This was tested when leptin replacement to women with hypogonadotrophic hypogonadism due to anorexia nervosa or excessive exercise was found to restore normal periods (501). Estrogen deficiency in these women results in major implications for bone health, ultimately contributing to increased osteopenia or osteoporosis (502). 

 

Body weight has been shown to predict testosterone levels in men (503); and obesity, specifically central adiposity, is associated with low testosterone levels (504). Increased AT also leads to elevated estradiol, resulting in reduced circulating testosterone through feed-back inhibition of gonadotrophs (504,505). A common medical condition in women at the crossroads of dysfunctional AT and reproduction is polycystic ovary syndrome (PCOS), which in roughly 50% of affected women is associated with increased central obesity and metabolic health risk. PCOS is commonly defined using the consensus of Rotterdam, which requires two of three criteria: polycystic ovaries on ultrasonography, hyperandrogenism, or amenorrhea. Studies of PCOS generally show that adiponectin levels are lower in these patients (506). Another burgeoning area of research is the study of excessive AT and reproductive malignancies, as obesity is known to increase the risk of breast, uterine, cervical, and prostate cancers (507). Studies have reported inverse relationships between leptin and adiponectin levels with breast, endometrial, ovarian, and prostate cancers (508,509).

 

EMERGING AREAS IN ADIPOCYTE BIOLOGY

 

Critical considerations in the study of fat tissue are its cellular complexity and heterogeneity. AT depots can exist in close association with other organs and act physiologically as metabolic “sinks” that store excess energy as lipid in a protective manner, or they can promote systemic metabolic dysfunction by secreting excess lipid or inflammatory adipokines. As the recognition of distinct AT depots increases, so does our understanding of their diversity. A recent review considers the locations and functions of several depots, ranging from facial AT to cardiovascular AT as well as the presence of adipocytes in bone marrow, within and between muscle beds, and joints (510). Currently, we are experiencing a new and exciting period in AT research with the focus shifting toward recognizing neglected AT depots, the expanding types of adipocytes, and the complex developmental and sex-regulated origins of adipocytes. Adipocytes are critical secretory cells that contribute a variety of circulating proteins, including endocrine hormones. Of course, adipocytes also produce lipids and can release genetic material that can have profound systemic functions.

 

Much remains to be discovered about the types of nerves present in fat tissue and how they vary according to AT type and location. How these AT nerves act to regulate metabolic homeostasis is a current focus of fat cell biology. Recent advances in whole tissue AT imaging and studies on brain-adipose communication suggest we are just beginning to uncover the capabilities and function of AT nerves, and there are many unanswered questions in this field (511). Research on the molecular pathways that connect AT innervation to insulin action in obesity and diabetes may provide insight into our understanding of the pathogenesis of metabolic disease states.

 

Another developing area of fat cell biology is the effects of exercise on adipocyte function. Recent studies have shown that transplantation of subcutaneous AT from exercise-trained mice improves glucose tolerance and insulin sensitivity in recipient, non-exercised mice (512), and strongly suggest that exercise favorably remodels AT to improve systemic metabolic health. Recently, an AT-derived lipid was shown to increase fatty acid uptake in skeletal muscle (513). The importance of AT to whole-body energy metabolism is well established; yet, the impacts of different types of endurance or resistance exercise on adipose tissue dynamics remains largely understudied, particularly in the context of obesity and other metabolic disease states.  

 

A newly discovered pathway shows that lipids can be released by adipocytes in the form of exosome-sized, lipid-filled vesicles (514). This process occurs independently of canonical lipolytic pathways, and adipocyte exosomes deliver excess lipid to local macrophages in obesity (514). Other novel pathways of paracrine regulation have also been demonstrated in AT. These paradigm-shifting observations demonstrated that extracellular vesicles (EV) from endothelial cells in adipose tissue can provide lipids and proteins to adjacent adipocytes. This EV communication between endothelial cells and adipocytes within AT is bi-directional and is regulated by fasting/refeeding and in conditions of obesity (515). These very recent observations reveal the highly complex signaling mechanisms that exist in AT.  

 

Though it was once considered a mere energy storage site, AT is now considered an important endocrine organ and site of inflammatory cell signaling that governs not only survival but also plays critical roles in reproduction and in glucometabolic homeostasis. As scientific methods for the study of AT continue to rapidly evolve, so does our understanding regarding the metabolic, biomechanical, immune, and secretory functions of AT in normal physiology and metabolic disease.

 

           ACKNOWLEDGEMENTS

 

The authors are grateful to Anik Boudreau and Christina Zunica for their assistance in editing and referencing the chapter. This work was supported by National Institutes of Health Grant R01 DK052968.

 

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Nephrolithiasis

ABSTRACT

 

Kidney stones are concretions of different mineral salts mixed with an organic matrix that form in the upper urinary tract. As a stone moves from the kidney to the ureter, it can present with renal colic symptoms, and may cause urinary tract obstruction and/or infection. In fact, acute passage of a kidney stone is one of the leading reasons for visits to an emergency room. Over the past four decades, the lifetime prevalence of nephrolithiasis has more than doubled in the United States (and several developed countries), afflicting around 11% of men and 7% of women. Unless the underlying etiology of stone formation is adequately addressed, kidney stones can recur at a rate of around 50% ten years after initial presentation. The evaluation of a kidney stone former requires an extensive medical history (to identify environmental, metabolic, and/or genetic factors contributing to stone formation), imaging studies to evaluate and track stone burden, and laboratory studies (serum and urinary chemistries, stone composition analysis) to guide lifestyle and pharmacological therapy. The majority of kidney stones are composed of calcium (calcium oxalate and/or calcium phosphate), either pure or in combination with uric acid. Calcium oxalate stones can be caused by hypercalciuria, hyperoxaluria, hyperuricosuria, hypocitraturia, and/or low urine volume. Calcium phosphate stones occur in patients with hypercalciuria, hypocitraturia, an elevated urine pH, and/or low urine volume. In addition to lifestyle changes (increasing fluid intake, reduction in salt intake, moderation of calcium and animal protein intake), pharmacological therapy directed at the underlying metabolic abnormality (thiazides for hypercalciuria, potassium citrate for hypocitraturia, xanthine oxidase inhibitors for hyperuricosuria) can significantly reduce calcium stone recurrence rate. Pure uric acid stones account for 8-10% of all stones, although their prevalence is significantly greater in stone formers with type 2 diabetes and/or the metabolic syndrome. Uric acid stones are primarily caused by an excessively acidic urine, and urinary alkalinization with medications such as potassium citrate can dissolve uric acid stones and prevent recurrent uric acid nephrolithiasis. Cystine stones result from inactivating mutations in genes that encode renal tubular transporters that reabsorb the amino acid cysteine, typically present in childhood, are highly recurrent, and require aggressive control of cystinuria with specific pharmacological therapy. Infection (struvite) stones often present as staghorn, and require careful surgical removal of all of the stone material.

 

OVERVIEW

 

Urinary stone disease is a common clinical condition that afflicts 1 in 11 individuals in the United States, and is increasing in incidence. It typically presents with renal colic symptoms following the passage of a calculus from the kidney to the ureter. The pathophysiology of kidney stone formation is diverse, and includes a combination of genetic and environmental factors. In fact, kidney stone formation should not be viewed as a diagnosis but rather a symptom of an underlying abnormality. Evaluation of a kidney stone former with a detailed history, and appropriate laboratory and imaging studies helps to identify risk factors for stone formation and provides an opportunity to institute therapy that reduces the risk of stone recurrence. This chapter reviews the epidemiology of kidney stone disease, discusses the pathophysiology underlying the most commonly encountered stone types, and details the evaluation and management of patients with nephrolithiasis.

 

EPIDEMIOLOGY AND NATURAL HISTORY

 

Epidemiology

 

Nephrolithiasis is a common clinical condition encountered in both developed and developing countries. Its prevalence in the United States has more than doubled over the past 4 decades from 3.8% in 1976-1980 to 5.2% in 1988-1994 to 8.8% in 2007-2010 (1, 2). Similar increases in the prevalence of nephrolithiasis have been reported in other developed countries (3). Factors underlying this rising prevalence include changes in diet and fluid intake, greater use of medications and procedures that predispose to stone formation, the association of stone disease with the rampant epidemics of obesity and type 2 diabetes, climate change, and increased use of abdominal imaging (2). In the United States, stone disease afflicts Caucasians at a greater frequency than Hispanics (Odds Ratio: 0.60 vs. Caucasians) and African Americans (Odds Ratio: 0.37 vs. Caucasians) (2). It is also more prevalent in men than women, although recent reports suggest a narrowing in this gender gap, with the greatest increase in stone incidence in recent years occurring in younger women (4). A marked geographic variability in the prevalence of nephrolithiasis is also reported, with 20-50% higher prevalence of stone disease in the U.S. Southeast (“stone belt”) than the Northwest, primarily due to differences in exposure to temperature, humidity, and sunlight (5). Recent predictions suggest a likely northward expansion of the present-day U.S. “stone belt” as an unanticipated result of global warming (6). In 2000, the total cumulative costs for caring for patients with urolithiasis were estimated at US $2.1 billion (7). The rising prevalence of obesity and diabetes, together with population growth, is projected to contribute to dramatic increases in the cost of urolithiasis by an additional $1.24 billion/year estimated by 2030(8).

 

Natural History

 

RECURRENCE

 

The natural history of stone disease was studied in detail in all validated cases presenting with incident kidney stones in Olmsted County, Minnesota between 1984 to 2003 and followed for stone recurrence through 2012 (9). For the first episode, 48% of patients passed their stone spontaneously with confirmation, 33% required surgery for stone removal, 8% presumably spontaneously passed their stone (without confirmation), and 12% had no documentation of passage (9). This cohort was followed for a median of 11.2 years with recurrence occurring in 11%, 20%, 31%, and 39% at 2, 5, 10, and 15 years, respectively. The stone recurrence rates per 100 person-years were 3.4 after the first stone episode, 7.1 after the second episode, 12.1 after the third episode, and 17.6 after the fourth or higher episode (10). Independent risk factors for incident stone recurrence include younger age; male sex; higher body mass index; family history of stones; pregnancy; history of a brushite, struvite, or uric acid stone; number of kidney stones on imaging; and diameter of the largest kidney stone on imaging (10). These studies have led to the development and refinement of the Recurrence Of Kidney Stone (ROKS) tool to predict the risk of symptomatic recurrence by using readily available clinical characteristics of patients with kidney stones (9, 10).

 

POTENTIAL COMPLICATIONS

 

Urinary Tract Infection

 

There is a bi-directional relationship between urinary tract infections (UTI) and nephrolithiasis, as chronic UTIs lead to the formation of struvite stones, and stone disease increases the risk of UTI. Struvite stones typically occur in patients infected with urea-splitting organisms, and their pathogenesis and management are described in more detail in a later section. Gram-negative bacilli are the most common pathogen in UTI in patients with urolithiasis. Independent risk factors for UTI among patients with kidney stones include female gender, older age, presence of obstruction, and higher number of kidney stones (11). Low fluid intake is a reversible risk factor for both UTI and nephrolithiasis (12). Infectious (13) as well as non-infectious stones (14) can harbor bacteria inside, making the bacteria resistant to antimicrobial therapy. In patients with recurrent UTIs, removal of non-struvite non-obstructing stones is associated with elimination of further UTI recurrence in nearly 90% of cases (15).

 

Chronic Kidney Disease and End-Stage Renal Disease

 

Loss of renal function in patients with kidney stones may occur as a complication of obstruction by a stone lodged in the ureter, a complication of the urological procedure to remove a stone, or from the disordered pathophysiology underlying some stones. Staghorn stones caused by uric acid nephrolithiasis, cystinuria, renal tubular acidosis (RTA), or chronic infection are well-recognized causes of decreased renal function. Additional risk factors for the development of chronic kidney disease in stone formers include a solitary kidney, ileal conduit, neurogenic bladder, and development of hydronephrosis (16). In the Olmsted County cohort, end-stage renal disease incidence in patients with recurrent symptomatic kidney stones was twice that of the general non-stone forming population even after adjusting for baseline hypertension, diabetes mellitus, dyslipidemia, gout, obesity, and chronic kidney disease (17). Still, the absolute risk of ESRD from kidney stone disease was low.

 

Stone Disease in Pregnancy

 

Pregnancy-related mechanical and physiological changes alter risk factors for kidney stone formation, and management of acute nephrolithiasis in pregnancy is significantly more complicated than in non-pregnant women, at least in part due to imaging limitations and treatment restrictions (18). In an observational study, stones in pregnancy were associated with recurrent abortions, mild preeclampsia, chronic hypertension, gestational diabetes mellitus, and cesarean deliveries (19). Urinary tract infections and pyelonephritis and signs of ureteral obstruction including hydroureter and hydronephrosis were common, while premature rupture of membranes and preterm delivery were not more frequent. The newborns also were affected by perinatal complications including low birth weight, lower Apgar scores, and perinatal mortality. The majority of stones during pregnancy are calcium phosphate with a lesser number of calcium oxalate, in contrast to non-pregnant women in whom calcium oxalate stones are most common (20).

 

ASSOCIATION WITH OTHER CONDITIONS

 

Traditionally, nephrolithiasis was thought of as a condition caused by poor diet and abnormal renal handling of electrolytes, with complications limited to the kidneys and the urinary tract. However, recent investigations suggest that kidney stones may in fact be a systemic disorder associated with serious disorders including osteoporosis and greater fracture risk (21), metabolic syndrome features including diabetes, hypertension, and dyslipidemia (22), and greater incidence of cardiovascular disease (23).

 

CLINICAL MANIFESTATIONS

 

Symptoms and Signs

 

A classical episode of renal colic has a sudden onset, with fluctuation and intensification over 15 to 45 minutes. The pain then becomes steady and unbearable and often is accompanied by nausea and emesis. As the stone passes down the ureter toward the bladder, flank pain changes in a downward direction toward the groin. As the stone lodges at the ureterovesical junction, urinary frequency and dysuria appear. The pain may clear as the stone moves into the bladder or from the calyceal system into the ureter. Hematuria, generally microscopic but occasionally frank, frequently accompanies stone passage. The presence of bleeding alone does not predict a more severe outcome. Episodes of rapid onset of pain, bleeding, and then rapid clearing, often called ‘passing gravel’, is the result of passing a large amount of crystals of calcium oxalate, uric acid, or cystine. “Non-classical” presentations of kidney stones include dull low back pain, gastrointestinal symptoms such as diarrhea, isolated microscopic hematuria, asymptomatic urinary obstruction with renal insufficiency, recurrent urinary tract obstruction, or incidental discovery on abdominal imaging.

 

Likelihood of Passage

 

The size, number, and metabolic composition of new stones strongly influence the natural history and complication rates. Smaller stone size and more distal location in the ureter at presentation predict greater likelihood of spontaneous stone passage. Furthermore, the clinical presentation can be in part classified by metabolic type (Table 1). Spontaneous stone passage may occur with calcium oxalate, calcium phosphate, uric acid, and cystine stones. Rarely does a struvite stone or a staghorn stone of other composition (cystine, uric acid) pass spontaneously.

 

Table 1. Clinical Manifestations of Stones by Composition

Clinical feature

Calcium

Uric acid

Struvite

Cystine

Crystalluria

+

+

-

+

Stone passage

+

+

-

+

Small discrete stones

+

+

-

+

Sludge and obstruction

-

+

-

+

Radiodense

+

-

+

+

Staghorn

-

+

+

+

Nephrocalcinosis

+

-

-

-

 

EVALUATION

 

History

 

Evaluation of kidney stones starts with a detailed history focusing on medical conditions associated with higher risk of kidney stones (e.g. primary hyperparathyroidism, gastrointestinal disorders or surgeries, frequent urinary tract infections, gout, metabolic syndrome, etc.), family history suggestive of genetic causes of kidney stones (e.g.cystinuria, idiopathic hypercalciuria, young age at onset, etc.), dietary history (e.g. intake of fluid, salt, protein, dairy products, oxalate-rich foods (Table 2) (24), etc.), medications associated with increased risk of kidney stones (e.g.topiramate, zonisamide, excessive vitamin C, etc.)  (25, 26), and medications that directly precipitate and form stones (e.g. indinavir, triamterene, etc.) (Table 3).  This history can provide guidance on the biochemical evaluation and management of kidney stones. 

 

Table 2. Oxalate content of foods (24)

Food category

High in oxalate

Low in oxalate

Fruits

Figs, raspberries, dates

Apples, oranges, peaches, raisins, mango

Vegetables

Spinach, okra, beans, beets

Lettuce, asparagus, carrots, avocado, corn

Grains

Whole grain products

White rice, pasta, white bread

Nuts

All nuts (peanuts, almonds...)

 

Other

Chocolate, cocoa, black tea

Coffee, milk products, meat products

 

 

Table 3. Drugs associated with kidney stones (25, 26)

Drugs associated with increased risk of stones

Drugs that precipitate and form stones

Hypercalciuria (predispose to calcium stones):

Excessive calcium and vitamin D supplement

Loop diuretics

Glucocorticoids

 

Hypocitraturia and high urine pH (predispose to calcium phosphate stones):

Carbonic anhydrase inhibitors (acetazolamide, topiramate, zonisamide)

 

Hyperoxaluria (predispose to calcium oxalate stones):

Frequent use of antibacterial agents

Excessive vitamin C supplement 

 

Hyperuricosuria (predispose to uric acid stones and calcium oxalate stones):

Uricosuric drugs (probenecid, losartan)

 

High urine ammonium (predispose to ammonium urate stones):

Laxative abuse

Anti-viral:

Indinavir

Atazanavir

Nelfinavir

Tenofovir disoproxil fumarate

Raltegravir

Efavirenz

 

Antibiotics:

Sulfadiazine

Sulfamethoxazole

Ciprofloxacin

Amoxicillin

Ampicillin

Ceftriaxone

 

Others:

Triamterene

Sulfasalazine

Allopurinol

Guaifenesin / Ephedrine

Magnesium trisilicate

 

Laboratory Testing

 

The extent of biochemical evaluation depends on the risk of stone recurrence and patients’ interest.  For first-time stone formers, a basic evaluation including urinalysis, urine culture, and basic metabolic panel should be obtained.  For high risk stone formers (Table 4) and interested first-time stone formers, a thorough evaluation (Table 5) is warranted which includes 24-hour urine stone risk profile (27-29).

 

BLOOD

 

The recommended blood testing of kidney stone formers includes assessment of renal function, serum electrolytes (including potassium, calcium, phosphorus, and magnesium), and serum uric acid. Assessment of serum PTH may be needed in patients with suspected primary hyperparathyroidism.

 

Table 4. High Risk Stone Formers (27-29)

Recurrent stone formers:

·       Recurrent kidney stones

·       Bilateral or multiple kidney stones

 

History suggestive of genetic causes of kidney stones:

·       Early onset of kidney stones (age < 18 years)

·       Family history of kidney stones

 

Stone types that are more commonly associated with metabolic abnormalities:

·       Pure calcium phosphate stones

·       Non-calcium stones (uric acid, cystine, struvite, and magnesium ammonium phosphate stones) 

·       Any stone requiring percutaneous nephrolithotomy

 

History of medical conditions associated with increased recurrence risk: 

·       Hyperparathyroidism

·       Sarcoidosis

·       Bowel disease (e.g. inflammatory bowel disease, chronic pancreatitis, chronic diarrhea)

·       Bowel resection (e.g. colon resection, ileostomy, small bowel resection, Roux-en-Y gastric bypass)

·       Cystic fibrosis

·       Metabolic syndrome (e.g. obesity, type 2 diabetes mellitus, dyslipidemia, hypertension)

·       Gout

·       Spinal cord injury

·       Neurogenic bladder

·       Recurrent urinary tract infection

·       Osteoporosis

·       Renal tubular acidosis

·       Polycystic kidney disease

·       Cystinuria

·       Primary hyperoxaluria

·       Anatomical abnormalities that result in impaired urine flow (e.g. medullary sponge kidney, ureteral stricture, horseshoe kidney, etc.)

 

At high risk for complications of kidney stones:

·       Solitary kidney (functional or anatomical)

·       Chronic kidney disease

·       Complicated stone episodes that resulted in severe acute kidney injury, sepsis, or complicated hospitalization

 

At high risk occupations (e.g. pregnancy, pilots, police officer, military personnel, firemen, etc.)

 

Table 5. Biochemical Evaluation for High Risk Stone Formers and Interested First Time Stone Formers (27-29)

Serum: Basic metabolic panel, albumin, phosphate, magnesium, uric acid, PTH

 

Urinalysis and urine culture

 

24-hour urine: Volume, creatinine, pH, calcium, citrate, oxalate, uric acid, sodium, potassium, magnesium, chloride, sulfate, phosphate, ammonium and cystine (if cystine stone is confirmed or suspected)

 

Stone analysis

 

URINE

 

The initial 24-hour urine sample should be collected on the patient’s typical random diet.  It is controversial whether one or two 24-hour urine collections should be obtained (30-34).  At least one collection is needed, but two collections are preferred (27-29) . It is important to provide detailed instructions to patients to ensure an adequate collection and proper storage of urine sample.  Patients start urine collection after their first morning void and end collection with the first morning void the next day.  Storage of urine sample varies according to the instructions of the urine collection kits.  Urine sample should be sent to a reliable lab for 24-hour urine stone risk profile analysis.  24-hour urine stone risk profile should be repeated at 8-16 weeks after dietary changes or if pharmacotherapy is initiated to monitor response to therapy and allow dose adjustment as needed (28). Once the therapeutic target is achieved, 24-hour urine stone risk profile is repeated annually (28).  

 

24-hour urine stone risk profile typically provides information on 24-hour urine volume, creatinine, pH, calcium, citrate, oxalate, uric acid, sodium, potassium, magnesium, sulfate, phosphate, ammonium and cystine (if requested).  In addition, relative supersaturations with respect to calcium oxalate, calcium phosphate and uric acid are reported. Relative supersaturations are calculated accounting for multiple factors including promotors and inhibitors associated with crystallization (35). Higher relative supersaturation is associated with higher likelihood of being stone formers and correlate with stone composition (36, 37).

 

STONE ANALYSIS

 

Knowledge of stone composition may help direct the appropriate choice of urological procedures, evaluation of potential underlying metabolic abnormalities, and medical interventions to prevent stone recurrence (27-29, 38, 39). Current guidelines recommend obtaining stone analysis when feasible for all first-time stone formers (27-29). Stone composition may change in the same individual over time (40, 41)  Discordant stone compositions may also coexist in the same individual with bilateral kidney stones (42, 43).  Therefore, repeat stone analysis should be obtained in recurrence under pharmacological treatment, early recurrence after urological intervention and late recurrence after a prolonged stone-free period (28).

 

Multiple analytical techniques are available for stone analysis.  The currently preferred methods are X-ray diffraction and Fourier transform infrared spectroscopy (44).  X-ray diffraction uses monochromic X-rays to create a unique diffraction pattern of the crystalline structure of the stone (45).  Fourier transform infrared spectroscopy uses infrared radiation to create a unique energy absorption band pattern of the molecular structure of the stone (45).  These patterns can then be matched to a reference database to determine the stone composition.  Both X-ray diffraction and Fourier transform infrared spectroscopy are very accurate in identifying pure stones; however, the majority of kidney stones in clinical practice are mixed stones (44, 45).  Both methods have limitations in identification of certain mixed stone compositions.  X-ray diffraction cannot identify non-crystalline structures thus is prone to high rates of error in the detection of apatite component in mixed stones which is mostly pseudo-amorphous (44).  It is also time-consuming and expensive which limit its broad use in clinical practice (45).  Fourier transform infrared spectroscopy is quick and less expensive, but it cannot reliably detect small amounts of components in certain mixed stones (e.g. whewellite (hydrated calcium oxalate) in whewellite/uric acid stones and struvite in struvite/apatite stones) (44-47).  The accuracy of stone analysis by Fourier transform infrared spectroscopy depends on the quality of the reference database and trained personnel (46, 47).  While pure stones are reliably identified, there is variability in reporting the components of mixed stones in commercial laboratories which needs to be kept in mind when interpreting stone analysis results (47, 48). 

