Anti-Müllerian hormone as a marker of steroid and gonadotropin action in the testis of children and adolescents with disorders of the gonadal axis
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In pediatric patients, basal testosterone and gonadotropin levels may be uninformative in the assessment of testicular function. Measurement of serum anti-Müllerian hormone (AMH) has become increasingly widespread since it provides information about the activity of the male gonad without the need for dynamic tests, and also reflects the action of FSH and androgens within the testis. AMH is secreted in high amounts by Sertoli cells from fetal life until the onset of puberty. Basal AMH expression is not dependent on gonadotropins or sex steroids; however, FSH further increases and testosterone inhibits AMH production. During puberty, testosterone induces Sertoli cell maturation, and prevails over FSH on AMH regulation. Therefore, AMH production decreases. Serum AMH is undetectable in patients with congenital or acquired anorchidism, or with complete gonadal dysgenesis. Low circulating levels of AMH may reflect primary testicular dysfunction, e.g. in certain patients with cryptorchidism, monorchidism, partial gonadal dysgenesis, or central hypogonadism. AMH is low in boys with precocious puberty, but it increases to prepubertal levels after successful treatment. Conversely, serum AMH remains at high, prepubertal levels in boys with constitutional delay of puberty. Serum AMH measurements are useful, together with testosterone determination, in the diagnosis of patients with ambiguous genitalia: both are low in patients with gonadal dysgenesis, including ovotesticular disorders of sex development, testosterone is low but AMH is in the normal male range or higher in patients with disorders of androgen synthesis, and both hormones are normal or high in patients with androgen insensitivity. Finally, elevation of serum AMH above normal male prepubertal levels may be indicative of rare cases of sex-cord stromal tumors or Sertoli cell-limited disturbance in the McCune Albright syndrome.
KeywordsTestis Sertoli Cryptorchidism Puberty Disorders of sex