 

Imaging

 

A number of imaging modalities are available to evaluate stone number, size, and location in patients with nephrolithiasis (Table 6). Abdominal computed tomography (CT) without contrast is the initial imaging test of choice for suspected stone disease due to its high sensitivity and specificity, along with its widespread availability and the rapidity of scan time. One downside to CT scan use is exposure to ionizing radiation, which may increase long-term cancer risk. Lower radiation doses are effective in the diagnosis of nephrolithiasis in most patients, leading to greater recent adoption of “low-dose” and “ultra-low dose” CT scan protocols for evaluation of stone disease (49, 50). Use of ultrasonography as the initial test in patients with suspected nephrolithiasis in the emergency department (ED) may reduce cumulative radiation exposure without significantly increasing subsequent serious adverse events, pain scores, return visits to the ED, or hospitalizations (51). Abdominal X-rays (KUB) may be used as an alternative to CT scan and ultrasonography for follow-up of stone burden, although this modality misses radiolucent stones such as uric acid stones. Magnetic resonance imaging is capable of identifying kidney stones, but cost and limited availability make it a less attractive imaging modality for nephrolithiasis.

 

Table 6. Comparison of Different Imaging Modalities in the Assessment of Nephrolithiasis

 

Availability

Cost

Ionizing Radiation

Other Advantages

Other Drawbacks

CT Scan

Wide

Moderate

Highest

-Detects extra-renal pathology

-Useful in identifying uric acid stone composition

-Major drawback is radiation

Ultrasound

Wide

Moderate

None

-Portable US available

-Use in children, pregnancy

-Does not visualize ureteral stones

-Large body habitus limits visualization

X-ray

Wide

Low

Low

-Useful in follow-up of known radiopaque stones

-Visualizes kidneys, ureters, and bladder

-Misses radiolucent and/or small stones

-Overlying bowel gas, and extra-renal calcification impact stone visualization

MRI

Limited

High

None

-Use in children, pregnancy

-Contrast risk in CKD patients

-Cannot distinguish stone from blood clot

IVP

Limited

Moderate

High

-Occasional use in

preoperative planning

-Contrast use

 

SURGICAL MANAGEMENT

 

An obstructive stone in the setting of urinary infection is a urological emergency and requires urgent decompression with a ureteral stent or nephrostomy tube (28, 38). Patients should have urine and, if appropriate, blood cultures obtained and be started on broad spectrum intravenous antibiotics until culture results are available. These patients often require fluid resuscitation and monitoring in an intensive care setting. Definitive stone treatment is delayed until infection resolves. 

 

Patients who present with a ureteral stone up to 10mm can be offered a trial of passage if they have no signs or symptoms of urinary tract infection, their renal function is at their baseline, and their pain is well controlled. The likelihood of ureteral stone passage is influenced by stone size and location with smaller, more distal stones having the highest chance of passage (52).  Furthermore, smaller stones tend to pass quicker than larger stones (53).  For those patients attempting spontaneous passage, a trial of medical expulsive therapy with pain control and a-blocker for 4-6 weeks can be offered in uncomplicated cases (i.e. in the absence of infection, uncontrolled pain, obstruction, renal insufficiency, or renal anatomy associated with low likelihood of spontaneous stone passage) (28, 38, 54-56).  Nonsteroidal anti-inflammatory drugs (NSAIDs) including intravenous ketorolac are the treatment of choice for pain control. (57)  Opioids are used as rescue therapy for pain refractory to NSAIDs. (58)  In emergency room setting, IV lidocaine is a useful non-opioid option for pain control with close cardiac monitoring if there are no contraindications. (59, 60)  Alpha-blockers inhibit basal tone and decrease peristaltic frequency and amplitude in the lower ureters, decrease intraureteral pressure and increase fluid transport, thus they are proposed to be useful in stone expulsion. (61)  However, the effectiveness of a-blockers as medical expulsive therapy remains controversial.  A recent meta-analysis showed the use of a-blockers is associated with increased stone clearance and decreased time to stone passage with little major adverse events compared to standard therapy without a-blockers, but the quality of evidence is low. (62)  The benefit of a-blockers was mainly demonstrated in individuals with ureteral stones with sizes 6-10mm.  Little effect was found in stones measuring 5mm or smaller likely because these stones frequently pass spontaneously even without medical expulsive therapy. (62)  Potential side effects of a-blockers include orthostatic hypotension, dizziness, tachycardia, palpitations, headache, and abnormal ejaculation in males.  In large clinical trials, these were not found to be more frequent in individuals treated with a-blockers than placebo with the exception of ejaculatory dysfunction in males. (62, 63)   

 

Outpatient referral to urology is indicated for stones larger than 10mm, stones smaller than 10mm that fail to pass with medical expulsive therapy, and stones causing obstruction at the ureteropelvic junction, renal pelvis, or renal calyces especially in symptomatic patients and those at high risk for potential complications. Urological referral should also be considered for high risk stone formers (Table 4). Surgical intervention for stone treatment depends on symptoms, stone composition, size, and location (Table 7) (28, 38, 64).

 

Table 7. Surgical Management of Kidney Stones (28, 38, 64)

Stone

Surgical procedure 

Urological emergency:

 

Obstructive stone with infected urine

·       Obtain urinalysis, urine culture and start empiric antibiotics and IV fluid resuscitation

·       May need ICU care

·       Urgent decompression (ureteral stent or percutaneous nephrostomy tube)

·       Delay definitive stone treatment until infection resolves

Ureteral stones:

 

Uncomplicated ureteral stone £ 10mm

·       Trial of medical expulsive therapy with pain control (NSAIDS with narcotics if needed) and a-blocker for up to 4-6 weeks with reassessment of pain control, renal function and stone passage

Proximal ureteral stone < 10mm

·       SWL*

·       URS# if cystine stone or uric acid stone

Proximal ureteral stone > 10mm

·       URS

Mid ureteral stone of any size

·       URS

Distal ureteral stone of any size

·       URS

Renal stones:

 

Asymptomatic renal stone < 15 mm

·       Conservative therapy with surveillance of symptoms and imaging at 6 months and then annually for stone growth and new stone formation

Symptomatic non-lower pole renal stone £20mm

·       SWL or URS

Symptomatic non-lower pole renal stone > 20mm

·       PCNL

Symptomatic lower pole renal stone £ 10mm

·       SWL or URS

Symptomatic lower pole renal stone > 10mm

·       URS or PCNL

Staghorn stones

·       PCNL

Involved kidney with negligible function

·       Consider nephrectomy if recurrent infection or pain

     

#URS: ureteroscopy;

†PCNL: percutaneous nephrolithotomy

*SWL: Shockwave lithotripsy.  Calcium oxalate monohydrate, brushite and cystine stones are hard and resistant to fragmentation by SWL, thus alternative methods of stone removal are considered (28).  SWL has a lower rate of complications and morbidity, but a lower stone free rate in a single procedure than URS.

 

MEDICAL MANAGEMENT

 

General Measures for All Patients with Kidney Stones

 

Some risk factors (e.g. low urine volume, hypocitraturia, high sodium intake, and high animal protein intake) are shared among different types of stones. General dietary measures (summarized in Table 8) targeting these risk factors can be recommended for stone prevention. These can be especially useful when stone analysis and/or 24-hour urine stone risk profile are not available. Results from a 24-hour urine collection (Table 9) can further refine these recommendations.

 

Low urine volume is a risk factor for nephrolithiasis. High urine volume leads to urinary dilution of lithogenic constituents and reduced crystallization of calcium oxalate, calcium phosphate and uric acid (65, 66).  Several prospective studies have demonstrated high urine volume achieved with high fluid intake is associated with reduction in incident stones and recurrent stones (67-69).  Fluid intake of 2.5 to 3 liters per day or achieving a urine volume of at least 2-2.5 liters per day is recommended (27-29). Regarding the types of fluid other than water, orange juice, lemonade, coffee (caffeinated and decaffeinated), tea and alcohol have been associated with reduced risk of stone formation although with some controversial results (5, 68, 70-72).  Cola and grapefruit juice have been associated with increased risk of stone formation (70).

 

A high dietary sodium intake is associated with increased risk of nephrolithiasis likely by causing increased urinary calcium and decreased urinary citrate (28, 66, 73, 74).  For every 100 mmol/day increase in dietary sodium intake, urinary calcium increases by an average of 40 mg/day in non-stone forming adults and by up to 80 mg/day in hypercalciuric stone formers (66, 74-76).  A low sodium diet reduced urinary calcium and recurrent stones in hypercalciuric stone formers (76, 77).  Stone formers are therefore recommended to limit their dietary sodium intake to less than 2300 mg/day (or 100 mmol/day) which is equivalent to 5.9 grams of salt (sodium chloride) (27-29).

 

A high dietary animal protein intake (meat, fish and poultry) is a risk factor for nephrolithiasis in general (69, 78).  It is associated with increased urinary calcium, uric acid, phosphate and reduced urinary citrate and pH (79). On average, urinary calcium increases by 1 mg/day for every 1 g/day increase in dietary animal protein intake (80, 81).  In a randomized clinical trial of recurrent calcium oxalate stone formers, a diet with limited animal protein (52 g/day) and sodium (50 mmol/day) but normal calcium (1,200 mg/day) reduced stone recurrence by about 50% at 5 years when compared to a low-calcium diet (400mg mg/day) in hypercalciuric stone formers (77).  It is recommended to limit dietary animal protein intake to 0.8 to 1.0 g/kg weight per day (28, 82).

 

Urinary calcium excretion increases with increased dietary calcium intake which can be more pronounced in individuals with hyperabsorptive idiopathic hypercalciuria (66, 83).  However, a diet restrictive in calcium has not been demonstrated to prevent nephrolithiasis.  On the contrary, several studies showed a lower dietary calcium intake is associated with a higher risk of both incident and recurrent stones than a higher dietary calcium intake in men and women (69, 73, 77, 84).  A restricted calcium diet increases enteric absorption of oxalate and urinary oxalate which increases supersaturation of calcium oxalate (85, 86).  In addition, a low calcium diet may lead to negative calcium balance and bone loss. Therefore, a normal calcium diet with 1,000-1,200 mg/day is recommended as a dietary measure for stone prevention (27-29). Dietary sources of calcium are preferred.  However, if supplemental calcium is needed, it is best taken in divided doses with meals to reduce enteric absorption of oxalate (27-29).

 

A diet rich in fruits and vegetables is associated with a decreased risk of incident kidney stones (87) and current guidelines on medical management of nephrolithiasis also recommend a diet rich in fruits and vegetables for prevention of stone recurrence (27-29).  In normal individuals, elimination of dietary fruits and vegetables decreased urinary potassium, magnesium, citrate and oxalate, and increased urinary calcium, ammonium and relative supersaturation of calcium oxalate and calcium phosphate (88). In hypocitraturic stone formers, introduction of fruits and vegetables in the diet increased urinary potassium, magnesium, citrate, volume and pH, and decreased relative supersaturation of calcium oxalate and uric acid (88). 

 

Table 8. General Dietary Measures for All Stone Formers (27-29)

Dietary measures

Targeted risk factors

Fluid intake:

·       Fluid intake of 2.5 to 3 liters per day

·       Achieving urine output of 2-2.5 liters per day

 

Low urine volume

Salt intake:

·       Sodium intake less than 100 mEq (2,300 mg) per day

 

Hypercalciuria

Animal protein intake:

·       0.8-1.0 grams / kilogram body weight per day

 

Hypercalciuria

Hyperuricosuria

Hyperphosphaturia

Hypocitraturia

Low urine pH

Calcium intake:

·       Calcium intake of 1,000-1,200 mg per day divided into 2 doses taken with meals (prefer dietary source over supplemental calcium)

 

Hyperoxaluria

Fibers, vegetables and fruits:

Hypocitraturia

Low urine pH

 

Table 9. 24-hour Urine Stone Risk Profile Interpretation (89)

Urine parameter 

Reference range

Risk factor for stone types

Interpretation

Volume

> 2-2.5 L/day

All

·       Reflect fluid intake and extra-renal fluid loss

·       Goal is above 2-2.5 L/day

Creatinine

·       Male: 20-25 mg/kg body weight/day

·       Female: 15-20 mg/kg body weight/day

---

·       Assess adequacy of urine collection

pH

5.7-6.3

·       High pH: calcium phosphate and struvite

·       Low pH: uric acid and cystine

·       High pH: distal renal tubular acidosis (dRTA) or UTIs

·       Low pH: excessive animal protein intake, chronic diarrhea, or idiopathic

Calcium

·       Male: < 300 mg/day

·       Female: < 250 mg/day

·       Either sex: < 4 mg/kg body weight/day

Calcium oxalate

Calcium phosphate

·       Hypercalciuria (see details in “Hypercalciuria”) 

Oxalate

< 45 mg/day

Calcium oxalate

·       Enteric hyperoxaluria

·       > 100 mg/day, consider primary hyperoxaluria

Citrate

> 320 mg/day

Calcium oxalate

Calcium phosphate

·       Hypocitraturia (see details in “Hypocitraturia”) 

Uric acid

< 700 mg/day

Calcium oxalate

Uric acid

·       High purine intake or production

Phosphorus

< 1,100 mg/day

Calcium phosphate

·       Excessive protein intake

Sodium

< 200 mmol/day

Calcium oxalate

Calcium phosphate

·       Excessive salt intake

Chloride

< 200 mmol/day

Calcium oxalate

Calcium phosphate

·       Varies with sodium and potassium intake

Sulfate

< 40 mmol/day

Calcium oxalate

Calcium phosphate

Uric acid

·       Excessive animal protein intake

Ammonium

< 40 mmol/day

---

·       Excessive animal protein intake

·       Non-dietary acid load (e.g.diarrhea)

Potassium

> 40 mmol/day

---

Low urine potassium:

·       Low alkaline intake

·       Potassium loss (e.g.diarrhea)

Magnesium

> 80 mg/day

---

Low urine magnesium:

·       Low magnesium intake

·       Malabsorption

Cystine

< 40 mg/day

Cystine stone

·       Cystinuria 

 

Calcium Stones

 

Approximately 80% of kidney stones are calcium stones (calcium oxalate and/or calcium phosphate) (44). The initiating events of stone formation are controversial (90-94), but there are three proposed pathways of stone formation: 1) Randall’s plaque (interstitial calcium phosphate deposit at the renal papilla) grows and erodes the urothelium and becomes a nidus for crystal growth in urine supersaturated with respect to calcium oxalate; 2) Randall’s plug formed by fixed particle mechanism in which a crystal nidus is attached to the apical epithelium of the collecting duct and allows crystal growth in urine supersaturated with respect to the constituents of the stone; 3) Randall’s plug formed by free particle mechanism in which a crystal nidus forms through homogenous nucleation in the lumen of the nephron in the supersaturated environment (92).  Randall’s plaque is a prominent feature in idiopathic calcium oxalate stone formers and patients with primary hyperparathyroidism; although plugging is also observed (93, 94).  Randall’s plug formed by fixed particle mechanism is seen in brushite stone formers and patients with dRTA and primary hyperparathyroidism.  Randall’s plug formed by free particle mechanism is seen in cystinuric stone formers and intestinal bypass patients (92).

 

Calcium stones can be idiopathic or associated with systemic diseases (see Table 10) (89, 95).  Idiopathic calcium stones formers may exhibit various urinary risk factors for calcium oxalate and calcium phosphate stones (Table 11) (89).

 

Table 10. Systemic Conditions Associated with Calcium Stones (89, 95)

Systemic diseases:

·       Primary hyperparathyroidism

·       Sarcoidosis

·       Bone diseases (e.g. fractures, multiple myeloma)

·       Immobilization

·       Hyperthyroidism

·       Distal renal tubular acidosis

·       Polycystic kidney disease

·       Bowel disease (e.g. inflammatory bowel disease, chronic pancreatitis, chronic diarrhea)

·       Bowel resection (e.g. colon resection, ileostomy, small bowel resection, Roux-en-Y gastric bypass)

·       Cystic fibrosis

·       Primary hyperoxaluria

·       Gout

·       Anatomical abnormalities that impair urine flow (e.g. medullary sponge kidney, ureteral stricture, horseshoe kidney, etc.)

 

Medications

·       Carbonic anhydrase inhibitor (e.g. topiramate, acetazolamide, zonisamide)

·       Calcium and vitamin D supplements

·       Vitamin C supplement

·       Loop diuretics

·       Uricosuric agents (e.g. probenecid, benzbromarone)

 

Table 11. Risk Factors for Calcium Stones (89)

Risk factors for calcium oxalate stones

Risk factors for calcium phosphate stones

Low urine volume

Low urine volume

High urine calcium

High urine calcium

Low urine citrate

Low urine citrate

High urine oxalate

---

---

High urine pH

High urine uric acid

---

 

PATHOGENESIS AND RISK FACTORS

 

Hypercalciuria

 

Hypercalciuria is the most common risk factor of calcium stones and found in 30-60% of calcium stone formers (96). Hypercalciuria is classically defined as 24-hour urine calcium greater than 300 mg/day in men, greater than 250 mg/day in women, greater than 4 mg/kg body weight/day in either sex, or urine calcium > 140 mg calcium/gram creatinine/day (75).  Although threshold values are provided to define hypercalciuria, there is no threshold value that predicts risk of stone incidence or recurrence. Rather, risk of stone incidence and recurrence increases progressively with higher urinary calcium excretion (97). 

 

Environmental (diet, supplement, and medications) and metabolic disorders can contribute to hypercalciuria.  One way to determine causes of hypercalciuria is to divide it into three broad categories: hypercalcemic hypercalciuria, normocalcemic hypercalciuria, and hypocalcemic hypercalciuria (Table 12). 

 

Table 12. Causes of Hypercalciuria

Hypercalcemic hypercalciuria

·             PTH-dependent causes

Primary hyperparathyroidism

o   Lithium-induced hyperparathyroidism

·             PTH-independent causes

o   Granulomatous diseases (e.g. sarcoidosis, tuberculosis, histoplasmosis, coccidoimycosis, lymphoma)

o   Vitamin D toxicity

o   Low level or activity of vitamin D 24-hydroxylase

o   Hypercalcemia of malignancy (e.g. bone metastases, lymphoma, PTHrP, multiple myeloma) 

o   Immobilization

o   Hyperthyroidism

o   Paget’s disease of bone

o   Vitamin A toxicity

o   Milk alkali syndrome

 

Normocalcemic hypercalciuria

·             Absorptive

o   Excessive calcium intake (diet and/or supplement)

o   Excessive animal protein intake

o   Sarcoidosis

o   Idiopathic hypercalciuria

·             Resorptive

o   Excessive animal protein intake

o   Hyperthyroidism

o   Immobilization

o   Paget’s disease of bone

o   Osteoporosis

o   Glucocorticoid excess

o   Distal renal tubular acidosis

o   Malignant tumors

o   Idiopathic hypercalciuria

·       Renal leak

o   Excessive salt intake

o   Loop diuretics

o   Mineralocorticoid excess

o   Glucocorticoid excess

o   Distal renal tubular acidosis

o   Idiopathic hypercalciuria

 

Hypocalcemic hypercalciuria

·       Autosomal dominant hypocalcemia (activating mutation in CaSR or GNA11)

 

Idiopathic Hypercalciuria

 

Idiopathic hypercalciuria is found in up to 50% of idiopathic calcium stone formers (98).  It appears to be familial which suggests a genetic basis (99). It presents with a pattern of variable inheritance, and is likely polygenic in most stone formers, with described base changes in some candidate genes including CaSR, VDR, TRPV5, TRPV6, CLCN5, ADCY10, and CLDN14 (75, 96). The pathophysiology of idiopathic hypercalciuria involves increased intestinal calcium absorption, renal leak of calcium, and increased bone resorption especially when challenged with a restricted calcium diet (75, 96).

 

Intestinal calcium hyperabsorption is the most common abnormality in idiopathic hypercalciuria (100). It can be 1,25(OH)2D-dependent or independent. In patients with 1,25(OH)2D-dependent absorptive hypercalciuria, there is generally an increased production of 1,25(OH)2D when compared to normal individuals (101), although some patient with CYP24A1 mutations exhibit reduced 1,25(OH)2D catabolism. The exact mechanism leading to increased 1,25(OH)2D production remains unclear. There was a suggestion that renal tubular phosphate handling may play a role; however, others found that regulators of 1,25(OH)2D production (PTH, serum phosphorus and renal tubular reabsorption of phosphate) in idiopathic hypercalciuric stone formers were comparable to non-stone formers (102-105). In patients with 1,25(OH)2D-independent absorptive hypercalciuria, intestinal absorption of calcium remains elevated despite a normal 1,25(OH)2D level. Again, the mechanism is unclear.  Animal studies demonstrated an increased abundance and half-life of vitamin D receptors (VDR) in the intestines of genetic hypercalciuric rats (106, 107).  In male calcium oxalate stone formers with idiopathic hypercalciuria, an elevated level of VDR in monocytes was found when compared to non-stone formers (108).  Both suggest increased tissue VDR may contribute to absorptive hypercalciuria in individuals with a normal 1,25(OH)2D level.

 

Idiopathic hypercalciuric stone formers also display abnormal renal calcium handling with a lower postprandial renal calcium reabsorption than normal individuals without a difference in filtered load (109). There is evidence of defective renal calcium reabsorption in both proximal tubule and distal nephron (110-112). The reduced renal calcium reabsorption could not be explained by sodium excretion and PTH levels (109). The underlying mechanism of decreased renal calcium reabsorption remains to be elucidated. 

 

Hypercalciuric stone formers were found to have lower bone mineral density (BMD) than non-stone formers even in those with absorptive hypercalciuria (21, 113). The decreased BMD is more pronounced in trabecular bone than cortical bone (21, 113).  Nephrolithiasis was associated with an increased risk of vertebral fractures in both men and women in a population-based retrospective cohort study (114), and with an increased risk of prevalent vertebral and wrist fractures in men in a cross-sectional study in NHANES III (115).  Prior bone histomorphometry studies demonstrated increased bone resorption in fasting hypercalciuria and decreased bone formation in absorptive hypercalciuria (113). The pathophysiology underlying bone loss in idiopathic hypercalciuria is not exactly clear; however, several risk factors have been associated with bone loss in this population. A restricted calcium diet sometimes used by patients or physicians to reduce urine calcium may generate negative calcium balance leading to increased bone resorption and bone loss without reducing risk of kidney stones (77, 116, 117). High dietary salt and protein intake increase urinary calcium excretion and create a subtle metabolic acidosis both of which may contribute to bone loss (21, 75, 113, 118).  Inflammatory cytokines such as IL1, IL6, TNF-α and GM-CSF have been associated with hypercalciuria and bone loss by increased bone resorption (113).  Idiopathic hypercalciuric stone formers were also found to have increased bone expression of RANKL and decreased expression of TGF-β which may be the mediators for increased bone resorption and decreased bone formation and mineralization, respectively (119). High 1,25(OH)2D and/or increased expression of VDR found in idiopathic hypercalciuria may also increase bone resorption and decrease bone formation (21).

 

Hypocitraturia

 

Urinary citrate is an endogenous inhibitor of calcium stone formation.  It forms a more soluble calcium citrate complex than calcium oxalate and calcium phosphate (89, 96).  It reduces urinary supersaturation with respect to calcium oxalate and calcium phosphate (89, 96).  Hypocitraturia is generally defined as urine citrate less than 320 mg/day and is a well-described reversible risk factor that is present in 20-60% of calcium stone formers (89, 96). Extracellular and intracellular pH affects renal citrate excretion. Systemic acidosis increases urinary citrate reabsorption and leads to hypocitraturia (120).  Intracellular acidosis increases intracellular citrate metabolism in the cytosol and mitochondrial via the TCA cycle (120). Thus, hypocitraturia occurs mainly in conditions with extracellular or intracellular acidosis (89).  Table 13 summarizes common causes of hypocitraturia (89, 96, 120). 

 

Table 13. Causes of Hypocitraturia (89, 96, 120)

Systemic diseases:

·       Complete dRTA (hypocitraturia, frank metabolic acidosis, hypokalemia, hypercalciuria, high urine pH)

·       Incomplete dRTA (hypocitraturia without frank metabolic acidosis, high urine pH)

·       Hypokalemia

·       Chronic diarrhea

·       Chronic kidney disease

·       Primary hyperaldosteronism

·       Idiopathic hypocitraturia

Dietary:

·       High animal protein intake

·       High sodium intake

·       Low fruit / vegetable intake

Medications:

·       Carbonic anhydrase inhibitor (e.g. topiramate, acetazolamide, zonisamide)

·       Angiotensin converting enzyme inhibitors

·       Angiotensin II receptor blockers

·       Diuretic-induced hypokalemia

 

Hyperoxaluria

 

High urinary oxalate increases urinary supersaturation with respect to calcium oxalate (96). Hyperoxaluria is generally defined as urinary oxalate greater than 45 mg/day (0.5 mmol/day) (89).  It is encountered in 8-50% of calcium stone formers (121). Etiologies of hyperoxaluria can be divided into three categories: 1) increased endogenous oxalate production due to inborn error of metabolism, 2) increased intake of foods rich in oxalate or its precursors (Table 2) or increased intestinal bioavailability of oxalate, and 3) increased intestinal oxalate absorption (89, 121).  Table 14summarizes causes of hyperoxaluria.

 

Primary hyperoxalurias (PH) are a group of rare autosomal recessive disorders involving overproduction of oxalate which results in markedly increased urinary oxalate excretion. There are three genetic forms: PH1 due to mutations in AGXT (encodes for a pyridoxal-5’-phosphate-dependent hepatic peroxisomal alanine-glyoxylate aminotransferase, AGT), PH2 due to mutations in GRHPR (encodes for glyoxylate reductase and hydroxypyruvate reductase, GRHPR) and PH3 due to mutations in HOGA1 (encodes for hepatic mitochondrial 4-hydroxy-2-oxoglutarate aldolase, HOGA) (122, 123).  Deficiency in AGT in PH1 results in decreased conversion of glyoxylate to glycine. The accumulated glyoxylate is in turn converted to oxalate by lactate dehydrogenase (LDH) leading to increased production of oxalate (123). Deficiency in GRHPR in PH2 results in decreased conversion of glyoxylate and hydroxypyruvate to glycolate and D-glycerate, respectively. The accumulated glyoxylate and hydroxypyruvate are converted to oxalate and L-glycerate respectively by LDH (123). Mutations in HOGA1 in PH3 result in decreased enzymatic activity of HOGA which converts 4-hydroxy-2-oxoglutarate (HOG) to pyruvate and glyoxylate. This results in accumulation of HOG which inhibits mitochondrial GRHPR (which is the deficient enzyme in PH2) activity, thus increasing oxalate production (122, 124).  Primary hyperoxalurias should be suspected if urinary oxalate is greater than 90 mg/day (1 mmol/day) (125). Measurement of other urinary metabolites including glyoxylate and L-glycerate can be helpful, but genetic testing is required for definitive diagnosis of primary hyperoxaluria (123).

 

Dietary oxalate is estimated to range between 50 and 1,000 mg/day (121).  Oxalate is absorbed mainly in the small intestine and to a lesser extent in the colon (121).  Intestinal absorption of oxalate varies between 10% and 72% (121). On a normal calcium diet (1,000 mg/day calcium), urinary oxalate increases by 2.7 mg/day with every 100 mg/day increase in dietary oxalate between 50mg to 750mg/day (126). Table 2 provides a list of foods rich in oxalate and some alternative options with low oxalate content (24).  Although there is considerable variation in the reported oxalate content in foods among the available online sources, the simplest strategy is to avoid the foods with high oxalate content (127).  Attention should be paid to portion size even with foods with low to moderate oxalate contents.  Excessive intake of vitamin C more than 1,000 mg/day is associated with increased urine oxalate because vitamin C is metabolized into oxalate in the body (128, 129).

 

A restricted calcium diet increases enteric oxalate availability for absorption and results in increased urinary oxalate (85, 86). Patients with chronic diarrhea, pancreatic insufficiency, inflammatory bowel diseases, or small bowel resections may have malabsorption of bile acids and/or fatty acids which can complex with luminal calcium in the intestine, resulting in increased bioavailability of oxalate to be absorbed and subsequently excreted in the urine (89, 96). This is also termed enteric hyperoxaluria.  Increased bioavailability of luminal oxalate can also result from decreased colonization by Oxalobacter formigenes which is a Gram-negative obligate anaerobe that utilizes oxalate as the sole energy source. O. formigenes also increases secretion of endogenous oxalate from plasma to the gut lumen which results in decreased urinary oxalate (126). Colonization by O. formigenes is associated with decreased bioavailability of intestinal oxalate for absorption, decreased urinary oxalate, and reduced risk of calcium oxalate stones (130, 131).  Use of certain antibiotics to which O. formigenes are sensitive (macrolides, tetracyclines, chloramphenicol, rifampin and metronidazole) within the past 5 years is associated with a reduction in colonization when compared to non-users (132), and has been separately associated with greater incidence of stone disease (133). Furthermore, patients with cystic fibrosis or inflammatory bowel disease who receive frequent antibiotic courses were found to have lower prevalence of colonization by O. formigenes, which may contribute to their higher oxalate excretion and increased kidney stone formation (134, 135). 

 

Table 14. Causes of Hyperoxaluria

Primary hyperoxaluria

·       Primary hyperoxaluria type 1 (mutations in AGXT gene)

·       Primary hyperoxaluria type 2 (mutations in GRHPR gene)

·       Primary hyperoxaluria type 3 (mutations in HOGA1 gene)

Secondary hyperoxaluria

·       High intake of oxalate rich foods (See Table 2)

·       Vitamin C intake > 1,000 mg/day

·       Low calcium intake

·       Pancreatic insufficiency

·       Inflammatory bowel disease (Crohn’s disease)

·       Small bowel surgeries

·       Cystic fibrosis

·       Decreased colonization by Oxalobacter formigenes

 

Elevated Urine pH

 

Urine pH higher than 6.7 is a risk factor for calcium phosphate stones. The pKa for monohydrogen phosphate (HPO42-) is ~ 6.7.  At a pH higher than the pKa, there is an increased abundance of HPO42- which complexes with divalent cation calcium (Ca2+) to form brushite (CaHPO4.2H2O) and eventually to hydroxyapatite [Ca10(PO4)6(OH)2](96). Table 15 summarizes potential causes of high urine pH.

 

Table 15. Causes of High Urine pH (89, 96)

·       Distal renal tubular acidosis (RTA)

·       Urinary tract infections

·       Primary hyperparathyroidism

·       Carbonic anhydrase inhibitors

·       Alkali therapy 

 

Hyperuricosuria

 

Hyperuricosuria is defined as urinary uric acid greater than 800 mg/day in men and 750 mg/day in women (136).  It is found in 40% of calcium stone formers and associated with increased risk of calcium oxalate stones (96). There are three main proposed mechanisms by which hyperuricosuria promotes calcium oxalate crystallization: 1) calcium oxalate precipitation on monosodium urate crystals through heterogeneous nucleation (137, 138), 2) removal of calcium oxalate crystallization inhibitors by colloidal urate particles (139), and 3) increased urate concentration decreases the solubility of calcium oxalate and leads to precipitation of calcium oxalate from solution by a salting-out mechanism (140). Hyperuricosuria is generally caused by increased purine intake, increased production of uric acid, or increased urinary excretion primarily from acquired conditions, although inherited causes of hyperuricosuria are also described (Table 16) (89, 136).

 

Table 16. Causes of Hyperuricosuria (89, 136)

Acquired Conditions:

Increased intake:

·       High purine rich diet (e.g. red meat, fish and poultry)

Increased production:

·       Gout

·       Myeloproliferative and neoplastic disorders

Increased urinary excretion:

·       Uricosuric drugs

 

Inherited Conditions (rare):

Disorders of uric acid metabolism:

·       Lesch-Nyhan syndrome

·       Glycogen storage disease type 1A

Disorders of renal uric acid reabsorption:

·       Renal hypouricemia

 

MANAGEMENT OF CALCIUM STONES

 

Lifestyle Measures

 

A diet high in fluid (fluid intake of 2.3-3 liters per day or achieving urine volume of at least 2-2.5 liters per day), rich in fruits and vegetables, low in sodium (less than 2300 mg/day or 100 mmol/day), animal protein (limit to 0.8 to 1.0 g/kg body weight/day) and oxalate (less than 100 mg/day) and normal in calcium (1,000 to 1,200 mg/day preferably from dietary source) is recommended for calcium stone prevention (27-29, 95, 96).  These were previously addressed in section “General measures for all patients with kidney stones” (Table 8). 

 

Pharmacotherapy

 

Thiazide and thiazide-like diuretics:

 

Hypercalciuria is the most common risk factor for calcium stones. Thiazide (hydrochlorothiazide or HCTZ) and thiazide-like diuretics (indapamide and chlorthalidone) can reduce urinary calcium by two proposed mechanisms: 1) blockage of NaCl symporter in distal convoluted tubule leads to decreased distal sodium and water reabsorption and volume contraction, which results in increased sodium and water reabsorption in the renal proximal tubule, resulting in increased calcium reabsorption by passive transport (141), and 2) increased distal tubular calcium absorption by increased abundance of transport proteins TRVP5 and calbindins (142, 143). The hypocalciuric dose-response to HCTZ has been studied in six healthy adults which demonstrated hypocalciuric effect at 12.5mg, 25mg, and 50mg daily; however, it was subtherapeutic for 12.5mg and 25mg daily when compared to 50mg daily (144). Several randomized controlled trials on thiazide diuretics with an average follow up of ~ 3 years showed reduction in the risk of recurrent stones in both hypercalciuric and normocalciuric stone formers (145). This underscores the lack of threshold effect of urinary calcium in predicting stone risk. Rather, the risk of stone formation increases progressively with increasing urinary calcium excretion even within the “normal range” (97).  It also supports the empiric use of thiazide diuretics in recurrent calcium stone formers, even those with normocalciuria (27, 146). The doses used in these trials were HCTZ 25mg BID, 50mg and 100mg daily, indapamide 2.5mg daily and chlorthalidone 25mg and 50mg daily (145).  A recent retrospective cohort study suggests lower doses of thiazide diuretics (HCTZ or chlorthalidone ≤ 12.5 mg daily or indapamide ≤ 1.25 mg daily) appear to have a similar protective effect against stone formation as higher doses in older adults (147).

 

Thiazide diuretics have also been shown to improve bone health. They reduce urinary calcium excretion resulting in positive calcium balance and reduced PTH which may reduce bone turnover (96). They have also been shown to stimulate osteoblast differentiation and function and inhibit osteoclast differentiation in vitro (21, 115, 148).  HCTZ 50mg daily improved cortical BMD in healthy postmenopausal women without baseline hypercalciuria (149, 150). In postmenopausal women with osteoporosis and hypercalciuria, addition of indapamide 2.5mg daily to alendronate 70mg weekly resulted in a reduction in urinary calcium and an additional increase in BMD at the lumbar spine over 12 months compared to alendronate single therapy (151). A meta-analysis of five observational cohort studies showed thiazide diuretics use was associated with a reduction in risk of hip fractures (152). Although there is no randomized placebo-controlled trial available, a secondary analysis of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) showed a reduction in hip and pelvic fracture risk in patients on chlorthalidone 12.5mg to 25mg daily compared to those on lisinopril or amlodipine (153).

 

Thiazide diuretics use may be associated with hypokalemia which may induce hypocitraturia, thus potassium supplements are often needed. Potassium citrate is superior to potassium chloride given its ability to increase urinary citrate and pH and to further lower urine calcium excretion (154). Combination of thiazide diuretics with a potassium sparing diuretic (e.g. amiloride) can also be considered.

 

In summary, thiazide diuretics are recommended to patients with recurrent calcium stones with and without hypercalciuria (27).  

 

Alkali therapy:

 

Potassium citrate treatment increases urine pH and urine citrate, decreases urine calcium, and decreases urinary supersaturation with respect to calcium oxalate (155). Several placebo-controlled randomized trials demonstrated potassium citrate and potassium magnesium citrate treatment reduced recurrent stone events in calcium stone formers with and without hypocitraturia (156-158).  

 

Potassium citrate treatment may also prevent bone loss.  Potassium citrate treatment increased BMD at the spine in idiopathic calcium stone formers (159) and increased BMD at the spine, femoral neck, and total hip in healthy elderly men and postmenopausal women without osteoporosis (160).

 

The proposed mechanisms by which potassium citrate improves BMD include systemic alkalization, increased osteoblastic activity, and reduced osteoclastic activity demonstrated by bone turnover markers, and positive calcium balance created by reduced urinary calcium excretion (21, 160, 161).

 

Currently, potassium citrate therapy is recommended for patients with recurrent calcium stones with and without hypocitraturia (27).

 

Xanthine oxidase inhibitors: Allopurinol and Febuxostat:

 

There is only one published randomized placebo-controlled trial on allopurinol in prevention of calcium nephrolithiasis. In this study, calcium oxalate stone formers with hyperuricosuria and normocalciuria treated with allopurinol 100mg TID had a lower rate of stone events than those treated with placebo over 24 months (162).  Allopurinol is recommended for patients with recurrent calcium oxalate stones with hyperuricosuria and normocalciuria (27).

 

Febuxostat was studied in a randomized controlled trial comparing febuxostat 80mg daily with allopurinol 300mg daily or placebo on the effect of stone prevention in calcium stone formers (calcium oxalate and/or calcium phosphate) with hyperuricosuria and normocalciuria over 6 months (163). Febuxostat led to a greater reduction in urinary uric acid than allopurinol or placebo, but percent change in stone size was similar to allopurinol or placebo. This study was not powered to detect difference in stone events in the three groups.  Currently, there is insufficient evidence to support the routine use of febuxostat for stone prevention in hyperuricosuric calcium stone patients, except in those who may be allopurinol-intolerant (29).

 

Pyridoxine:

 

Pyridoxine (vitamin B6) supplementation is helpful in primary hyperoxaluria type 1 (PH1) with specific mutations, namely Gly170Arg, Phe152Ile and Ile244Thr.  A trial of pyridoxine for 3 months with a starting dose of 5 mg/kg body weight/day titrated to a maximum of 20 mg/kg body weight/day can be attempted in patients with suspected primary hyperoxaluria.  Response to therapy is defined as more than 30% reduction in urinary oxalate from baseline (123).

 

Uric Acid Stones

 

Uric acid stones generally represent around 10% of all stones analyzed, although their prevalence has markedly increased in recent years, in parallel with the diabetes and obesity epidemics (164, 165). In a series of 2,464 calculi, the proportion of uric acid stones was 35.7% in patients with type 2 diabetes and 11.3% in patients without type 2 diabetes (166). Reciprocally, the proportion of patients with type 2 diabetes was significantly higher among uric acid than among calcium stone formers (27.8 versus 6.9%) (166).  In fact, several epidemiological and metabolic studies have reported an association of uric acid stone disease with various features of the metabolic syndrome including obesity, type 2 diabetes, hypertension, dyslipidemia, hyperglycemia, hepatic steatosis, and greater visceral adiposity (167).

 

PATHOGENESIS AND RISK FACTORS

 

The three main factors implicated in the development of uric acid nephrolithiasis are low urine pH, hyperuricosuria, and low urine volume (Table 17) (168). Of these, low urine pH is the primary determinant of uric acid nephrolithiasis, as acidic urine favors the protonation of urate, forming relatively insoluble uric acid which precipitates in this overly acidic urinary environment. In fact, a decline in urine pH from 6.0 to 5.0 increases urinary uric acid concentration six-fold, whereas states of increased urate production typically increase urate excretion two-fold. Therefore, uric acid stone formation is more determined by pH than by urine volume or urine uric acid concentrations. Low urine pH may result from excessive intake of animal proteins (81), gastrointestinal alkali loss (from chronic diarrhea or laxative abuse), or may be idiopathic as frequently observed in patients with obesity, type 2 diabetes, and/or the metabolic syndrome (164, 168). Human metabolic studies have identified greater acid excretion and reduced urinary buffering by ammonia as the two culprits of aciduria in uric acid nephrolithiasis (169). Hyperuricosuria is less frequently encountered in patients with uric acid nephrolithiasis, and may result from inherited and/or acquired conditions (Tables 16 and 17). Finally, low urine volume due to extra-renal fluid losses contributes to increased urinary saturation with respect to uric acid, leading to stone formation.

 

Table 17. Risk Factors and Etiological Conditions Associated with Uric Acid Nephrolithiasis

Urinary Risk Factor

Type of Abnormality

Etiological conditions

Low urine pH

Inherited Conditions

Inherited uric acid lithiasis (unknown genetic abnormality)

Acquired Conditions

Metabolic syndrome, obesity, diabetes, CKD, high animal protein intake, gastrointestinal alkali loss

Medications

Laxatives

Hyperuricosuria

Inherited Conditions

Disorders of uric acid metabolism (e.g. Lesch-Nyhan);

Disorders of uric acid excretion (e.g. renal hypouricemia);

Glycogen storage disorder type 1A (glucose-6-phosphatase deficiency)

Acquired Conditions

Gout, myeloproliferative disorders, hemolytic disorders, high purine intake

Medications

Uricosuric agents: Losartan, Probenecid, Benzbromarone

Low urine volume

Acquired Conditions

Chronic diarrhea, excessive perspiration, low fluid intake

Medications

Laxatives

 

MANAGEMENT

 

Since uric acid stone formation is more determined by urine pH than by urine volume or urine uric acid concentrations, the cornerstone of therapy is urinary alkalinization.

 

Lifestyle Changes

 

Dietary restriction of animal protein intake is helpful in decreasing ingestion of proton sources, which reduces aciduria and aids with urinary alkalinization. At the same time, animal protein restriction also lowers uric acid excretion through a reduction in purine intake. Conversely, greater ingestion of alkali-rich fruits and vegetables aids in raising urine pH. Finally, higher fluid intake in general aids in raising urine volume, while intake of certain fruit juices such as orange juice can increase urine pH (along with the concomitant rise in urine volume). Still, one should be cautious about the sugar load imparted by fruit juices in uric acid stone formers with pre-diabetes or frank diabetes.

 

Pharmacological Therapy

 

Medical dissolution therapy of uric acid stones with alkali therapy (potassium citrate, to raise 24-hour urine pH to 6.0 to 6.5) is the cornerstone of uric acid management. Alkali therapy is well-tolerated by most uric acid stone formers and effectively dissolves stones, potentially avoiding the morbidity of urological interventions (170, 171). In patients who cannot tolerate potassium citrate, alternative alkali regimens include sodium bicarbonate and potassium bicarbonate. Occasionally, xanthine oxidase inhibitors (allopurinol or febuxostat) are added to potassium citrate in patients with hyperuricosuria whose uric acid stones recur despite alkali therapy. A recent study has suggested that the thiazolidinedione pioglitazone may aid in raising urine pH in uric acid stone formers (172), although the risk/benefit ratio of this medication needs to be considered.

 

Cystine Stones

 

PATHOGENESIS AND RISK FACTORS

 

Cystine represent around 1-2% of stones in adult patients, but account for 5-8% of stones in pediatric patients. Cystine stones result from inactivating mutations in genes that encode renal tubular transporters that reabsorb the amino acid cysteine (173). The complexation of two molecules of the dibasic amino acid cysteine results in the formation of cystine which is relatively insoluble. Cystine normally appears in urine in small amounts that are insufficient to cause supersaturation, crystalluria, or stone formation. Due to defects in renal cysteine reabsorption, patients with cystinuria exhibit greater than a 10-fold increase in urine cystine excretion (as well as greater excretion of the other dibasic amino acids lysine, ornithine, and arginine). As a result, the solubility limit of cystine in the urine (250 mg/L) is exceeded. Homozygous inheritance results in more severe phenotype, whereas heterozygous inheritance is associated with variable increases in amino acid excretion and an intermediate increase in cystinuria. Cystine stones form in the upper urinary tract as early as the first decade of life, and tend to be large, staghorn, bilateral, and highly recurrent (173). Stone formation may manifest as obstruction, infection, hematuria, and renal failure. Cystine stones are visible on standard abdominal radiographs because of the relative density of the sulfur constituent of cysteine (Table 6).

 

MANAGEMENT

 

The goal of therapy in cystinuria is to reduce cystine excretion and increase urinary cystine solubility (173, 174). This is accomplished using a combination of lifestyle changes and pharmacological interventions.

 

Lifestyle Changes

 

Large urine volumes of 3-4 liters per day may be effective at reducing cystine concentration and reducing stone recurrence in some patients, although this is difficult to institute in children and even adult patients. Dietary protein restriction to around 1 g protein/kg body weight/day reduces cysteine intake, and may cause small decreases in cystine synthesis (175), although this should be avoided in growing children and adolescents. A low sodium intake can also contribute to reduced cystine excretion (176).

 

Pharmacological Therapy

 

When fluid and dietary therapy fail, then pharmacologic therapy may be effective. Alkaline pH in the 7.0-7.5 range will reduce cystine solubility and can be achieved by the addition of alkali therapy such as potassium citrate (177). Tiopronin (Thiola®) and D-penicillamine reduce cystine formation in urine by preventing cysteine-cysteine complexation and the formation of more soluble thiol-cysteine disulfides that are more readily excreted in the urine. Both agents however have potentially serious side effects (proteinuria, abnormal LFTs, others) and therefore they are not used as first-line treatment (174).

 

Struvite (Infection) Stones

 

PATHOGENESIS AND RISK FACTORS

 

Struvite (magnesium ammonium phosphate) stones form only in the presence of bacteria that produce urease. Common urease-producing bacteria that may populate the urinary tract are proteus, klebsiella, pseudomonas, and enterococci. Urease-mediated splitting of urea and the generation of ammonium results in an alkaline urine. Urine pH above 7.0 normally is associated with very low urine ammonium levels of less than 10 mEq/day. However, urine ammonium excretion exceeding 30 mEq/day along with 24-hour urine pH > 7.0 virtually make the diagnosis of struvite stones. Other constituents of the stone may include calcium carbonate and brushite (calcium phosphate), which form crystals in the very alkaline urine. Patients who form struvite stones do not pass them spontaneously, but rather are at high risk for bleeding, obstruction, and decreased renal function. Some infection stones begin as calcium oxalate stones that become infected with a urease-producing bacterium. Spread of infection to the contralateral kidney may occur.

 

MANAGEMENT

 

Because untreated staghorn calculi will require nephrectomy in 50% of patients, definitive treatment is indicated (178). Growth of infection stones and their progressive damage to kidney tissue may be limited by shockwave lithotripsy and percutaneous nephrolithotomy (PCNL); however definitive treatment of struvite stones is surgical removal. Extended antibiotic therapy has proven ineffective in eradicating the infection and does not substitute for complete removal of even the smallest particulate of the stone (178). Management with PCNL followed by careful follow-up and medical management minimizes stone recurrence and maintains kidney function in the majority of patients (179). Larger stone burden pre-operatively, residual stones after surgery, and presence of medical comorbidities are independent risk factors for stone recurrence or residual stone-related events (179). Acetohydroxamic acid inhibits urease produced by the bacteria and has been shown to be effective in eradicating chronic infection of struvite stones (180). Use of the drug has been limited, however, as it is associated with potentially serious side effects such as hemolytic anemia and venous thromboembolic disease.

 

CONCLUSIONS

 

In conclusion, urinary stones are common, morbid, and highly recurrent. The pathophysiology of kidney stone formation is diverse, and includes a combination of genetic and environmental factors. Several endocrinological disorders increase the risk of stone formation. Metabolic evaluation of patients with kidney stones helps to identify the underlying etiological factors and provides an opportunity to institute preventive lifestyle and/or pharmacologic measures to reduce stone recurrence risk.

 

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Performance Enhancing Hormone Doping in Sport

ABSTRACT

 

Sport is the organized playing of competitive games according to rules. Hence doping represent drug cheating, a fraud on competitors, the sport, and the public. The charter of the World Anti-doping Agency (WADA) forms a harmonized Code that authorizes an annually updated list of prohibited doping substances and methods as well as accrediting national anti-doping labs around the world. Sports performance has 4 major components: skill, strength, endurance, and recovery, with each sport employing a distinct combination of these elements. These performance characteristics also correspond to the most potent and effective forms of doping. Sports requiring explosive power are most susceptible to androgen doping through their effect on increasing muscle mass and strength whereas sports that require endurance are most enhanced by hemoglobin (blood) doping which increases oxygen delivering capacity to exercising tissues. Performance in contact sports and those involving intense physical activity or training may also be enhanced by growth hormone and its secretagogues through speeding of tissue recovery from injury. Hormones remain the most potent and widely detected doping agents being responsible for about 2/3 of anti-doping rule violations detected by increasingly sophisticated detection methods. At present, the vast majority of positives are still due to a wide variety of androgens, including marketed and illicit nutraceutical, designer, specific androgen receptor modulator (SARM)) synthetic androgens as well as exogenous natural androgens, while the peptide hormones (erythropoiesis stimulating agents, growth hormone and its secretagogues) and autologous blood transfusion remain difficult to detect.

 

INTRODUCTION

 

Across the world, sport is a ubiquitous human social activity that forms an unique intersection of health, recreation, entertainment, and industry (1). It is both a major economic activity as well as a profound influence on social behavior of individuals at home, work, and play. One practical and concise definition of sport is the organized playing of competitive games according to rules. In that context, rule breaking is cheating to achieve an unfair competitive advantage whether it involves using illegal equipment, match fixing, banned drugs, or any other prohibited means.

 

The illicit use of banned drugs (doping) to influence the outcome of a sporting contest, constitutes a fraud against competitors, spectators, sport, sponsors, and the public no different from other personal, professional, or commercial frauds. While performance enhancement is almost invariably the intent of cheating, impairing performance is also well known in horse racing and even, rarely, in human elite sports (e.g. drink-spiking of banned drugs, injurious physical assaults). Rules of sporting contest may change by agreement, but once set, represents the boundaries of fair competition. Nevertheless, fairness is an elastic, socially constructed concept which may change gradually over time. For example, a century ago deliberate training itself was considered an ungentlemanly breach of fairness as competition was then envisaged as a contest based solely on natural endowments. Similarly, some sports once maintained a distinction between amateurs and professionals. The philosophical foundations of the concept of fairness is a deep and complex issue (2,3) where the focus has been mainly on distributive justice with an implicit goal of equality of outcomes. Less attention has been given to the philosophical basis of fair competition in sport where the prior distribution of talent and training and the outcome of contest are intended to provide equality of opportunity, but not of outcome, between contestants.

 

Naïve arguments are made that deny doping is cheating, or unsafe or violates the spirit of sport and asserting that drugs should be freely available or under medical supervision (4-6). However, removing prohibition on doping would immediately render drug taking as pervasive as training in elite sport extending to promising underage and sub-elite athletes. Ensuing demands on doctors to prescribe excessive, often massive, drug doses without medical indications would be unprofessional, unethical, and unsafe. This could convert sporting participation into a potentially dangerous rather than a healthful activity. In practice, creating enforceable boundaries for drugs in sport is unavoidable whether it is prohibition or, even under the most idealistic libertarian scenarios, by age or dosage. Within the limitations of unverifiable self-report regarding an illicit activity (7,8), surveys indicate athletes support antidoping testing mainly to prevent cheating but also to promote safety (9-11). Motivating factors for, and routes by which athletes get involved in doping are complex but include the use of non-banned nutritional supplements as a gateway to doping (12) and the suspicion of athletes or their entourage that their rivals may be using illicit drugs, the so-called “false consensus belief” (7,13-15). These philosophical issues are not considered further here and, recognizing that sport requires concrete, practical decisions, the establishment and enforcement of agreed rules is the basis of fair competition. An excellent discussion of the logic and morality of a decisive antidoping approach from an ethicist with extensive experience in sports anti-doping is recommended (16).

 

It is well understood that individual human genetic endowments are unequal and, among these, sporting prowess is at least partly genetically determined (17). However, little is still known of the genotype-phenotype correlations that underlie beneficial genetic endowments for sports performance. Natural genetic advantages are recognized in height (tallness for basketball, shortness for jockeys and motor-cycle riders) and hereditary erythrocytosis where a high circulating hemoglobin due to a high affinity erythropoietin (EPO) receptor (18)) for endurance sports, or conversely genetic disadvantage such as the common α-actinin-3 deletion genetic polymorphism which limits anaerobic, explosive power (19). More examples of genetic (dis)advantages for sports performance are likely to be identified as genomics continues to expand our understanding of the biological basis of health, including natural human sporting prowess. In the context of sports doping, however, a person’s genetic endowment is a given creating a natural boundary whereby the use of exogenous drugs or chemicals (including DNA) may constitute drug cheating or doping.

 

WORLD ANTI-DOPING AGENCY (WADA) AND THE GLOBAL ANTI-DOPING REGULATORY ENVIRONMENT

 

Cheating is as old as sport itself, yet the present endemic of doping using pharmaceutical drugs to boost sports performance is largely a Cold War legacy. Eastern European national doping programs were established by governments aiming to achieve a short-cut propaganda victory over their Western rivals, a challenge quickly reciprocated and then taken up by individual coaches and athletes. Starting with power sports (20), the epidemic became entrenched as an endemic in sufficiently affluent circles. In 1967, following the introduction of anti-doping rules by some sports federations, the International Olympic Committee (IOC) established its Medical Commission, which published their first list of prohibited substances. During the 1970’s the IOC Medical Commission took an increasingly active role by banning androgens which required developing standardized, valid methods to detect and deter androgen doping. After discarding alternatives such as immunoassays and blood sampling, in the 1980s mass spectrometry (MS)-based tests became (21) and remain the standard for detecting synthetic androgens in urine.

 

In 1999, the IOC established the WADA based in Montreal to be equally supported by governments and sporting organizations with its charter, the WADA Code, representing a harmonized set of global anti-doping rules introduced in 2004, revised in 2009 and 2015 and will be again revised in 2021(22). WADA also publishes an annually updated Prohibited List of Substances and Methods, accredits national anti-doping labs together with their operational anti-doping testing framework, and established the Court for Arbitration in Sport (CAS) to settle anti-doping legal disputes as sport’s “Supreme Court”. The WADA Code has been adopted by over 660 sporting organizations including all Olympic and Paralympic organizations and National Anti-Doping Organizations as well as most non-Olympic International and National Sports Federations. The WADA Code prohibits substances or methods which meet 2 of 3 criteria comprising:

(i) enhance performance (cheating),

(ii) harmful to health (safety) or

(iii) violate the spirit of sport (unsporting).

 

Although the primacy of penalizing cheating is widely understood, implementing these criteria encounter ethical and practical difficulties in proving ergogenic effects of increasing numbers of illicit and/or non-approved substances. These substances have unknown safety so that human testing is not feasible making athlete safety an important consideration. Crucially, the Code imposes strict liability on individual athletes so that a positive anti-doping test (including refusal or avoidance of testing or possession, attempts, trading and tampering with banned drugs) constitutes an anti-doping rule violation (ADRV), regardless of intent or negligence. Sanctions involve suspension from any elite competitive sport and extend to support personnel and teams. Suspensions, once 2 years are now 4 years since adoption of the 2015 Code. This is generally believed to be longer than the ergogenic benefits of doping, although recent evidence suggests that androgen effects on muscle may create durable or even permanent effects (23) which might argue for much longer or permanent banning of androgen doping violators.

 

The Prohibited List bans, at any time either in or out of competition, the use of performance enhancing hormones, including androgens, EPO and growth hormone and related substances or drugs which stimulate endogenous production of these hormones (Table 1). Among the 15 categories of prohibited substances (12) and methods (3), hormones feature prominently in S1 (anabolic agents, mainly androgens), S2 (peptide hormones, growth factors, related substances and mimetics), S4 (hormone and metabolic modulators), and S9 (glucocorticoids) with S1 and S2 having important “catch-all” provision for unnamed but related substances “with similar chemical structure or biological effects”. In addition, the S0 category bans non-approved substances, those without current regulatory approval for human therapeutic use. The prominence of hormones is reinforced by the WADA laboratory statistics for anti-doping tests where hormones remain the most frequently detected banned drugs (Table 2). In 2017, of over 322,000 anti-doping tests ~1.5% were positive with 61% due to hormones, the vast majority (~99%) due to androgens. These findings confirm that the detection of androgen doping is effective whereas the low rate of detection of hemoglobin or growth hormone doping may reflect the limitations of available tests for peptides and peptide hormones which require blood rather than conventional urine sampling and feature low sensitivity and brief windows of detection, rather than their lack of abuse. Further use of out-of-competition testing and blood samples together with more sensitive detection tests with longer windows of detection are required particularly for peptide hormones.

 

Table 2. Performance Enhancing Hormone Tests in WADA Labs

 

2005

2009

2013

2017

ACCREDITED LABS

33

35

33

31

TOTAL TESTS

183,337

277,928

269,878

322,050

POSITIVES

(% OF TOTAL)

3,909

(2.1%)

5,610

(2.0%)

5962

(2.2%)

4756

(1.5%)

HORMONES

(% OF +VE)

55%

73%

57%

61%

ANDROGENS

3893

5541

3352

1813

BLOOD/EPO

16

68

63

85

GH/PEPTIDES

0

1

0

19

Source: WADA website report on laboratory testing figures. See http://www.wada-ama.org/en/Science-Medicine/Anti-Doping-Laboratories/Laboratory-Testing-Figures/

 

The prevalence of doping in elite sports as an illicit activity with drastic consequences for athletes admitting guilt remains difficult to quantify using laboratory-based testing, inference from performance, or self-report questionnaires (24). The most promising methods appear to be questionnaires using the unrelated question random response methodology (25). This methodology was developed to estimate the prevalence of sensitive, disapproved, or illicit activities by asking the sensitive personal questions masked by mixing them with unrelated non-sensitive questions in an anonymized framework. This provides overall prevalence self-report estimates of the target activities without allowing for individual identification of answers. One study of two elite athletic competitions provided estimates of 43.6% and 57.1% for recent (last year) and 70.1% of ever use of banned doping methods (26). However, another study using the same methodology found markedly lower prevalence estimates of 0.7% to 11.9% for recent use of banned doping (27). The discrepancies between these prevalence estimates requires further clarification. A significant limitation of these methods is their reliance on athlete’s perception of banned methods. For example, whether “banned drugs” are interpreted as including the widely used (but non-banned) nutritional supplements which athletes are urged to avoid for fear of adulteration with unlabeled banned substances.

 

Therapeutic Use Exemption (TUE)

 

In rare cases, an elite athlete with a genuine medical need for therapeutic use of a prohibited drug may be granted a TUE (28). This exempts the athlete from the Code’s strict liability provision and permits them to compete during ongoing necessary medical treatment. WADA provides medical guidelines that standardize the evaluation and management of TUE applications for a range of medical illnesses. A TUE is granted by a national anti-doping organization based on an independent, expert review of valid, documented diagnosis, appropriate clinical indications and dose for hormonal treatment with a view to facilitating essential medical treatment but avoiding unjustified use or over-dosage. After stringent review TUE’s may be granted for treatment with testosterone, glucocorticoids, and insulin but there are very rarely any valid medical indications for EPO or, in adults, for growth hormone or IGF-1 in elite athletes. For example, TUE’s are usually justified for young male athletes with genuine androgen deficiency, occurring in ~1:200 men (29),  due to organic pituitary-testicular disorders with an established pathological basis (e.g. bilateral orchidectomy, severe mumps orchitis, Klinefelter’s syndrome, etc.) who require life-long testosterone replacement therapy (30). The TUE will approve, subject to regular review, a standard testosterone replacement regimen, including dosage and monitoring, with changes to regimen requiring approval. TUEs are not granted for men with functional decreases in blood T due to non-reproductive disorders including stress (“over-training”) or ageing (“andropause”, “LowT” “late-onset hypogonadism”), or for women.

 

In principle, detection of prohibited substances is ideally aimed at identifying a xenobiotic substance or its distinctive chemical signature(s) which do not occur naturally in the body, thereby distinguishing it categorically from normal body constituents. Such identification of a non-natural substance that can’t be of endogenous origin is congruent with the strict liability onus in proving an anti-doping rule violation (ADRV). Proving an ADRV is more difficult to achieve with administration of natural hormones or their analogs which must be distinguished from their endogenous counterparts. In this situation, the alternative requires developing valid biomarkers to prove the use of banned substances through their distinctive effects on the body and tissues. It is a formidable challenge to validate an indirect biomarker as proof of an ADRV capable of withstanding vigorous medico-legal challenge when a proven ADRV would prevent an athlete from pursuing their profession. Proof of an ADRV based on a doping detection test requires rigorous standardization and harmonization of every stage of the anti-doping tests from sample collection, chain-of-custody, storage, and analysis including accounting for any fixed (genetic, gender, age, ethnicity) or variable (exercise, hydration, masking vulnerabilities) factors which may impact on proposed test metrics.

 

COMPONENTS OF SPORTS PERFORMANCE AND DOPING

 

Sports performance has 4 major dimensions – skill, strength, endurance and recovery (Figure 1). High performance in any sport requires a characteristic blend of these dimensions although individual sports differ widely in that balance. Similarly, the major ergogenic drug classes have distinctive effects aligned predominantly along one of these dimensions so that the most effective ergogenic drug classes used in doping are dictated by these dimensions of sports performance (Figure 2). While every sport requires an acquired skill, some are largely or solely based on skill and concentration (e.g. board games, target shooting, car driving, and motor-cycle riding) and may benefit from drugs that reduce anxiety, tremor, inattention or fatigue. Sports that are highly dependent on explosive, short-term anaerobic power (sprinting, throwing, boxing, wrestling), typically ones which favor a stocky, muscular build, are most susceptible to androgen-induced increases in muscle mass and strength. Other sports with an emphasis on aerobic effort and endurance (e.g. long distance or duration events), characteristically favored by a lean build, may be boosted by hemoglobin doping (blood transfusion, erythropoietin (EPO) and its analogs or mimetics. Finally, sports that depend on recovery from major injury or recurrent minor injury during intensive training, notably contact sports, may benefit from tissue proliferative and remodeling effects of growth hormone and various growth factors.

Figure 1. Components of Sports Performance

Figure 2. Drugs that Enhance Sports Performance

ANDROGENS

 

Although the ergogenic effects of androgens were discovered empirically soon after the identification of testosterone as the principal male androgen of testicular origin in 1935 (31), their applications to elite sport performance were mainly developed during the Cold War by trial and error experiments undertaken on unknowing elite athletes (20,32,33); however, the scientific basis of androgen doping was only objectively proven in the 1990’s. Until that time, the settled consensus was that exogenous androgens had no effect in eugonadal men whose androgen receptors were already saturated by endogenous testosterone (T) (20,34,35). The then alleged benefits of androgen doping were misattributed placebo responses together with training and nutritional effects. Using an exemplary placebo-controlled, randomized clinical trial design with a wide range of testosterone doses, Bhasin et al showed that T increased muscle mass and strength in eugonadal young men to a similar extent as exercise alone and with additive effects when combined with exercise (36) (figure 3). Subsequent dose-response studies showed that administration of T increased muscle mass and strength by 10% without and 20-37% with exercise (where exercise alone increased them by 10-20%) together with additive effects from 3% increase in circulating hemoglobin. These benefits extended from below to well above physiological T doses or blood levels without evidence of plateau (37,38) and regardless of age (39).

Figure 3. Biological Basis of Androgen Doping

Androgen doping may be either direct or indirect (Table 3, figure 4). Direct androgen doping involves administration of testosterone, natural or synthetic androgens whereas indirect androgen doping includes a variety of non-androgenic drugs which increase endogenous T. Direct androgen doping originally involved all pharmaceutically marketed natural (T, DHT, nandrolone) and synthetic androgens but has extended to non-marketed designer and nutraceutical androgens as well as pro-androgens (androstenedione, DHEA) and the new class of non-steroidal androgens (selective androgen receptor modulators, SARM (40)). Indirect androgen doping involves use of hCG, LH, anti-estrogens (estrogen receptor blockers, aromatase inhibitors), opioid antagonists, and neurotransmitters involved in neuroendocrine regulation of endogenous LH and T secretion (41-44).

Table 3– Direct and Indirect Androgen Doping and Detection Methods

 

Substance

Detection method

Direct

 

Synthetic androgens

L/GC-MS

Natural androgens

L/GC-MS, T/E, CIRMS

Designer & nutraceutical androgens

L/GC-MS (bioassay)

Indirect

 

hCG (urinary or recombinant)

hCG immunoassay or LC-MS

hLH (recombinant)

hLH immunoassay or LC-MS

Anti-estrogens

L/GC-MS

GnRH analogs

L/C-MS

Opioid antagonists & neurotransmitters

L/C-MS

 

Figure 4. Direct and Indirect Androgen Doping

Detection of direct androgen doping using steroids of known chemical structure is highly effective using gas or liquid chromatography MS (45-47). Traces of synthetic androgens or their metabolites may remain detectable for periods up to months after last administration (48). Recent developments including the identification of long-term metabolites has further widened the detection windows for synthetic androgens (49-55). Challenges to detection of synthetic androgens have included the development of non-marketed designer and nutraceutical androgens, the use of natural androgens, and pro-androgens, masking methods, restricting use to out-of-competition training or micro-dosing. Designer and nutraceutical androgens are typically non-marketed synthetic androgens based on structures and synthesis methods recovered from expired patent literature of the 1960-70’s. These are now synthesized by unregulated chemical manufacturers without Good Manufacturing Practice (GMP) licensing advertising and sold over the internet or over-the-counter as nutritional supplements, which may contain undeclared steroids (56). However, once the chemical structures of any synthetic androgens are known, they are easily detectable although the sheer profusion of such chemicals represents an ongoing challenge. Nevertheless, despite their novelty, there is little evidence designer androgens have been used after they are discovered so that there is a high likelihood of detection. As a result, virtually all ongoing androgen ADRVs are still due to conventional marketed synthetic androgens.

 

Distinguishing Between the Exogenous and Endogenous Steroids

 

Administration of natural androgens (T or DHT) or pro-androgens (androstenedione, DHEA), raises the problem of distinguishing between the exogenous and endogenous steroids. Exogenous T administration can be detected by the urine T/E ratio, the ratio in urine of T to its 17α-epimer epitestosterone (E), operating as a sensitive screening test. In males, both T and E are co-secreted by Leydig cells and excreted in urine consistently so that the urine T/E is usually stable for any individual over time, being typically around 1. Administration of exogenous T, which is not converted to E, increases the urine T/E ratio and, when it exceeds a specified threshold, is evidence for administration of exogenous T. The urine T/E ratio thresholds were originally population-based, set initially at 6 and then subsequently lowered to 4. However, the urine T/E ratio is not an effective screening test for testosterone doping in females (57)because, unlike males whose circulating testosterone originates from as single source subject to strong negative hypothalamic feedback, circulating testosterone in females originates from three steroidogenic sources (adrenal, ovary, extraglandular conversion) none of which are subject to strong negative feedback by testosterone. Furthermore, the possibility of false negatives and false positives of population-based thresholds are limitations which may require further analysis to confirm or refute T doping in individual cases. These considerations have led to establishment of the steroid module of the Athletes Biological Passport (ABP), a compendium of serial observation of any individual’s tests which creates adaptive individual-specific T/E ratio threshold (58). This substitution of an individual’s own person-specific, in place of the population-based, thresholds allows for more sensitive and accurate detection of individual deviations in urine T/E ratio as evidence of T doping.

 

One limitation of the urine T/E ratio is a genetic polymorphism of the uridine 5'-diphospho-glucuronosyltransferase(UGT) 2B17 gene which encodes a phase II hepatic enzyme that glucuronidates T rendering it more hydrophilic to facilitate urinary excretion. This polymorphism comprises a genetic deletion which, in homozygotes, produces a non-functional enzyme that reduces urinary T (but not E) excretion to near zero producing an extremely low T/E ratio (<0.1). While this genetic polymorphism has no apparent biological effect on T action, it is unevenly distributed geographically being much more frequent in South East Asian populations (59). This biological false negative means that administration of exogenous T will be greatly reduced and may not exceed the usual population-based T/E ratio thresholds (60). On the other hand, it will exceed any individual’s own specific urine T/E ratio threshold so that genotyping and/or Bayesian profiling of serial T/E ratio in an ABP program provide complementary evidence (61-63).

 

Administration of exogenous T may also be identified by carbon isotope ratio MS (CIRMS) that can distinguish endogenous from exogenous T according to the C13/C12 ratio of urinary T (64,65). Commercially, steroids are manufactured from plant sterols produced by photosynthesis that exhibit distinctly lower C13/C12 ratio (typically, -26‰ to -36‰ relative to the global standard) compared with mammalian T biosynthesis (between -16‰ to -26‰) (66,67). Hence, a significantly lowered (“depleted”) C13/C12 ratio of urinary T, exceeding 3‰ relative to endogenous reference steroids, indicates that urinary T originates at least partly from exogenous chemical manufacture from plant sterols. CIRMS can also be applied to detect administration of other natural androgens or pro-androgens including DHT and DHEA (68), androstenedione, or even attempted masking by administering E (to lower urine T/E ratio) (66). A few T products (<5% (69)) have recently emerged with a lower, more mammalian-like C13/C12 ratio for urine T (70) creating a challenge for CIRMS detection. Nevertheless, extended isotope profiling of other steroid precursors and metabolites provides additional complementary reference biomarkers (71). A longitudinal application of CIRMS along the lines of another module of the ABP has been proposed (72). Furthermore, development of hydrogen ion ratio mass spectrometry has further enhanced the ability to distinguish between endogenous and exogenous steroids even when the carbon isotope ratio is non-informative (73-75). Suppression of urine (or serum) LH excretion may also provide corroborative evidence for the use of exogenous T or other synthetic androgens (63,76-78).

 

While MS is highly effective for detecting specific androgens, it requires knowledge of the chemical structure to be detected and otherwise cannot be applied. This principle applies to never-marketed designer or nutraceutical androgens sold over the internet or in unregulated over-the-counter nutritional supplements with unlabeled steroid content. A potential solution is the modern in vitro androgen bioassay that incorporates the human androgen receptor together with a convenient transactivation chemical read-out signal into a host yeast or mammalian cell (79). This has the generic capacity to detect all bioactive androgens regardless of structure due to their direct activation of the androgen receptor. Constructed in vitro androgen bioassays feature a sensitive dose-response signal proportional to the potency of the bioactive androgen (80-83). Yeast host cells have high specificity for detecting androgens but are less sensitive than mammalian cells, which express native steroid mechanisms including steroidogenic enzymes and/or other steroid receptors. Mammalian in vitro androgen bioassays can also detect pro-androgens, steroids lacking intrinsic androgenic bioactivity but which are converted into androgens within the mammalian cell. Hence, while mammalian host cells sacrifice specificity for higher sensitivity, they can also detect pro-androgens (79). Hence yeast and mammalian in vitro androgen bioassays are complementary in detecting both androgens and pro-androgens. The limitations of in vitro androgen bioassays are their susceptibility to matrix effects and difficulties in standardizing bioassay-based test. Consequently. they are best deployed to characterize products and substances for androgens or pro-androgen content rather than to detect androgens in complex biological samples. Hence the yeast androgen bioassay was decisive in the first conviction for use of a designer androgen by proving that tetrahydrogestrinone (THG) was a potent androgen (84) and has also been used to screen synthetic progestins to show that, unlike the original androgen-derived progestins, the modern generation of progestins are not androgenic (85).

 

Additional underutilized options to detect androgen doping is the use of alternative biological matrices such as hair, skin or nails as well as saliva and exhaled breath (86). Hair has the advantages of minimally invasive sampling with simple, convenient storage and the potential for very long window of detection, according hair growth rates (87). MS-based methods have been reported to detect exogenous (88-102) and endogenous (91,103-106) androgens in human hair following long-term, but not single dose (107), exposure. However, hair analysis tests have yet to undergo the rigorous standardization and validation required to become acceptable anti-doping tests in their medicolegal context. Problems that remain to be fully overcome include matrix effects, low recovery and limited sensitivity as well as the impact of age, hair color, alopecia, and shaving or passive chemical (cosmetic) contamination of hair.  Additionally nails and skin could also provide analogous information on past androgen exposure with relatively long windows of detection but suitably rigorous tests are yet to be convincingly developed (108). Saliva sampling has also been considered (109,110) for anti-doping application analogous to the use of salivary cortisol measurement for diagnosis of hypercortisolism (111). While potentially applicable to xenobiotic drugs, salivary testosterone immunoassay is not sufficiently accurate (112) and is not suitable to detect testosterone doping because even microscopic blood contamination (e.g. gingivitis, chewing hard food, tooth brushing) produces anomalous high readings. The existence of these renders salivary testosterone testing for antidoping purposes as unreliable by providing opportunity for claims of false positive for any adverse findings. Exhaled breath testing has also been investigated for certain small molecular weight chemicals (113). In theory, androgen-induced gene expression in circulating leukocytes might provide an additional biomarker of androgen action if specific and reproducible signatures can be defined (114); however, as direct detection of androgens is feasible and preferable for proving an ADRV, a role for gene expression biomarkers of androgen action remains to be established for anti-doping.

 

Indirect Androgen Doping

 

This doping strategy aims to increase endogenous T production and thereby evades detection by routine screening tests for exogenous T such as urine T/E ratio or CIRMS. Urine hCG is detected by commercial hCG immunoassays using immunoassays specific for intact heterodimeric hCG (including its nicked variant) which, if positive by exceeding a detection threshold (>5 IU/L), requires confirmation by a second immunoassay for intact heterodimeric hCG which is required to prove hCG use. A highly sensitive LC-MS method to detect urine hCG (115) is more specific than immunoassays (116) and has a lower threshold for a positive result in male athletes (117). A key issue is to distinguish a positive hCG urine test, presumptively indicating hCG doping, from early trophoblastic tumor or immunoassay artefacts. As hCG doping is not effective in women and urine hCG screening can detect early pregnancy, an unwarranted privacy intrusion, hCG testing is restricted to male athletes (43). Although direct LH doping is an implausible doping threat (118), suppressed (63,76-78,118) or elevated urine LH may be useful for confirming direct or indirect androgen doping (42,43,76,119). Anti-estrogens (estrogen receptor antagonists) or aromatase inhibitors, which can cause reflex increases in serum and urine LH and testosterone (42), are detected by MS-based chemical detection methods.

 

Overall, detection of direct androgen doping is now so effective that in WADA-compliant elite competitions it is restricted to the ill-informed, often using counterfeit or unlabeled products (120). Yet the potency of androgen doping in power sports continues to prompt development of novel androgen doping strategies. These will include use of undocumented synthetic androgens, novel indirect androgen doping methods and micro-dosing of natural androgens during out of competition training. The retreat to using micro-dosing inherently reduces the dose-dependent ergogenic benefits of doping while maintaining the risk of detection and disqualification. There remains a need to maintain deterrence by effective detection methods for evolving new androgen doping threats.

 

HEMOGLOBIN (BLOOD) DOPING

 

Hemoglobin doping involves either direct blood transfusion or indirect methods of increasing hemoglobin via stimulating erythropoiesis by administration of erythropoietin, its analogs or mimetics (see excellent reviews (121,122)) (Table 4). Boosting hemoglobin is advantageous in aerobic, endurance sports such as road cycling, distance running and cross-country skiing. Maximal oxygen consumption (Vo2), a rate-limiting factor in aerobic exercise, principally determined by cardiac output and blood oxygen transfer with a lesser contribution from tissue oxygen transfer (123). Experiments on exercise tolerance and blood transfusion were first reported in 1945 (124,125)but the scientific basis of hemoglobin doping via enhanced tissue oxygen transfer was firmly established in 1972 by the work of Ekblom et al reporting experiments in healthy volunteers who underwent venesection and/or re-transfusion of 1, 2 or 3 units (400 mL) of blood with repeated testing of maximal exercise-induced oxygen consumption before and after each procedure (126). This proved unequivocally that the maximal oxygen consumption was highly correlated with acute changes in hemoglobin (figure 5). Subsequently, during the 1970-80’s before its banning in 1988, blood transfusion became a prevalent surreptitious practice in road cycling and cross-country skiing and the apparently low prevalence among distance runners may be an underestimate (127). Modelling of historical performance in European road cycling from 1993 onwards shows a unique progression averaging an improvement of 6.4% corresponding closely with the performance enhancement (6-7%) due to rhEPO administration, which is sustained for at least 4 weeks after administration (128-130).

Table 4. Direct and Indirect Hemoglobin Doping and Detection Tests

Doping Mechanism

Detection

Direct (Blood transfusion)

 

Heterologous

Flow cytometry: bimodal population of blood group antigens

Autologous

No direct detection tests.

Athletes Biological Passport Biomarkers:

Urine phthalate excretion

Total hemoglobin mass

Indirect (Erythropoiesis stimulation)

Direct

 

rhEpo & biosimilars (>100) Epo analogs

Urine double immunoblot, (LC-MS)

Indirect

 

Hypoxia altitude training, hypoxic sleep area

Not banned

Hypoxia-mimetics: hypoxia-inducible factor & stabilizers, iron chelation, cobalt, 2,3 DPG analogs

LC-MS/MS

Artificial O2 carriers: HbOC, perfluorocarbons

LC-MS/MS

 

Figure 5. Direct and Indirect Hemoglobin Doping

Blood Transfusion

 

Transfusion may involve either another person’s (homologous) or the athlete’s own (autologous) blood administered prior to a contest to acutely increase circulating hemoglobin. Homologous blood can be transfused at any convenient time to enhance performance in competition but when performed by untrained personal in non-clinical environments risks transfusion reaction, blood-borne infectious disease, and iron overload. By contrast, autologous transfusion reduces health risks but requires complex coordination as venesection itself is detrimental to performance, and it requires balancing recovery from blood withdrawal and loss of erythrocyte viability during long-term cryostorage with training and competition schedules. Although blood transfusion was first banned by the IOC in 1986, the first practical approach to banning blood doping was the introduction of hematocrit testing in 1997 by the international skiing and cycling federations. These regulations excluded athletes on health grounds from entering competition on the day if their hematocrit exceed a safety threshold (0.50). However, this encouraged hematocrit titration to just below threshold and only prevented competing until hematocrit returned under that threshold, which could be quickly accomplished by venesection. The first ADRV’s for blood manipulation involving hematocrit threshold and titration were in 2001.

 

Homologous blood transfusion creates a bimodal population of blood group antigens which is detectable by flow cytometry using a panel of 12 minor blood group antigens (131), from the wider array of blood group antigens (132), which can detect a <5% contamination of exogenous erythrocytes. Subsequent refinements simplified and improved test sensitivity so that a panel of 8 antigens can detect contamination comprising a minor admixture population of 0.3-2.0% with no false positives but high sensitivity (~80%), the latter depending on the magnitude of the minor contaminating mixture (133,134). Alternatives based on genotyping for the admixture population of leukocytes have also been proposed (135,136). As a test proving unequivocally the presence of non-endogenous erythrocytes in the circulation, this method is definitive if performed to the required standard. A remotely hypothetical defense against a positive test, based on stable marrow chimerism from a vanished twin, was raised by a cyclist who subsequently admitted transfusion (135). Based on risk of detection as well as to health risks, homologous transfusion has now largely disappeared in favor of autologous transfusion (137).

 

Autologous Transfusion

 

The biggest gap in current anti-doping tests is the lack of a specific test to detect autologous transfusion (138). Research to identify robust physico-chemical or biological markers for direct identification of a subpopulation of ex-vivo aged erythrocytes is underway using flow cytometry  (139) but the dilution and rapid clearance of effete erythrocytes make for challenging detection problems (140). In the interim, other indirect methods have been developed. These include measuring urinary excretion of phthalates, plasticizers that leach out from the polyvinylchloride blood packs used to store venesected blood (141). This test has brief window of detection (2 day) so will detect auto-transfusion during or immediately before events (characteristic in road cycling, according to convicted dopers) but may miss earlier auto-transfusion. Furthermore, the ubiquity of low-level environmental phthalate exposure requires establishing detection thresholds and non-plastic blood containers can be used. An alternative is the measurement of total hemoglobin mass (142), a measure with good stability and reproducibility even during exercise and circumvents influence of variations in plasma volume such as due to dehydration or dilutional masking (142,143). However, as this requires inhalation of carbon monoxide, which has transient detrimental effects on performance, it is not ideal for routine anti-doping use and its sensitivity may be insufficient to detect all EPO micro-dosing (144,145). Nevertheless, alternative methods for serial measurement of total hemoglobin mass remain attractive. Other hypothetical methods include the detection of microRNA (146) or immune reactions to transfusion (147) but the sensitivity and specificity of these proposed tests remains to be fully evaluated.

 

The best detection test for autologous hemoglobin doping at present is the hematological module of the ABP introduced in 2009 (148). Conceptually, it is a biomarker test which adopts a Bayesian approach of creating serially-adaptive, person-specific reference limits, based on using all prior testing, to supplant population-based thresholds. Combining all of an individual’s previously collected hematological data creates a probabilistic test of whether any new result deviates significantly from that individual’s personal reference limits (149). These person-specific thresholds allow for ongoing refinement and reinforcement by further testing. The thresholds are calculated by a variety of algorithms incorporating routine hematological parameters, notably hematocrit and reticulocyte counts. Those were developed over the last two decades to create the ABP hematological model which is sensitive to both direct and indirect hemoglobin doping (150). The first attempts to regulate hemoglobin doping in the late 1990’s sought to prevent road cyclists or cross-country skiing athletes competing on health risk grounds when their hematocrit exceeded pre-determined, population-based safety criteria (e.g. hematocrit 0.50 or hemoglobin 170 g/L for cycling). However, while this excluded extreme hemoglobin doping only until the short period when the safety threshold was no longer exceeded, it allowed an increase in an athlete’s natural hematocrit, typically averaging ~0.45, up to the permitted ceiling threshold which fostered titrated hemoglobin doping and manipulations like hemodilution by saline or plasma volume expander infusions to avoid detection (151). More sophisticated hematological algorithms were then developed to detect hemoglobin doping initially for the Sydney 2000 Olympics (152,153), the first generation of algorithms developing validated tests for ongoing and for recent cessation of hemoglobin doping, using a combination of biochemical variables related to erythropoiesis physiology. This approach was simplified by a second generation algorithm using only routine hematological parameters (hemoglobin, reticulocytes) (154), and was subsequently combined with the concept of a sequential development of individual-specific reference ranges (155)  into a third generation algorithms (156,157) which were refined for the ABP (148,149). The hematological module of the ABP currently employs an algorithm involving 8 parameters derived from routine hematological profile (hemoglobin, hematocrit, erythrocyte count, reticulocyte count and percentage, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration) (158). This is capable of detecting any form of hemoglobin doping, whether direct or indirect, with good but imperfect sensitivity (143-145) and using only routine hematological tests. The reported increasing use of very low EPO doses (“micro-dosing”) would markedly reduce the magnitude of any dose-dependent ergogenic benefits (145) while still carrying risks of detection, disqualification, and disgrace.

 

Stimulation of Erythropoiesis

 

Indirect methods to increase hemoglobin include administration of recombinant human EPO or its analogs as well as hypoxia-mimetic drugs (hypoxia-inducible factor stabilizers, iron chelation, cobalt, 2,3 diphosphoglycerate analogs) or artificial oxygen carriers (perfluorocarbons, hemoglobin-based oxygen carriers). Related but non-banned methods include altitude training or its simulation by sleeping in hypoxic rooms which are less effective than hemoglobin doping (129).

 

The identification of the human EPO gene in 1985 led to the marketing of recombinant human EPO (rhEPO) between 1987-9. Despite the IOC’s prohibition of EPO’s use in sports in 1990, the commercial availability of rhEPO created powerful new opportunities for indirect hemoglobin doping which were soon proven experimentally (159). A drug, which circulates for hours to days, but with potent and long-lasting ergogenic effects after its disappearance due to the 4-month lifespan of erythrocytes, is both attractive for doping and a challenge to anti-doping testing. Expiry of the rhEPO patent in 2004 allowed marketing of a profusion of generic EPO (“biosimilar”) products, estimated globally at over 80 (160), as well as modified EPO analogs (darbepoeitin, pegylated EPO, peginesatide, EPO fusion proteins). A fatal cluster involving deaths of 18 Dutch and Belgian road cyclists, presumably due inadvertent over-dosage during empirical attempts to maximize ergogenic effects of illicit rhEPO, was reported (161), although difficult to verify (162). A similar excess of unexpected deaths of road cyclists was also reported again in 2003-5 when novel EPO analogs and EPO biosimilars were marketed.

 

Detection of EPO in urine is difficult because of the prevailing low concentrations and need to distinguish exogenous recombinant from endogenous EPO. The first effective method for rhEPO in urine was a double immunoblot (163,164)which was capable of detecting urinary excretion of a variety of exogenous EPO products and analogs according to their differences in glycosylation side-chains, and differences in primary amino acid sequence where they exist, while distinguishing them from endogenous EPO. Although further refined (165) and extended to other EPO analogs (166), the immune-electrophoresis test is sensitive but relatively laborious and provides only a short window of detection of up to a week post-administration (167). More sensitive methods based on proteomics (for EPO analogs with differences in primary structure) together with glycomics (for biosimilars and analogs which have host-cell specific variations in side-chain glycosylation but unchanged natural EPO primary structure (168)) are possible but not yet approved. Additional applications to detect EPO and analogs using dried blood spots have been reported (169). Similarly, preliminary investigations have proposed a EPO-induced gene expression signatures as a biomarker to detect EPO administration but specificity relative to exercise and other physiological effects remain to be clarified (170).

 

Other EPO mimetics such as hypoxia mimetic drugs including hypoxia-inducible factor (HIF) stabilizers and related small molecules represent growing threats as potential indirect hemoglobin doping agents (171). These non-peptide chemicals interfere with various steps of the molecular oxygen sensing mechanism to mimic renal hypoxia and thereby induce EPO secretion resulting in increased blood hemoglobin. As a convenient orally active alternative to the lucrative pharmaceutical market for injectable erythropoiesis-stimulating peptides (~$7-8 billion (172)) to counteract anemias of chronic renal failure or marrow failure due to myeloproliferative disease or cytotoxic cancer therapy, they constitute a very active area of pre-clinical patent-based clinical drug development (171). Experience suggests that such innovator products can enter the doping black market before marketing approval (150,171). Despite the profusion of pre-clinical leads, they represent families of related chemical structures disclosed in patents for which LC and/or GC-MS detection tests should, in principle, be effective. Understanding the metabolism of these drugs when they come to market may identify long-lasting metabolites that can extend the windows of detection. Coupled with evidence from the ABP, manipulation of the EPO pathway may be detected in conjunction with corroborative measurement of inappropriately suppressed or elevated endogenous EPO for the prevailing hemoglobin level.

 

HIF is a key generic biological mechanism for tissue sensing of hypoxia and triggering local (neovascularization, angiogenesis) and systemic (EPO) defensive reactions. The promoter of the EPO gene contains enhancer and inhibitor regions with the hypoxia-responsive element which binds HIF and a GATA binding site which enhance and inhibit, respectively, EPO gene transcription. HIF is a heterodimer formed by constitutively expressed subunits with the β subunit in excess and availability of α subunit limiting formation of bioactive HIF. The 3 HIFα subunit isoforms are subject to hydroxylation of specific proline residues by prolyl hydroxylase enzymes which inactivate HIFα by ubiquitination, a tag which targets it to proteasomal degradation. HIFα subunit inactivation is strongly dependent on tissue oxygenation being active during normoxia but reduced during hypoxia when persistence of HIFα stabilizes the HIF heterodimer. Notably, during hypoxia the expression of HIFα in renal cortical cells stimulates EPO gene expression so that HIF stabilization by prolyl hydroxylase inhibitors leads to increased EPO secretion and circulating hemoglobin. Hence inhibiting prolyl hydroxylase activity via blocking its required cofactors (ascorbate, ketoglutarate, iron) using cobalt, nickel, iron chelation, ketoglutarate analogs or mechanism-based chemical inhibitors can result in increased hemoglobin via stimulation of EPO secretion (171). Similarly, small molecule GATA inhibitors potently stimulate circulating EPO, hemoglobin and performance in mice (173) although none have yet been marketed so their human efficacy and safety remain to be determined.

 

Another approach to increase oxygen delivery to muscle has been to exploit the ability of 2,3 diphophoglycerate (2,3 DPG), whose binding to hemoglobin reduces its affinity for oxygen with the left-shift of its oxygen dissociation curve as an oxygen unloading mechanism in tissues. 2,3 DPG analogs, developed as radiation sensitizers for hypoxic radio-resistant tumors, enhance tissue oxygen delivery in vivo (174,175) but would feature only short-term, acute effects readily detectable by mass spectrometry (176,177).

 

Adverse effects from use of rhEPO or its analogs are well known in medicine but poorly recognized in doping. They include immunogenicity (with risk of EPO autoantibody mediated pure red cell aplasia) (178,179), cardiovascular complications (including venous thromboembolism, stroke, hypertension and myocardial infarction) and premature death (180-183). In routine clinical use of EPO to correct renal anemia, the goal is a gradual increase to subnormal hemoglobin targets so that the excessive and/or rapid rises in hematocrit and blood viscosity (184) may explain the excess unexplained deaths among young European road cyclists in the late 1980s. In addition, use of rhEPO may deplete iron stores which limits hemoglobin synthesis so that athletes may also use oral or injectable iron supplements, which carry their own risks such as iv iron supplementation’s potentially adverse effects in enhanced tissue oxidative damage and excess mortality in chronic kidney disease (185). Although clinical safety experience with ESAs is restricted to patients with serious medical disorders, there is evidence from the general community that higher natural hematocrit is associated with worse long-term cardiovascular health outcomes (186-188).

 

GROWTH HORMONE

 

Growth hormone (GH) is a tissue growth promoter in children but after puberty it is predominantly a metabolic hormone although latent tissue growth promoting effects may be unleashed under non-physiological circumstances, such as during recovery from tissue injury. There is consistent anecdotal evidence that GH has been used in elite sports for decades (189). Nevertheless, ergogenic effects of GH remain unproven and largely speculative as discussed in excellent recent reviews (190-192). Claims of GH benefits in sport have included increases in muscle mass and strength, especially in conjunction with androgens, and/or improved tissue healing with more rapid recovery from either major injuries or minor repetitive injuries, such as from intense physical training allowing for more effective training. The biological basis of ergogenic effects of GH have been tested in these two different scenarios with largely inconclusive findings.

 

Evidence for direct enhancement of athletic performance by GH has been investigated in two well controlled RCTs with a primary focus on athletic performance. In one study, 96 recreational sub-elite athletes (63 male, 33 female, mean age 28 years) were administered 8 weeks of daily sc injections of GH or placebo with the men also having weekly im injections of T enanthate or saline placebo for the last 5 weeks (193). GH increased lean (muscle) mass (by +2.7 kg) and reduced fat mass (by -1.4 kg) while T increased lean mass (alone by +2.4 kg, by +5.8 kg with GH). The effects of GH were marginally significant for anaerobic sprint capacity (by +3.9%, p=0.05) when pooling male and female participants but this was due to significant effects in men only (by +5.5% alone and +8.3% with GH). However, there were no significant effects on maximal Vo2 consumption, dead lift, or jump height (193). A second study involved 30 healthy non-athletes (15 male, 15 female, mean age 25 years) who were administered daily sc injections of GH at high (4.6 mg/day) or low (2.3 mg/day) doses or placebo (194). There was no significant effect on muscle mass or maximal Vo2 consumption. Additional controlled studies of GH effects but with less focus on athletic performance have also shown that (a) a single dose of GH (~0.8 mg) in 9 recreational athletes did not affect maximal Vo2 or power output in repeated 30 min bursts of bicycle ergometry (195), (b) short term (6 days), low dose GH (~1.7 mg/day) treatment of 48 male androgen abusers withdrawn from androgens for 12 weeks significantly increased maximal Vo2more than placebo (196), (c) daily sc injections of a GH receptor antagonist (pegvisomant) or placebo for 16 days to 20 sedentary men did not change maximal Vo2 although time to exhaustion at 90% maximal Vo2 was reduced (197)and (d) 4 weeks of daily sc injections of GH (~5 mg/day) increased whole body protein synthesis (198), lipolysis and glucose uptake (199) with uncertain significance for athletic performance. Overall, these studies indicate that GH has, at most, a modest ergogenic effect in men only and through enhancing T effects. That is consistent with the fact that young women have markedly greater growth hormone secretion than young men so that growth hormone cannot explain the sex differences in athletic performance (200).

 

It is also claimed that GH may enhance injury healing, thereby facilitating more intensive training and/or recovery from muscle, connective tissue or bone injury, notably in contact sports. This claim is difficult to evaluate and no well controlled studies of recovery from sports injuries or tolerance of training intensity in elite athletes are reported. The most germane surrogate evidence available arises from investigations on the use of GH in recovery from injuries due to burns, fracture, or for wound healing. A recent Cochrane meta-analysis review of GH treatment effects on recovery from burns injury and healing of donor skin graft sites suggests that GH has a small benefit in skin healing with large burns and reduced hospital stay but there was no benefit in reducing mortality or scarring and adverse effects, notably hyperglycemia, were increased (201). In practice, the increased mortality due to administration of high dose GH in critical illness (202) has led to GH treatment not being widely adopted in clinical practice of treatment of burns. Similarly, the only well controlled study of GH effects on bone healing from fracture reported that, among over 400 patients with tibial fractures treated for up to 16 weeks with GH (1, 2 or 4 mg/day) or placebo, there was no benefit of GH for overall healing (203). Finally, while there are numerous experimental studies of GH or growth factors on wound healing in animal models a wide variety of findings are reported with detrimental, neutral, or beneficial effects but no well-controlled human studies are available. In summary, the available evidence for improved tissue repair or regeneration is minimal.

 

Important caveats on interpreting these few well designed studies are that the effects of higher GH and T doses, as used in doping, have not been studied so that more potent higher dose and/or interactive effects cannot be excluded in the absence of well controlled, high dose, placebo-controlled studies. Nevertheless, the hypothesis that high dose GH exposure would enhance muscular function is inconsistent with the experience of acromegaly in which patients experience much higher (25-100 times) growth hormone exposure than doses that can be ethically administered to healthy human volunteers (204), yet characteristically display muscular weakness rather than increased muscle size or strength (205). Anti-doping science history suggests that caution is required before rejecting evidence for claimed ergogenic effects without investigations replicating the pharmacological doses used.

 

Furthermore, safety analysis is not feasible based on the few, small, short-term studies of GH’s potential ergogenic effects; however, there are significant safety concerns about the long-term risk of cancer following GH administration. Even standard therapeutic GH doses administered to GH deficient children are associated with increased risk of second cancers in some (206-208) but not all (209) follow-up studies although these risks appear largely confined to survivors of childhood cancers and its treatment which render them GH deficient (210-213). Although the significant cancer risk based on uncontrolled observational cohort data using standard GH doses remains contentious (214,215), the long-term risks of much higher GH doses used illicitly by athletes must be viewed with significant concern.

 

Detection of GH doping remains difficult (216). A major challenge is the non-glycosylated primary structure of recombinant and endogenous 22 kDa GH, that lack the distinctive side-chain carbohydrate differences of exogenous glycoproteins EPO or hCG which provide a convenient basis for sensitive molecular detection tests. Nevertheless, minor infidelities in commercial manufacturing of GH may incorporate distinctive non-natural chemical features proving an exogenous origin (217-219) although these findings have not been developed into detection tests. Challenges to the detection of GH doping arise from the physiological pattern of endogenous GH secretion with its intermittent, pulsatile pattern subject to prominent influence of exercise, stress, and nutritional effects together with GH’s brief circulating half-life and low urine concentrations (220,221). Like other major doping classes, there are both direct and indirect forms of GH doping, involving either direct administration of GH or IGF-I or their analogs and indirect GH doping involving drugs that aim to increase endogenous GH and IGF-I secretion (Table 5).

 

Table 5. Growth Factors, Growth Hormone Related and Other Peptides

Growth Factors

Growth Hormone Related Peptides

Other Peptides

 

GHRH analogs

Ghrelin analogs

Other

 

FGFs

GHRH

Lenomorelin (ghrelin)

IGF-1 & analogs (MGF, long R3IGF-1)

Thymosinß4

HGF

CJC-1295

GHRP-1

IGF-2

 

MGF

CJC-1293

GHRP-2 (pralmorelin)

Insulin & analogs

 

PDGF

Sermorelin

GHRP-3

AOD-9604

 

VEGF

Tesamorelin

GHRP-4

hGH 176-191

 

 

 

GHRP-5

 

 

 

 

GHRP-6

 

 

 

 

Hexarelin

 

 

 

 

Ipamorelin

 

 

 

 

Alexamorelin

 

 

 

 

Anamorelin

 

 

 

 

Macimorelin

 

 

 

 

Tabimorelin

 

 

 

 

Examorelin

 

 

 

The first test to detect administration of exogenous GH, the 22kD recombinant form of human GH, was based on blood sampling to measure the ratio of circulating isoforms of GH recognizing the fact that the pituitary secretes not only the major 22 kD isoform (65-80%) but also a variety of minor isoforms including a wide variety of minor isoforms and their multimeric variants (222). Administration of exogenous GH suppresses endogenous pituitary GH secretion leading to a predominance of circulating 22 kD GH. This is the basis for the GH isoform ratio test whereby a serum sample is measured by two different GH immunoassays, one with predominant 22 kD GH specificity (“rec” assay) and the other recognizing the broad spectrum of pituitary GH isoforms (“pit” assay) and the ratio of results (“rec”/”pit” ratio) is an index to detect administration of exogenous recombinant GH (220,223). This ratio test then serves to detect administration of exogenous recombinant human 22kD GH analogous to detection of exogenous T by the urine T/E ratio and exogenous insulin by analysis of serum C peptide (224). The differential GH isoform ratio test has undergone extensive validation involving standardization of the two GH immunoassays with distinctive immunoreactivities to quantify 20kD and 22kD epitopes as well as its application to various populations of elite athletes and evaluating physiological factors which might impact on the validity of test read-out. A strength of this test is that it is aimed at the exogenous doping agent itself, although it cannot definitively distinguish it from its endogenous counterpart. The major limitations of this differential isotope ratio test are its narrow window of detection (24-36 hr post administration) and its inability to detect indirect GH doping. While pituitary-derived human GH might not be detected, human pituitary GH, once obtained from national scale pituitary collection and purification programs, has not been available since 1985 when its risks of Creutzfeldt-Jakob disease were identified (225,226) with recombinant human GH replacing pituitary-extracted GH worldwide. This differential isoform test was first introduced for the 2004 Olympics (227) and led in 2010 to the first successful detection of out of competition GH doping (228).

 

A complementary detection test with a wider window of detection has been developed based on biomarkers of GH action. This uses two serum biomarkers of tissue GH effects, circulating IGF-1 as a short-term marker of hepatic GH action, and N-terminal peptide of procollagen type III (PIII-NP) as a long-term marker of GH-dependent collagen synthesis. In a study of 102 recreational athletes (53 male, 49 female, mean age 25 years, from 4 different European cities) randomly assigned to self-inject 2.7 mg or 5.4 mg GH or placebo once daily, measurement of serum IGF-1 and PIII-NP by specific immunoassays were able to correctly classify 86% of samples from males and 60% of samples from female using an empirical linear discriminant analysis of log-transformed serum IGF-1 and PIII-NP at the specificity of 1:10,000 required for a WADA biomarker threshold (229). Subsequent studies have shown that additional collagen biomarkers, N-terminal propeptide and C-terminal telopeptide of type I collagen, further widen the window of detection for GH administration (230,231). This multiplex biomarker test, based on using standardized immunoassay antibodies, requires establishment of reliable reference range with specificity (false positive detection rate) of no more than 1:10,000 incorporating the impact of gender and age, although exercise, injury, ethnicity and sports type appear not to be confounding influences but is not yet in routine use by WADA anti-doping labs. The two GH doping test, the differential isoform and biomarker approaches, are considered ultimately complementary (232).

 

IGF and Insulin Doping

 

IGF-1 is a circulating marker of hepatic GH effects and mediator of GH action so the marketing in 2005 of recombinant human IGF-I alone, and later with its major binding protein recombinant human IGF binding protein 3 (IGF-BP3) (233), for treatment of diabetes, insulin or GH insensitivity or motor neuron disease, together with the availability of IGF-1 analogs for laboratory use, creates the possibility of IGF doping (234). Time-series analysis of elite sports performance (235) is consistent with the occurrence of IGF-1 doping but its prevalence is unknown (56). As the biological basis for ergogenic effects of IGFs is due to its GH-like effects, this remains largely speculative and accompanied by the same safety concerns. IGF-1, IGF-2 and their analogs (236) as well as insulin and its analogs (237) are all readily detectable by LC-tandem MS and preliminary evidence suggests that biomarkers for IGF-1 administration (IGF-2, IGFBP2) may widen the window of detection (238). However, a specific test to detect IGF doping remains to be established (239).

 

MGF is a splice variant of IGF-I which, although not known to appear in the circulation, have any pharmacological effects, or be approved for human use (240), is advertised on the black-market and internet (241) for alleged anabolic or tissue repair/regeneration benefits. Like other short peptide with known structure, it is readily detectable using LC- tandem MS (241).

 

Insulin has long been used in doping and was prohibited in sports since 1999 (242,243). Other than its proper medical use in diabetics, the use of insulin and its analogs for doping is based solely on its easy availability coupled with anecdotal information from other drug users. There are no clinical studies showing any ergogenic effects of insulin or its analogs in non-diabetic individuals. The doping folklore appears to arise from the classification of insulin as being “anabolic”, in a loose generalization and mistaken analogy to androgens. In healthy non-diabetic individuals, insulin and its synthetic analogs stimulate weight gain via hypoglycemia and increasing appetite, but produce fat rather than muscle gain. The adverse effects include hypoglycemia, hypokalemia, injection-related infections and weight (fat) gain. Doping detection tests for insulin and its analogs continue to evolve and focus on highly sensitive and specific quantitative MS-based proteomics (244).

 

Growth Factors, GH Releasing and Other Peptides

 

For the unscrupulous in pursuit of the unlawful, the increasingly stringent detection of androgen and hemoglobin doping, the two most potent classes of ergogenic drugs, has led to new, highly speculative form of doping involving peptide growth factors and GH releasing peptides. These are within the size range of automated bulk custom peptide synthesis and are marketed cheaply by chemical manufacturers. While notionally sold solely for laboratory research, these unregulated products are available for purchase over the internet. Promoted by speculative fantasies on their mode of action coupled with testimonials to their efficacy but without objective testing or assurance of safety in humans, they are believed to be widely used by gullible and/or desperate athletes and their trainers. As unregistered drugs, this growing range of peptides appears to constitute a greater threat to athlete’s health than a risk of effective cheating.

 

The S2 category of Prohibited Substances lists, in addition to GH and IGF-1, GH fragments and releasing peptides, a wide array of growth factors and modulators and, crucially, a generic catch-all provision for unnamed growth factors and peptides which may affect connective, vascular, muscular, or regenerative tissues or energy utilization and other substances with similar chemical structure or biological effects.

 

The major category of oligopeptides used for doping is the class of GH releasing peptides analogs of the endogenous GH releasing peptides, GHRH and ghrelin, whereby their analogs aim to increase endogenous GH secretion and are therefore banned (table 5). Most of these peptide were developed in the pharmaceutical industry from the 1990s aiming to provide cheaper, orally active, non-peptide agonists with capacity for sustained stimulation of endogenous GH secretion to “rejuvenate the GH/IGF-1 axis” (245), an unusually explicit acknowledgment of the regular nexus between hormonal rejuvenation and doping (246). However, none of these hormonal peptides have been registered for human therapeutic use with only one (pralmorelin) registered for single-dose, diagnostic use (for GH deficiency) in Japan and unacetylated cyclin ghrelin marketed in Europe for Prader-Willi syndrome. Although they may stimulate GH release initially, many failed to achieve sustained GH release due to desensitization and none achieved meaningful clinical improvements in any target diseases. If their unproven ergogenic benefits are due to sustained GH release this renders them unlikely to be beneficial; nevertheless, the caveat on not accepting negative conclusions without direct testing are also relevant to this class of peptides. Like other short peptides, once chemical structures are known, detection is readily feasible using LC-MS (247,248). The illicit nature of this market raises the risks of counterfeit and unsafe products with attendant risks of infection and residual toxic contaminants unlike the purity pharmaceutical product manufacturers are required to demonstrate by batch release testing.

 

PROGRESS, GAPS, AND FUTURE PROSPECTS

 

Anti-doping science continues to make major progress over recent decades especially since the advent of WADA with its harmonization and focus on deterrence through standardized testing. Progress from improved MS-based testing methodologies and instrumentation, summarized annually by the editor (M Thevis) of the major antidoping science journal, Drug Testing and Analysis (249), is evident from the increasing numbers of ADRV findings among frozen stored urine samples now banked for 10 years from previous Olympics. Like any efforts to combat human malfeasance, the quest for drug-free and safe sport requires ongoing vigilance and continual renewal of intelligence-based detection testing. While great progress has been made in the two canonical forms of doping, androgen and hemoglobin doping, human ingenuity continually finds way to challenge the testing just as traditional frauds are supplanted by cyber-crime and ingenious computer hacking. It is important to bear in mind that the winning margin (defined as the difference in performance between gold and silver medals, getting a medal or not, making a final or not in the Olympic athletic or swimming events) is <1% (200) so even small systematic advantages may be important motives and unfair advantages for doping.

 

The major gaps remaining in anti-doping science are (a) the lack of a definitive test for autologous blood transfusion, (b) need for more sensitive detection tests for peptide doping with wider windows of detection and (c) more economical, affordable and robust sample handling and storage procedures including dried blood spot sampling. These challenges must be met by adapting novel technologies such as quantitative proteomics, genomics, and metabolomics as well as implementing more out of competition and blood testing. Such progress depends on innovative applied research which is supported by WADA, Partnership for Clean Competition and certain national anti-doping organizations together with regular peer-review research granting agencies. Finally, the development of effective forensic intelligence investigations, a slow, complex and costly process but which can have salutary effects (e.g. for road cycling in the Lance Armstrong case), is proving a valuable complementary approach as an adjunct to effective laboratory testing.

 

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Laboratory Assessment of Testicular Function

ABSTRACT

 

Since the symptoms of hypogonadism are nonspecific, and the signs of testosterone deficiency can be subtle and slow to develop, the assessment of testicular function relies heavily on laboratory testing. The laboratory diagnosis of hypogonadism is based on a consistent and unequivocally low serum total testosterone level measured in blood samples obtained in the early morning, but normal ranges vary with different methods and among laboratories. Moreover, many men who present with adult onset testosterone deficiency have a low level of sex hormone-binding globulin (SHBG) associated with obesity, insulin resistance, and type 2 diabetes. In these men, tests for the free (or non-SHBG/bioavailable) testosterone fraction testosterone) are helpful for an accurate diagnosis. If testosterone deficiency is confirmed, the next step is to differentiate between primary and secondary hypogonadism by measuring LH and FSH.  With many disorders, however, both the testes and the hypothalamic-pituitary unit are affected. Other tests such as estradiol, inhibin-B, and Mullerian inhibitory hormone, and provocative endocrine tests using hCG, GnRH or its analogs, or antiandrogens or antiestrogens, and semen analysis in the subfertile male are discussed.   

 

INTRODUCTION

 

The evaluation of men for suspected hypogonadism begins with a detailed medical history and a careful physical examination. Laboratory tests are an essential component of almost all evaluations, and proper interpretation of the results obtained requires an understanding of methodology as well as an awareness of the impact of endocrine rhythms, age, race, body composition, drug exposure, co-morbidities, other illnesses, and nutrition on tests of endocrine testicular function. Endocrinologists rely on clinical laboratories to provide accurate and precise, and in some cases highly sensitive assays for accurate diagnoses. We expect the reference ranges to be based on large normal control populations, and for some assays, normative data must be stratified by age. Unfortunately, these expectations are not always fulfilled. Endocrinologists should examine the protocols for each assay, and discuss them with the laboratory director. In recognition of these potential shortcomings, the U.S. Endocrine Society has spearheaded efforts towards standardizing the methods by which testosterone assays are validated (1), and several societies have provided guidelines and recommendations to physicians who order and interpret the results of androgen assays (2-4). The College of American Pathologists and the U.S. Centers for Disease Control havevoluntary Hormone Standardization Programs to help clinical, research, and public health laboratories maintain and enhance the quality and comparability of their results.

 

TESTOSTERONE

 

Testosterone, the major androgen in men, is necessary for fetal male sexual differentiation, pubertal development, and the maintenance of adult secondary sex characteristics and spermatogenesis. Testosterone regulates gene expression in most extra-genital tissues, including muscle and bone, and modulates the immune system. The testes are the source of more than 95% of the circulating testosterone in men although the adrenal cortex produces large amounts of the testosterone precursor steroids, dehydroepiandrosterone (DHEA) and androstenedione.

 

Following birth, there is a period of activation of the gonadotropin-releasing hormone (GnRH) pulse generator that stimulates testosterone secretion to peak levels of 200-300 ng/dl (7-10.5 nmol/L) at ages 1-2 months and lasts until age 4-6 months, and is often called mini-puberty (5). Thereafter, GnRH secretion declines, causing a fall in testosterone to very low levels until puberty begins.  LH and testosterone testing at mini-puberty provides an opportunity to confirm the diagnosis of hypogonadotropic hypogonadism (6).

 

In early puberty, LH and testosterone secretion increase dramatically during sleep which gradually transitions to sustained secretion throughout the day and night (7). The nocturnal rise in testosterone levels in early puberty can be used clinically to evaluate boys with delayed puberty because it may precede pubertal testis growth, and indicates that puberty has begun.  Figure 1 illustrates age-specific median values and ranges for testosterone in morning blood samples from 138 boys (8).  

Figure 1. Serum testosterone levels (median and range) by LC-MS in leftover samples from blood checks before minor surgery or for the exclusion of endocrine diseases. Data from Kulle et al (8). To convert total testosterone levels to nmol/L, multiply by 0.0347

Hypothalamic neurons that express kisspeptin, neurokinin-B, and dynorphin, are thought to activate and up-regulate GnRH and initiate pubertal development (9).  Adult testosterone levels are usually achieved by age 16 years, and generally range from 300-1000 ng/dL (10-35 nmol/L). 

 

The secretion of testosterone is periodic with oscillations occurring over hours, days, and months. Frequent sampling of peripheral blood in adult men reveals small frequent moment-to-moment fluctuations (10) whereas spermatic venous blood sampling reveals robust episodes of testosterone secretion occurring about once per hour (11).  Presumably because of this rapid pulse frequency and a plasma half-life of 60-90 min, only small testosterone fluctuations are generally observed in peripheral blood. Therefore. a single blood sample is usually an adequate assessment of testosterone production on a given day.  There is a substantial diurnal variation in testosterone levels in adult men, however, with highest levels in the early morning, followed by a progressive decline throughout the day, reaching lowest levels in the evening (Figure 2). Thus, the time of day of blood sampling is an important consideration, and a blood sample drawn in the morning between 0800 and 1000h is recommended because reference ranges are generally based on morning values. Nadir evening levels  in young men are generally 15-20% lower than morning values although the differences can be as great as 50% (12). The diurnal testosterone rhythm is blunted as men grow older (13) as well as in young men with primary testicular failure. On the other hand, the diurnal variation in testosterone is exaggerated in hypogonadal men with hyperprolactinemia (14), much like normal adolescents. There may be a seasonable variation in testosterone as well (15).

Figure 2. 24-hour testosterone concentration profiles in eugonadal men (n=5), untreated hypogonadal men with PRL-producing pituitary adenomas, and men with testicular failure (n=5). Adapted from Winters, SJ (14). To convert total testosterone levels to nmol/L, multiply by 0.0347.

There is also a slight decline in testosterone levels following a meal (16), so that some authors suggest obtaining a fasting level.  In general, the results of one abnormal test should be confirmed. In one study in which multiple blood samples were taken from middle-aged men over one year, there was a relatively good correlation (r=0.85) between the plasma testosterone level in the first sample and the mean value of seven subsequent samples; however, as many as 15% of men were misclassified, with either low or normal values that were not reproducible (17).

 

Testosterone deficiency can be an obvious clinical diagnosis, and laboratory tests are often merely confirmatory; however, the diagnosis of hypogonadism is sometimes less straightforward. Experts and international guidelines state that the diagnosis of hypogonadism requires the presence of symptoms of hypogonadism as well as a low serum testosterone level. Symptoms of hypogonadism lack specificity, however, and are subjective. Thus, the diagnosis by laboratory testing may be more objective and less controversial. On the other hand, as discussed below, laboratory test results may be inconsistent and suffer from methodological flaws.  While men with low testosterone levels tend to have symptoms of hypogonadism (18), many studies have shown that symptoms are often unrelated to testosterone levels, and many men with low testosterone levels do not complain of symptoms (19, 20).

 

The level of total testosterone in serum is the best single test to screen for hypogonadism because methodology has been optimized and normative data are widely available.  While early assays extracted plasma steroids into organic solvents and separated them by column chromatography, those research methods generated radioactive waste and are too costly for clinical purposes.  Currently most hospital laboratories use automated platforms with either a competitive or proportional two-site format. These platforms use small sample volumes to avoid matrix effects, and a monoclonal antibody with a chemi-luminescent label for detection. The accuracy of the result is highly dependent on the affinity and specificity of the antibody used, and the survey result summarized in Figure 3 demonstrates that platform immunoassays continue to provide variable results with a coefficient of variation (SD/mean) among methods of 12% at a potency of 256 ng/dL which is close to the lower limit of the usual reference range for normal men. Nevertheless, when the manufacturer provides a reference range which is based on a large number of samples, and results are age-adjusted, automated assays are probably sufficient for most clinical purposes in adult men. Assay modifications have reduced the impact of hemolysis, icterus, and lipemia on measured results.

Figure 3. Total testosterone levels (±SD) in a proficiency sample (Y-04) distributed by the College of American Pathologists, and assayed using 14 different instruments in 1556 participating laboratories in 2018. The mean (±SD) value was 256 ± 51 ng/dL. The most frequent method (#6) was used by 353 laboratories. Method #4 is mass spectrometry, and was 7% lower than the mean of all methods. To convert total testosterone levels to nmol/L multiply by 0.0347

There is a positive bias at low values in most immunoassays so that automated assays are not recommended for clinical purposes in women and children. Reference laboratories are increasingly employing liquid chromatography (LC) - tandem mass spectrometry (MS) methods (21, 22). This approach combines the resolution of chromatography with the specificity of MS, and is now viewed as a gold standard.  Larger aliquots of serum are extracted with organic solvents, steroid hormones are separated by chromatography, in some assays following derivatization, and values are determined by peak area integration of testosterone-containing fractions of the column eluate. Accuracy is gained by adding an internal standard as a stable isotope of the same compound being measured in order to correct for procedural losses.  LC-MS accurately detects testosterone concentrations as low as 1.0 ng/dL (23, 24). In the 2018 CAP survey (Figure 3), the coefficient of variation for the high range sample (419 ng/dL) was 10%, and was a very respectable 11% for both the mid- range (245 ng/dL) and low-range (72 ng/dL) samples among those laboratories (n=35) that used LC-MS methodology. The equipment needed is costly, however, and each LC-MS assay requires substantial analytical development and optimization, as well as highly trained personnel.

 

While one study of men ages 40-80 who self-reported good or excellent health found no age-related change in total testosterone levels (25),  most cross-sectional and longitudinal studies have found that testosterone levels peak in the third decade, and decline as men grow older (e.g. (26, 27). Therefore, most reference laboratories present results for testosterone as a function of age. Small differences based on race and ethnicity have been found (28). One reference range for total testosterone levels in normal volunteers assayed by high-turbulence flow liquid chromatography(HTLC)-MS (22) is shown in Table 1. The decline in total testosterone in this study was roughly 0.4% per year beyond age 20, and mean T levels were 31% lower in men older than age 80 compared to men in their 20’s

 

Table 1. Impact of Age on Total Testosterone by HPLC–MS/MS in Normal Men  

 Age (years)

20-29

30-39

40-49

50-59

60-69

70-79

>80

Mean (ng/dl)

590

546

573

534

559

417

404

SD

232

206

209

194

226

177

234

 95th percentile

1052

910

901

909

928

755

716

5th percentile

283

319

310

296

290

168

92

N

61

76

55

51

19

44

19

Results from Quest Diagnostics, San Juan Capistrano, CA, courtesy of M.P. Caulfield. To convert total testosterone levels to nmol/L multiply by 0.0347

 

Table 2 shows a second reference range with combined values from the Framingham Heart Study analyzed by LC-MS, and the European Male Aging Study and Osteoporotic Fractures in Men Study analyzed by GS-MS (29). Mean levels for men in their 80s were 20% lower than for men in their 20s representing a similar decrease of 0.33% per year. Overall values are, however, 20-40% higher than from the reference laboratory in Table 1. Thus age-dependent reference ranges are method-specific, and no generalizable “cut-point” for the diagnosis of testosterone deficiency seems possible.

 

Table 2. Impact of Age on Total Testosterone Levels Measured by LC-MS or GC-MS) from Bhasin et al (29)

Age (years)

20-29

30-39

40-49

50-59

60-69

70-79

80+

Mean (ng/dl)

713

656

617

611

569

567

570

95thpercentile

1104

1084

1001

1059

965

1097

1079

5th percentile

375

343

304

286

276

254

238

N

220

660

872

788

493

289

26

 To convert total testosterone levels to nmol/L multiply by 0.0347

 

SEX HORMONE BINDING GLOBULIN (SHBG)

 

Sex hormone-binding globulin (SHBG) is a glycoprotein of molecular weight 90-100,000 KDa that is produced by the liver. SHBG binds testosterone and other steroids, prolongs their metabolic clearance, and regulates their access to target tissues (24). SHBG had been measured indirectly by radio-ligand binding assays, but two site immuno-radiometric and enzyme-linked assay kits are now widely available, and automated versions have been developed. There is little effect of meals or time-of-day on SHBG

Figure 4. SHBG levels from birth to old age in males and females. Redrawn from Elmlinger et al (30).

A cross-sectional analysis of the impact of age on SHBG levels in males and females is shown in Figure 4. There is an increase following birth to peak values at ages 4-5 years followed by a decline that is greater in males than females (30). SHBG levels in adult male plasma range from 20-100 nmol/L, and rise slightly in old age (28); analysis by race revealed higher levels among younger AA males (31).

 

In normal men, SHBG binds 40-60% of the circulating testosterone with high affinity, and the level of SHBG is a major determinant of the total testosterone level (Figure 5). Single nucleotide polymorphisms in the SHBG gene influence the affinity of SHBG for testosterone and affect the level in plasma (32).

 

Figure 5. Levels of SHBG and total testosterone in 28 normal men whose BMI ranged from 23-40 kg/m2. From Winters SJ et al (33).

Various clinical conditions are associated with reduced or increased SHBG levels, and thereby tend to lower or increase total testosterone concentrations (Table 3).  High SHBG levels in men with HepC or hyperthyroidism may lead to very high total testosterone levels, and can be a diagnostic dilemma. Serum SHBG levels are low in obesity (34), in T2DM or the Metabolic Syndrome (MetS) (35). In fact, low SHBG is frequently used as a biomarker in epidemiology studies to predict the development of the MetS as well as type 2 diabetes mellitus (36).  SHBG levels rise with weight loss (37) and when insulin resistance improves even without weight loss, e.g. with exercise. The molecular mechanism linking low levels of SHBG with insulin resistance and obesity is partly through hyperinsulinemia (38, 39) and through liver fat and cytokines which together regulate the transcription factor HNF4 alpha, a proximate activator of the SHBG promoter (40, 41) and at least 50% of genes expressed in liver.

 

Table 3. Factors that Influence the Level of SHBG in Plasma

Increase

Decrease

hyperthyroidism

hypothyroidism

hepatitis C

insulin resistance/NAFLD

GH deficiency

growth hormone excess

alcoholic cirrhosis

glucocorticoids

acute intermittent porphyria

androgens

first generation anticonvulsants

progestins

estrogens, certain SERMS

nephrotic syndrome

mitotane

genetic polymorphisms

thinness

obesity

genetic polymorphisms

 

 

FREE AND NON-SHBG (BIOAVAILABLE) TESTOSTERONE

 

The free hormone hypothesis proposes that the biological activity of a hormone is engendered by its unbound (free) but not its high affinity protein-bound concentration in plasma. The hypothesis remains controversial, and was recently reviewed for testosterone (42).

 

While some authors propose that there is little extra benefit in measuring free testosterone, my view is that the total testosterone level is sometimes inadequate to determine whether testosterone deficiency or excess is present. This occurs with a borderline value, or when the clinical findings and the total testosterone concentration do not agree. I encounter this conundrum most often in obese men, those with type 2 diabetes, NAFLD, or the Metabolic Syndrome among whom total testosterone levels are generally low, and who present because they have symptoms consistent with hypogonadism. In the Boston Area Community Health (BACH) Survey, 57% of men younger than age 50 with low total testosterone levels reported “normal” sexual function compared to 33% of those with low free testosterone levels (19). Likewise, the European Male Aging Study found that men age 40-79 with low free but normal total testosterone levels more often had symptoms and signs of sexual dysfunction while men with low total but normal free testosterone concentrations were more obese but had physical or sexual symptom scores that were similar to men with normal values (20). Thus, free testosterone seemed to be more closely aligned with the symptoms and signs of adult hypogonadism than was the total testosterone level.

 

In older men, on the other hand, the total testosterone level may be within the normal range while free testosterones decline more dramatically. The total testosterone level may also be elevated, as in men with hyperthyroidism, hepatitis-C, and patients treated with first generation anticonvulsants. Each of these situations occurs primarily because the primary condition is associated with a decrease or increase in the circulating SHBG level. 

 

Therefore, an assessment of free or non-SHBG bound testosterone may assist in the accurate diagnosis of androgen deficiency. This approach stems from the hypothesis that the bioactivity of circulating testosterone is due to the small percentage (1-4%) of total testosterone that circulates unbound (free testosterone) as well as the 40-50% of testosterone that circulates loosely bound to albumin and is often designated as “bioavailable-testosterone” (non-SHBG testosterone). The dissociation of albumin-bound testosterone is very rapid, and a short dissociation time is thought to allow the albumin-bound fraction to be available for uptake by cells (43). However, there is considerable debate as to which method for estimating free testosterone most accurately reflects testosterone production (42, 44).

 

Equilibrium dialysis is the benchmark reference method for measuring free testosterone. With the two-step approach, the free testosterone concentration is calculated from the product of the total testosterone level and the percentage that is determined to be free. H3-testosterone is added to the serum sample, and is allowed to partition between two compartments, one containing the tracer and serum sample, and the second containing buffer or an albumin solution. The compartments are separated by a semi-permeable membrane with a low-molecular weight cut-off. The protein-bound testosterone is retained while unbound testosterone, including H3-testosterone, crosses the membrane. It may take several hours to reach equilibrium, and using centrifugal ultrafiltration accelerates dialysis by forcing the unbound steroid thorough the membrane. Overall, the result is dependent on the accuracy of the total testosterone assay, and there is potential error due to temperature effects, sample dilution, and tracer impurities, among other problems. This assay is limited to reference laboratories.

Liquid chromatograph tandem mass spectrometry (LC-MS) can reliably measure hormone concentrations as low 1 pg/mL, and has been adapted for the measurement of free testosterone.  With this approach, equilibrium dialysis of undiluted serum is performed against buffer overnight at 37C, or more rapidly using ultracentrifugation cartridges, and (free) testosterone  is measured directly in the low molecular weight dialysate by LC-MS (45). Many reference laboratories have embraced this approach but it remains too complex and costly for most hospital laboratories.  

 

A published reference range using the two-step equilibrium dialysis method (Table 4) reveals that free testosterone levels decline as men grow older with a difference of 36% between the youngest and oldest reference groups. Thus, the percentage change in free testosterone by this method with age (0.6%/year) exceeds the rate of decline in total testosterone (0.3-0.4%/year).

 

Table 4. Reference for Free Testosterone by Equilibrium Dialysis in Normal Men

 Age (years)

20-29

30-39

40-49

50-59

60-69

70-79

>80

Mean (pg/ml)

102

86

74

84

84

80

65

SD

29

33

22

30

22

27

26

95.00%

148

144

114

136

111

131

101

5.00%

57

45

37

35

55

47

34

N

49

55

48

36

14

36

17

Results from Quest Diagnostics, San Juan Capistrano, CA, courtesy of M.P. Caulfield. 3H-testosterone was added to a sample diluted 1:5 in assay buffer and incubated in a dialysis chamber for 20 h at 37 °C to allow the tracer to reach equilibrium with the endogenous testosterone and binding proteins. To convert total testosterone levels to nmol/L multiply by 0.0347.

 

Calculated Free Testosterone

 

A technically simpler approach used by many hospital laboratories is to calculate the free testosterone level from the levels of total testosterone and SHBG, using experimentally determined testosterone binding constants. The KD for testosterone for SHBG is most often defined as 1 x 109 L/M, and for albumin 3 x 104 L/M.  These are estimates, however, and different constants will produce different results (46). Differences in the level of albumin in the sample have little impact on the calculated free testosterone, and are usually ignored. Free and non-SHBG bound testosterone levels can be computed using an internet program (www.issam.ch/freetesto.htm). There is an excellent correlation between the level of free testosterone obtained by equilibrium dialysis and the calculated free testosterone level (47), and between the calculated free testosterone and non-SHBG testosterone levels. Calculated free testosterone concentrations vary with the binding constants and algorithms employed, however, and SNPs of the SHBG gene may influence the affinity of SHBG for testosterone (32), and thereby the accuracy of the calculated free testosterone level. It has also been suggested that ligand binding to the SHBG dimer is allosteric such that occupancy of one site by a ligand alters the affinity of the second site. Furthermore, because the result is calculated from the levels of testosterone and SHBG, measurement error for either assay impacts directly on the calculated free and non-SHBG testosterone levels. A reference range (21) for free testosterone based on calculation revealed a decline of 61% from young to old adult men (Table 5), representing a rate of decline of 1%/yr.  In a second study (48) the difference between young and old was only 26%, but again far exceeded the fall in total testosterone with aging which was 1.2%.

 

Table 5. Influence of Age on Free Testosterone Levels in Community Men

Age (years)

20-29

30-39

 

40-49

 

50-59

 

60-69

 

70-79

 

>80

 

Mean (pg/ml)

148

132

116

99

82

72

58

5th Percentile

79

70

61

50

45

33

29

95thPercentile

229

212

199

164

135

132

93

n

220

660

872

788

493

289

26

Total testosterone was measured in community dwelling men in Framingham, MA using LC-MS.  SHBG was measured using a two-site immunofluorometric assay from DELFIA-Wallac, Inc. Free testosterone was calculated by the law-of-mass-action equation using of K SHBG-T of 0.998 × 109 L/mol and a K Alb-T of 3.57 × 104 L/mol. Data from Bhasin et al (29). To convert total testosterone levels to nmol/L multiply by 0.0347.

 

Bioavailable Testosterone

 

Non-SHBG-testosterone is called "bio-available” (BAT) because adding SHBG to an androgen-containing sample reduces its androgen receptor binding activity (49). This approach assumes that the testosterone bound with low affinity to albumin is active. The non-SHBG-testosterone (bioavailable) level can be determined by adding a tracer amount of 3H-testosterone to the serum sample, and selectively precipitating the SHBG-bound 3H-testosterone by adding 50% ammonium sulfate or concanavalin-A Sepharose. The 3H-testosterone that remains in the supernatant is presumed to be either free or albumin-bound, and is counted. The percentage of 3H-counts added that is in the supernatant is multiplied by the total testosterone level in order to determine the non-SHBG (bioavailable) testosterone. The assay is not readily automated, requires purified 3H-testosterone, and the complete separation of SHBG from albumin is presumed, but not verified.

 

Free Testosterone Index

 

The free testosterone index (FAI, free androgen index) represents the ratio: total testosterone/SHBG (both in units of nmol/L).  This value is easy to calculate, and may be valid in serum samples from women but is not valid in men (50)because most of the SHBG in men is bound to testosterone. Like the calculated free testosterone, and BAT, the FAI is dependent on accurate values for testosterone and SHBG

 

Direct Free Testosterone

 

The direct free testosterone assay was developed as a single-step, non-extraction method in which an125I-labeled testosterone analog competes with unbound testosterone in plasma for binding to a testosterone-specific antiserum that has been immobilized on a polypropylene assay tube. The basis for the test is that the analog has a low affinity for SHBG and for albumin.   Values for normal men with this method, as a percentage of the total testosterone (0.2-0.64%), are substantially lower than the 1.0-4.0% determined by other methods. While this difference alone does not cause a problem if adequate reference ranges are available, it immediately prompted speculation concerning the accuracy of the method.  Subsequent studies revealed that analog free testosterone assay results are positively correlated with the level of SHBG, much like total testosterone (33), and that free testosterone is un-measurable by analog assays in a dialysate of normal adult male serum (51).  Thus, the free testosterone level determined with analog assays appears to provide essentially the same information as the total testosterone level, is often misleading (tends to over diagnose hypogonadism), and is not recommended. The 2018 College of American Pathologists survey revealed that only 7% of participating laboratories continue to use this method, and it is has nearly disappeared from research articles related to testosterone.

 

Testosterone in Saliva   

 

The level of testosterone in saliva is positively correlated with the plasma free testosterone concentration. Salivary samples are easily collected, usually by a non-stimulated drool.  Both extraction and non-extraction immunoassay methods are available although LC-MS is being increasingly employed (44).   Usual values in adult men are 150-500 pmol/L (40-145 pg/ml).   Salivary testosterone assays are a useful research tool for field studies and other settings in which blood sampling is impossible or impractical.  While androgens are stable for a few days in untreated saliva, method artifacts may occur, and careful assay validation and quality control are essential.  Salivary testosterone assays have not been recommended for clinical purposes.

 

Cell-Based Reporter Bioassays

 

Cell-based reporter bioassays have been developed to analyze androgen bioactivity in biological samples. A stable cell line is created by transfection with plasmids encoding the human androgen receptor and a reporter system containing an androgen-responsive gene such as the mouse mammary tumor virus (MMTV)-luciferase reporter. When cells are stimulated with androgens, luciferase activity is increased dose-dependently (52), These assays remain investigational.

 

DIHYDROTESTOSTERONE

 

5 alpha reduction of testosterone to the more potent androgen dihydrotestosterone (DHT) is essential for fetal male genital development. T is converted to DHT by at least two steroid 5α-reductase (5AR) isoenzymes, 5AR types 1 and 2.   5AR-1 is found in liver, skin, brain, ovary, prostate, and testis whereas 5AR-2 is expressed in prostate, seminal vesicle, epididymis and skin. Approximately 20% of the circulating DHT in men is secreted by the testes, and the remainder is derived from the bioconversion of testosterone in tissues.  Because of a high level of expression of 5AR-2 in prostate, testosterone is effectively converted to DHT in that tissue, in which the level of DHT exceeds the peripheral blood concentration by 5-10-fold. The concentration of DHT in adult male serum is only about 10% of the value for testosterone, however. Therefore, an assay with negligible cross-reactivity with testosterone is needed for an accurate result.  An LC-MS method is now used by most reference laboratories. With gradient elution, the separation is totally complete (53).  Patients with 5α-reductase deficiency type 2 have ambiguous genitalia, and are generally detected as neonates, although a few patients have only microphallus or cryptorchidism.  A rise in testosterone but not DHT following hCG stimulation, producing a ratio of more than 10:1, is characteristic in most of these patients (54).  However, DHT production by 5AR1 can reduce the reliability of the ratio for diagnosis (55). The diagnosis is most often made by urinary steroid profiling by GC-MS, together with mutational analysis. DHT levels are often measured in epidemiological studies and in clinical research on prostate cancer and its treatments, and testosterone treatment results in a dose-dependent increase in serum DHT concentrations (56). Yet DHT levels are probably not useful in most clinical situations.

 

ESTRADIOL AND ESTRONE

 

Estrogens are important male hormones. They regulate the hypothalamic-pituitary-testicular axis, influence the function of  the testes and prostate, increase growth hormone and IGF-1 secretion, modify lipid metabolism and other hepatic proteins, and play an important role in male skeletal health, body fat, and perhaps sexual functioning  (57, 58).  Serum levels of estradiol and estrone are often measured in men with gynecomastia (59) or with unexplained gonadotropin deficiency. These conditions are rarely, but occasionally, due to estrogen-producing tumors, or to acquired (60) or genetic (61)   abnormalities in which estrogen production is increased. Moreover, the accurate measurement of estradiol may be helpful when SERMs, aromatase inhibitors, or hCG are used are used to increase testosterone levels, and in research on the role of estrogens in males (58, 62).  Estradiol is produced from testosterone, and estrone is produced from androstenedione, by aromatase P450, the product of the CYP19 gene. This enzyme is expressed in Leydig cells and in the adrenal cortex, as well as in adipose- and skin-stromal cells, aortic smooth muscle cells, kidney, skeletal muscle cells, and the brain. The promoter sequences of the P450 aromatase genes are tissue-specific, but the translated protein appears to be the same in all tissues. Increased aromatase expression in adipose and skin stroma with obesity is the most common cause for mild estrogen excess in men.  Interestingly, most studies of the age-associated decline in testosterone levels do not find a parallel fall in plasma estradiol levels perhaps because of increasing aromatase activity and fat mass as men grow older (63).

 

Because of low levels in males, traditional immunoassays for estrogens employed large volumes of plasma (2-5 ml) that were extracted with organic solvents. Because those assays are time-consuming and expensive to perform, non-radioactive automated methods that were optimized for the higher values normally found in pre-menopausal women were employed. However, the very small sample volumes used in automated assays may produce unexpectedly high values.  Results for estradiol in male plasma determined in platform assays are unreliable. The lack of agreement between laboratories is shown in Figure 6 which depicts the results for the low potency proficiency sample (mean 199 pg/mL) distributed in 2018 by the College of American Pathologists to 1450 participating laboratories. While many assays produced similar results, overall values ranged from 151 to 415 pg/ml (554-1523 pmol/L), with a coefficient of variation of 33%, compared to the goal of ±12.5%.  The mean value of the sample far exceeds normal levels in males, however, and testing at a lower potency would no doubt have produced even more dramatic differences.

Figure 6. Estradiol levels (±SD) in a mid-range proficiency sample (Y-05) distributed by the College of American Pathologists assayed using 15 different instruments in 1499 participating laboratories in 2018. The most frequently used (#6) was used by 341 laboratories. Mass spectrometry assays (n=11) are represented in column 4.

Mass spectrometry assays for estradiol and estrone, with a limit of detection of 1 pg/ml, have replaced conventional radio-immunoassays in most reference laboratories (24), and are highly recommended. The result with LC-MS is lower than with many immunoassays which presumably also detect interfering substances. Moreover, the between laboratory coefficient of variation for laboratories using MS methods (column 4) was also acceptably low at 10.3%.

 

Most of the circulating estradiol in men is loosely bound to albumin or is unbound (64), and only about 20% is thought to be bound to SHBG. Therefore, the serum level of SHBG was not predicted to appreciably influence the actions of estradiol. Measurement of free estradiol may be useful, however (65). In a series of studies, calculated non-SHBG-bound estradiol levels correlated more strongly with low bone mineral density and with indexes of high bone turnover in older men than did levels of total estradiol (66). On the other hand, increased mortality over 12 years of follow-up was seen among those with either low or high total as well as calculated free estradiol levels at baseline when compared to those in the middle tertile (67).  Akin to testosterone, non-SHBG (bioavailable) estradiol levels can be determined by ammonium sulfate precipitation using 3H-estradiol, or by equilibrium dialysis with or without using LC-MS.  Estimated levels are < 1 pg/mL. Calculated values require an accurate total estradiol assay, affinity constants are less certain than for testosterone, and the influence of testosterone and other steroids that also bind SHBG on the calculated estradiol level is no doubt important.  Age-specific free estradiol levels were reported but total estradiol was measured using an electro-chemiluminescence immunoassay (68); results using LC-MS methods are needed.  

 

GONADOTROPINS

 

FSH and LH, together with TSH and hCG, form a closely related family of heterodimeric glycoprotein hormones. Each consists of a common α-subunit that is non-covalently linked to a specific β-subunit. The α–β dimer is held together by a ‘seatbelt’ structure formed by the C-terminal amino acids of the β-subunit wrapped around the α-subunit.  Both subunits have asparagine-linked carbohydrate chains (2 for human α-subunit, 2 for FSH-β, and 1 for LH-β). The oligosaccharides project from the peptide skeleton, and by shielding of epitopes and altering the tertiary structure of the hormone, the sugars may impact receptor activation and bioactivity as well as antibody binding. Glycosylation also prolongs hormone clearance.

 

Most laboratories utilize fully automated, commercial assay systems for peptide hormones including LH and FSH.  Detection is generally by chemiluminescence, avoiding the use of radioactive tracers. Many assays achieve specificity by utilizing a biotinylated monoclonal antibody to the α-subunit as a capture antibody.  A second monoclonal antibody to the β-subunit is labeled with an organic ester that produces chemiluminescence in the presence of hydrogen peroxide (indicator antibody). Various pituitary and recombinant preparations are used for the standard curve but most assays are calibrated in terms of IU/L of International Reference Preparations (IRP) of highly purified human LH and FSH. The various standards have differing sugar sequences and branch patterns producing some variation in results between laboratories and assays, however, differences are relatively small.  When defined as a level of precision of replicate determinations of <10%, the sensitivity of these assays approximates 0.1 U/L.

Figure 7. Serum LH and FSH levels in normal males measured by an ICMA assay. Redrawn from Resende et al. (69).

Figure 7 shows mean levels of LH and FSH across the stages of pubertal development in boys measured with an automated ICMA (69). Values with an immunofluometric assay were slightly higher. FSH levels tend to exceed LH before puberty in boys, and both gonadotropins rise progressively during puberty with substantial overlap among the various pubertal stages.  

Prepubertal children have low amplitude but discrete pulsatile patterns of LH secretion that are amplified during sleep as puberty begins (70).  In adult men, LH is released in robust pulses every 1-2 h throughout the day and night with within subject variation in pulse height and between-pulse interval (Figure 8). In part because of pulsatility, the normal range for LH is wide, with typical ranges for adult men in terms of 2nd IRP of 1.6–8.0 IU/L for LH and 1.3–8.4 IU/L for FSH. Because of this pulsatility, pooling of 3 samples taken 20–30 min apart may provide a more accurate estimate of a person’s LH value than does a single sample.

 

Figure 8. Pulsatile patterns of LH secretion. Blood samples were drawn every 10 min for 12h starting at 0800 h from a 32-year-old normal man whose testosterone level was 474 ng/dL. Winters, SJ (unpublished).

Men with elevated levels of LH and FSH generally have testicular damage. Testes are usually small, there is oligo or azoospermia, and testosterone levels are low. Circulating estradiol levels are generally normal, however, in part because testicular aromatase is stimulated by the high level of LH. In some men with high LH and FSH levels, testosterone levels remain normal, sometimes because of elevated SHBG. The term “mild” or “compensated” hypogonadism is often applied (71). The diagnosis of gonadotroph adenoma is sometimes entertained, but long-standing infertility and small testes are a clue to the correct diagnosis. Much like the controversy surrounding thyroid hormone replacement in individuals with “subclinical” hypothyroidism, men with compensated hypogonadism who are not seeking fertility, mighty be viewed as candidates for testosterone replacement even though testosterone levels are within the reference range. This is often the case in teenagers with Klinefelter syndrome (72).

 

As men grow older, there is a decrease in sperm quality, the testes tend to be smaller, and there are fewer germ cells with more hyalinization and thickening of the tunica albuginea. Inhibin-B levels fall and FSH levels are often elevated. Mean LH levels also rise, but most often remain within the reference range (73).

 

Men treated with SERMs or aromatase inhibitors (74), and the rare men with inactivating mutations of the estrogen receptor-α (75) often have elevated LH and normal or elevated testosterone levels. These findings imply that estradiol is a major mediator of testicular negative feedback control of GnRH-LH secretion. Elevated LH and testosterone levels are also characteristic of men with androgen insensitivity syndromes (76) and men treated with anti-androgens (77), and reveal that there is additional negative feedback control of gonadotropins through the  androgen receptor.

 

FSH levels may be increased selectively because FSH production is regulated not only by GnRH and gonadal steroids but also by a paracrine control mechanism that involves pituitary activin and follistatin, and gonadal inhibin (see below).  When Sertoli cells fail to function normally, inhibin-B production declines, and the paracrine effects of pituitary activin stimulation of FSH-β gene expression is unopposed. Most men with high FSH levels have small testes and oligo- or azoospermia (78); however, FSH levels may sometimes be within the reference range (note that in some instances the reference range for FSH may be too high and some experts feel that the upper level for FSH is 8IU/L) in infertile men with severe damage to the germinal epithelium (79)  who may have either normal sized or small testes. In these cases, testicular biopsy is sometimes needed for a definitive diagnosis which is most often maturation arrest at the primary spermatocyte stage.

 

An elevated serum FSH level may rarely indicate an FSH-producing pituitary adenoma (80).  These tumors are generally macroadenomas that present with headache and a visual disturbance, and often co-secrete free α-subunit. The testes may occasionally  enlarge (81). The LH level is usually suppressed even when the tumors are LH-β immunoreactive, and testosterone levels are generally low.  LH secretion by a pituitary tumor is very rare (82, 83).  Most pituitary adenomas that are immunoreactive for LH or FSH are clinically silent with little or no hormone secretion.

 

The diagnosis of hypogonadotropic hypogonadism (HH) is based upon finding a low serum testosterone level with inappropriately low/normal LH and FSH concentrations. It is important to verify testosterone deficiency since a low SHBG level may result in the misdiagnosis of testosterone deficiency, and low LH may overlap with the reference range because of the pulsatile nature of LH secretion. Thus, the diagnosis of secondary hypogonadism (84) is sometimes problematic. The differential diagnosis of true hypogonadotropic hypogonadism is broad, and includes congenital HH due to a variety of genetic mutations including Kallmann syndrome (85), and acquired disorders such as prolactinoma, other sellar and suprasellar tumors or cysts, infiltrative diseases and vascular causes, iron overload, head trauma and others. HH in childhood limits pubertal development while in adults HH results in the regression of secondary sexual characteristics, albeit slowly.    

 

Isolated deficiency of FSH due to inactivating mutation in the FSH-β gene is a rare disorder (86-88). Affected men have been azoospermic, with borderline or low testosterone and increased LH levels. Pathological examination revealed narrowed seminiferous tubules, decreased Sertoli cell numbers, absence or aplasia of germ cells, and Leydig cell hyperplasia. Most mutations disrupt the ability of the mutant FSH- β to combine with the alpha-subunit. The low testosterone and increased LH levels may be explained by impaired signaling from the dysfunctional seminiferous tubules to Leydig cells (89). A few hypogonadal men with inactivating mutations of the LH-β gene have also been described (90-92).  Immunoreactive LH may be present or absent in serum depending on the nature of the mutation, but if present is bioinactive (92). Testosterone is low, and FSH is increased.  As placental hCG, rather than LH, stimulates male sexual differentiation, affected males have a normal male phenotype at birth while bioactive LH deficiency results in testosterone deficiency, and prevents normal puberty.

 

Two-site assays can occasionally be "too specific".  Polymorphisms that affect immunoassay detection by monoclonal antibodies may lead to misdiagnoses. For example, there is a relatively common polymorphism in the LH-β gene that is characterized by two point mutations in codons 8 and 15 resulting in two amino acid substitutions and an extra glycosylation site (88). Even though men and women with the LH variant appear to be normal and fertile, the serum LH level is low or undetectable using certain monoclonal antibodies whereas the result with other assays is normal. Clearly, when a man with a low or undetectable LH with a normal testosterone level is encountered, or a disparity between LH and FSH levels cannot be readily explained, a second assay method should be used.   

 

Various glycoforms of LH and FSH with structurally heterogeneous glycans are found in the pituitary and in the circulation, and glycosylation has been shown to influence hormone clearance and biological activity (93). Both sex steroids and activin have been reported to affect gonadotropin glycosylation. Highly sialylated glycoforms, with an acidic pH, tend to have a longer circulating half-life whereas the more alkaline forms tend to exhibit greater bioactivity in vitro. There is evidence that LH and FSH glycosylation is physiologically important. The ideal immunoassay would detect accurately only the total bioactive LH or FSH in the sample, but clearly this goal is difficult to accomplish. As an alternative approach, in vitro bioassays for LH and FSH can be used to assess the function of the gonadotropins. An in vitro bioassay based on the production of testosterone by cultured mouse or rat Leydig cells (94) was developed to assess LH function, and the production of estradiol by rat granulosa cells or immature Sertoli cells can be used to assay FSH bioactivity (95). Bioassays based on cAMP production by cell lines stably expressing gonadotropin receptors, with quantification using cAMP-responsive promoters linked to a luciferase reporter, have also been developed (34).  While useful for the study of the biological properties of recombinant or purified proteins, the clinical use of these assays is limited by the nonspecific effects of serum. In fact, many findings reported in patients using in vitro LH bioassays were found subsequently to be methodological artifacts.

 

Glycoprotein α-Subunit

 

Glycoprotein α-subunit is secreted in bursts that coincide with LH secretory episodes, implying pre-eminent regulation by GnRH (96) Accordingly, serum α-subunit levels increase at puberty (97) and are elevated in men with testicular failure and in postmenopausal women.  α-Subunit is also expressed in thyrotrophs, and levels are increased in patients with primary hypothyroidism (98).

Figure 9. Pulsatile patterns of α-subunit secretion. Blood samples were drawn every 10 min for 12h starting at 0800 h from a 32-year-old normal man whose testosterone level was 474 ng/dL. Winters, SJ (unpublished)

α-Subunit levels are low, but measurable, in normal children and in patients with congenital hypogonadotropic hypogonadism.  Because peak α-subunit levels after GnRH stimulation tend to be lower in IHH patients than in prepubertal boys, this test has been used to distinguish between these two patient groups, although some overlap occurs (99). α-Subunit is produced by as many as 20% of pituitary adenomas (100) often together with FSH or sometimes GH. Because it is cleared by renal excretion, α-subunit levels are high in patients with renal failure (101) .

 

INSULIN-LIKE FACTOR 3

 

Insulin-like factor-3 (INSL3) is a peptide hormone member of the relaxin-insulin hormone family that is secreted by Leydig cells following LH stimulation. INSL3 affects testicular descent through effects on the gubernaculum (102) and plays a role in spermatogonial differentiation. At the time of puberty, INSL3 levels rise in parallel with testosterone (103).  INSL3 concentrations in the blood of normal adult men are approximately 1 ng/mL (0.4-1.5 ng/mL).   Patients with Leydig cell dysfunction have lower levels, and the very low concentrations in men with hypogonadotropic hypogonadism increase following hCG administration (104) but are unaffected by testosterone treatment (105).

 

INHIBIN-B

 

Inhibin, a glycoprotein hormone produced by the testes as well as the ovaries, is responsible for the selective negative feedback control of FSH secretion, and functions as an intra-gonadal regulator (106). Most evidence supports the idea that inhibin decreases FSH-β mRNA levels by blocking pituitary activin-stimulated FSH-b transcription (107).  Inhibin is a 32-kDa heterodimer composed of an α-subunit, and one of two β subunits, βAor βB. Inhibin-B (α-βB) is the form produced by testicular Sertoli cells; inhibin-A (α-βA), produced by the corpus luteum and placenta, is undetectable in adult male plasma. Higher molecular weight forms of the uncombined inhibin α-subunit that lack bioactivity are released into the circulation in excess of dimeric inhibin-B.

 

The Gen II two site inhibin-B ELISA assay uses a capture antibody raised to a peptide from the βB-subunit, and a biotinylated detection antibody raised to a peptide from the inhibin α-subunit (108).  Wells are then incubated with a streptavidin-labeled horseradish peroxidase followed by the substrate tetramethylbenzidine which produces a colorometric signal that is proportional to the amount of inhibin-B in the sample. The assay is not automated and no performance statistics could be located.  Older assays were more complex, and added methionine and an oxidation step with hydrogen peroxide to facilitate capture, and heating with sodium dodecyl sulfate solution to enhance specificity.

 

Inhibin-B is produced by the fetal testis and is measurable in serum at term. Levels increase substantially in newborns coincident with the rise in gonadotropins and testosterone (mini-puberty), remain elevated for 2-4 months and then decline (109).  In contrast to barely detectable levels of gonadotropins and testosterone during childhood, however, circulating inhibin-B is readily measurable in sera from prepubertal boys, implying that production is partly gonadotropin-independent. Serum inhibin-B levels increase to adult values at the time of puberty, and decline as men grow older (110).  Serum inhibin levels are partly determined by gonadotropin stimulation (111) but based on studies in monkeys, also reflect Sertoli cell number (112). With the updated assay, results for normospermic men were (2.5-95thpercentile) 32-416 with a median value of 174 pg/mL (113).

Figure 10. Inhibin B levels in males in relation to age. Reprinted from Andersson A-M (114) with permission of the publisher (Elsevier).

Although there is no remarkable pulsatile fluctuation in circulating inhibin-B levels, there is a diurnal variation in adult men with the highest values in the morning and nadir values approximately 35% lower in the evening. This diurnal pattern parallels that of testosterone (115).

 

Inhibin has been extensively studied as a biomarker of spermatogenesis. In keeping with its function to suppress FSH production, circulating inhibin-B levels are inversely correlated with FSH levels in adult men, and are more strongly correlated inversely when values from men with primary testicular failure are included in the analysis (111).  Inhibin-B levels are higher in fertile than infertile men but there is substantial overlap between the groups (113, 116). There is also a demonstrable, albeit weak, positive correlation with sperm count (117), and with the germ cell score in testicular biopsy specimens among infertile men (118). Inhibin-B levels are low in men with testicular failure, and are very low in men with Klinefelter syndrome in whom seminiferous tubules are hyalinized and Sertoli cells are essentially absent (119). Low but measurable inhibin-B levels in men following chemotherapy (120) or testicular irradiation (121), some of whom develop germinal cell aplasia, suggest that germ cell factors regulate SC inhibin production. Low inhibin-B levels predict persistent azoospermia in men with testicular cancer who undergo orchidectomy, chemotherapy and irradiation (122). Serum inhibin levels changed little in normal men who participated in a male contraceptive clinical trial of testosterone together with a progestin, and developed azoospermia or severe oligospermia (123).

 

Inhibin-B levels have been studied as a biomarker in the male partners of infertile couples undergoing testicular spermextraction (TESE) as a predictor of success of intra-cytoplasmic sperm injection (ICSI). While mean inhibin B levels tend to be lower (and FSH higher) in men with no sperm found at TESE, there is no level of inhibin-B that reproducibly predicts either the presence or absence of spermatozoa in TESE samples, or successful in vitro fertilization (124). Thus, measurement of inhibin-B is not recommended in the decision making for fertility potential among azoospermic men undergoing TESE and ICSI. Further, very large doses of FSH are needed to increase circulating inhibin-B levels in men, so that an FSH stimulation test for inhibin-B is also not helpful in a clinical evaluation of hypospermatogenesis.

 

Inhibin-B levels are reduced in gonadotropin-deficient men (125).  In those with the complete form of congenital hypogonadotropic hypogonadism, values are lower than in men with partial gonadotropin deficiency who have some spontaneous pubertal development. This quantitative difference is partly due to a presumed larger mass of Sertoli cells (as reflected by larger testicular size) in the latter group of men. Low inhibin-B levels have been reported to differentiate boys with constitutional delay of puberty (CDP) from those with congenital hypogonadoptropic hypogonadism (CHH) with 80-100% sensitivity and specificity (126). While plasma inhibin B concentrations in CHH and CDP may overlap, in one study of boys age 14-18 years with delayed puberty, among those who were genital stage 1 (testis volume ≤ 3ml), inhibin-B levels <35 pg/ml predicted persistent hypogonadism (testosterone levels < 3 nmol/L), and presumably the diagnosis of CHH, after 2 years of follow-up (127).  Inhibin-B is undetectable in most boys with congenital anorchia as in castrates, and is therefore a useful test to help distinguish these patients from boys with intra-abdominal testes (128).

 

High inhibin-B levels have been reported in a few boys with FSH-producing pituitary tumors (129), in patients with Sertoli cell tumors (130) and in boys with McCune Albright syndrome with macro-orchidism and autonomous function of Sertoli cells (131).  Adrenal tumors often express the inhibin α-subunit, and there is one report of high serum dimeric inhibin level in an adult man with an adrenal neoplasm (132).

 

ANTI- MULLERIAN HORMONE

 

Anti-Mullerian hormone (AMH, also known as Mullerian inhibitory hormone) is a 140 KDa homodimeric member of the TGF-β family of growth and differentiation factors (133). It is produced by fetal Sertoli cells and causes regression of Mullerian structures during male sexual development (134). AMH production increases in response to FSH and is inhibited by androgens. AMH is readily detectable in the serum of prepubertal boys in concentrations of 10-70 ng/ml, and declines to levels of 2-5 ng/ml with entry into adolescence (135). AMH remains detectable at low values throughout adulthood (104) but how AMH functions in males beyond fetal life is uncertain. AMH levels are elevated in untreated men with congenital hypogonadotropic hypogonadism and decline following treatment with hCG (136). AMH is absent from the plasma of most prepubertal boys with congenital anorchia but is generally detectable in boys with bilateral cryptorchidism (134). Therefore, measuring AMH is useful in evaluating boys with non-palpable gonads. AMH levels are low in men with seminiferous tubular failure but overlap with normal in men with oligospermia, and do not predict testicular sperm retrieval for ICSI (137).  Levels tend be low in obesity (138),

Figure 11. Serum concentrations of AMH in healthy males from birth (cord blood) to age 69 years. From Aksglaede L et al (139). Red dots are values from men with bilateral anorchia. Republished with permission of The Endocrine Society.

FUNCTIONAL TESTS

 

hCG Stimulation

 

Human chorionic gonadotropin (hCG) can be used as a test agent to examine Leydig cell steroidogenesis in prepubertal boys who secrete little or no endogenous gonadotropins. A variety of protocols have been used. In one study, serum testosterone levels rose to greater than 300 ng/dL (10.5 nmol/L) in healthy prepubertal boys administered hCG 1500 IU intramuscularly every other day for seven doses. The effect of hCG to increase circulating levels of testosterone, precursor steroids and DHT can be used in conjunction with mutation analysis to help evaluate patients with a 46,XY karyotype who have ambiguous genitalia which may be due to androgen insensitivity, defects in testosterone biosynthesis, or 5α-reductase deficiency (140). When the basal and hCG-stimulated profile indicates accumulation of steroid precursors upstream of an enzymatic defect, sequencing can be performed to identify the disorder. A raised serum testosterone to DHT ratio would suggest the diagnosis of 5α-reductase deficiency type 2. However, DHT production by 5α-RD1 can reduce the reliability of the ratio for diagnosis (55).

 

Testosterone levels are generally unaffected by hCG in boys with congenital bilateral anorchia but increase in boys with bilateral intra-abdominal testes (141). Therefore, this test, together with measurement of inhibin-B and AMH, help establish which boys have intra-abdominal testes and should undergo laparoscopy, and orchidopexy or orchiectomy (128).

 

Adult men with primary testicular failure have elevated endogenous serum LH concentrations, and hCG will predictably increase serum testosterone levels less in these men than in eugonadal men. In gonadotropin-deficient men, the testosterone response to short-term administration of hCG is also blunted because Leydig cell steroidogenic enzymes are down-regulated. Overall, hCG testing nowadays provides little clinically useful information in either group of adult men.

 

Blockade of Steroid Hormone Biosynthesis and Action

 

Lowering circulating sex steroid levels with pharmacological inhibitors, or blocking steroid hormone action with receptor antagonists, can be used to assess the integrity of the hypothalamic-pituitary-testicular unit as a research tool, and clinically as a treatment for adult men with adult onset hypogonadism (142).

 

Ketoconazole, a competitive inhibitor of cytochrome P450 cholesterol side chain cleavage (P450scc) and C17/20 lyase in the biosynthetic pathway to testosterone is used in the treatment of endemic mycoses and off-label in other conditions including Cushing syndrome, but causes a dose-dependent reduction in circulating testosterone and estradiol levels. (143). Because ketoconazole also lowers cortisol production, glucocorticoids are co-administered with ketoconazole to prevent the symptoms of cortisol deficiency.

 

Estrogens produced by the testes, peripheral tissues, and the CNS play an important role in the physiological feedback regulation of gonadotropin secretion in men. Selective estrogen response modulators (SERMs) bind to estrogen receptors (ER-α and/or ER-β) and exert estrogen-like effects, or inhibit estrogen effects, in a tissue-specific manner. SERMs also have non-classical extra-nuclear (membrane) signaling mechanisms in certain cells. By blocking estradiol negative feedback, most SERMS, such as clomiphene (144) and tamoxifen, increase plasma LH and FSH levels. Similarly, aromatase inhibitors (145) such as anastrazole, reduce circulating estradiol levels, and increase LH in men.  Dosages of 25 mg/day of clomiphene produced a mean two-fold increase in serum testosterone levels in adult men (Helo 2015). (142)., and while higher doses are more effective, estradiol levels may increase, and gynecomastia and galactorrhea may occur (144).  Two-fold increments in LH were produced by anastrazole 10 mg daily within 3-4 days.  Both blocking estrogen negative feedback and decreasing estradiol production increased LH pulse frequency indicating an effect of estradiol on the GnRH pulse generator. A normal response implies functional integrity of GnRH-LH-testosterone pathways; however, responses among normal subjects are variable, and diagnostic tests using SERMS are not thought to be useful clinically.  So far, these agents are not FDA-approved for any use in men.

 

Nonsteroidal antiandrogens increase serum LH levels in men by blocking the androgen negative feedback effect on GnRH secretion (146) and can be used to test GnRH-LH integrity.

 

GnRH Test

 

GnRH is used as a research tool to examine the responsiveness of gonadotrophs to their physiological stimulus. In normal adult men, the intravenous administration of 100 µg of GnRH increases serum LH levels three- to- six-fold while serum FSH levels rise by about 50%. Generally, the total and incremental release of LH and FSH following GnRH administration is directly proportional to the basal hormone level, although exceptions do occur.

 

The GnRH test was introduced as a method to diagnose hypogonadism, and to distinguish hypogonadism due to hypothalamic from pituitary disorders. The LH and FSH response to GnRH is subnormal when gonadotrophs are damaged or destroyed by pituitary tumors or by other pathologies. However, the gonadotropin response to stimulation with GnRH is also diminished in patients with GnRH deficiency since GnRH up-regulates its receptor on gonadotrophs, as well as the expression level of each of the gonadotropin subunit genes and thereby LH and FSH production. Thus, the LH response to GnRH stimulation is attenuated in these patients even though the pituitary is essentially normal. Therefore, when evaluating gonadotropin deficient adult men, the GnRH test generally provides little information beyond that of the basal testosterone, LH and FSH levels, and is not recommended for clinical purposes.

 

GnRH analog (naferelin, leuprolide, buserelin, triptorelin) testing may help distinguish prepubertal boys with constitutional delay of puberty (CDP) from those with congenital hypogonadotropic hypogonadism (147-149). These agents bind the GnRH receptor with higher affinity and have a longer circulating half-life than does native GnRH. The LH and testosterone responses at 4h and 24h in boys with CDP generally exceed those of HH.  In one study, prepubertal boys age 13.7-17.5 years with testicular volume ≤ 4ml whose LH level at 4h following 0.1 mg sc triptorelin exceeded 5.3 U/L all progressed to testicular enlargement, testis size ≥ 8 ml, over 18 months. Those boys with lower LH basal ≤ 2U/L and stimulated ≤5.3 mIU/ml values did not progress to this level. The CHH group also had low levels of inhibin-B (<111 pg/ml) at baseline (127). Thus, the test seems to be of use in evaluating prepubertal boys with delayed puberty. In patients with CHH who are homozygotes or compound heterozygotes for mutations of the GnRH receptor gene, GnRH binding to its receptor may be either absent or reduced in affinity, or receptor signal transduction may be impaired, and the LH response to GnRH stimulation in these patients may be absent or reduced. The LH response in those with partial hypogonadotropic hypogonadism overlaps with the normal response (150), and while these tests are helpful so far there is no gold-standard diagnostic test to fully differentiate boys with CHH from those with CDP.

 

SEMEN ANALYSIS

 

Laboratory testing for men who present for an evaluation of infertility begins with the semen analysis (151). Most laboratories report the semen volume, the sperm density (million/mL), motility (% with progressive motion on the microscopic slide), and the sperm morphology (% with oval-shaped sperm heads), and follow WHO guidelines in their methods and reports (152). The test helps predict fertility potential but is not a test of fertility since there is substantial overlap between results for fertile and infertile men (153). Moreover, lack of complete adherence to standardized methods, and insufficiently skilled laboratory staff, may substantially limit the diagnostic value of the semen analysis. The sample can be collected by masturbation, or by interrupted intercourse, generally after 2-3 days of abstinence from ejaculation. Most of the sperm are in the first portion of the ejaculate so it is important to collect a complete sample. Condom collection is less desirable since condoms contain spermicides, but if so, the sample should be immediately placed into a sterile jar. It is important to analyze the sample within 60 min of collection, as motility decreases over time. In cold climates, the sample should be kept warm in transit by holding it within outer clothing adjacent to the body. Even with optimized collection methods, there is a physiological day-to-day variation in sperm production, so 2-3 samples may provide more insight that a single sample. A man can be considered to be sterile if there are no sperm in the ejaculate (azoospermia).

 

Table 6. WHO Guidelines for an Adequate Semen Analysis

Semen volume

 ≥1.4 mL

Sperm concentration

≥15 million/mL

Total sperm output

≥40 million sperm

Sperm progressive (total) motility

≥32 (≥40%)

Sperm morphology

≥4%

 

GENETIC TESTING

 

Assisted reproductive techniques have enabled azoospermic and severely oligospermic (<5 million/mL) men to reproduce. Therefore, there is a need to identify and discuss the consequences of possible mutations with the infertile couples. Genetic abnormalities are found in 3% to 5% of men with severe oligosperma, and 14% to 19% of men with non-obstructive azoospermia (NOA). The peripheral blood karyotype should be analyzed in these men unless there is a likely cause for hypospermatogenesis (e.g. cancer chemotherapy or orchitis). Of those with azoospermia, 2/3 have Klinefelter syndrome (47,XXY and mosaics) (154). Rarely, azoospermic men have a 46,XX chromosome constitution with Y-chromosome material detectable on the X-chromosome by FISH analysis using an antibody to the transcription factor encoded by the SRY gene. In studies of various ethnic groups, 4-15% of men with idiopathic non-obstructive azoospermic and 1–10% of men with severe oligospermia have microdeletions of the AZF regions of the Y chromosome (155, 156). Thresholds of 0.5-5 million sperm/mL have been suggested for genetic testing for higher specificity and overall lower cost (157) although some authors would like to see all infertile men tested.  Microdeletions (undetectable in standard karyotypes; YCMD) are detected by PCR and may partially or completely eliminate the azoospermia factor (AZF)a or the AZFb/c region including genes that are important for spermatogenesis. YCMD are detected by PCR. Couples are counselled that male offspring will inherit the AZF microdeletion, and will be at risk for infertility. In azoospermic men Y deletion testing is essential prior to considering surgical sperm retrieval because AZFa , AZFb or AZFB+c deletion are associated with very severe spermatogenic defects such as germ cell arrest or Sertoli cell only syndrome with an essentially zero prospect of success (160). On the other hand, even azoospermic men with AZFc deletions have ~ 50% chance of surgical sperm recovery (160).

 

Obstructive azoospermia may be recognized by a low-volume ejaculate with low pH. The testes of these men are generally normal in size, and FSH and inhibin-B levels are in the reference range. Obstruction can be demonstrated by ultrasound (158). If bilateral absence of the vas deferens (CBAVD) is found, approximately 75% of men will have cystic fibrosis, sometimes with little respiratory or pancreatic disease (159). When Cystic Fibrosis is diagnosed by genetic testing, the next step is to screen the female partner for Cystic Fibrosis gene abnormalities in order to assess the risk to any offspring.  

 

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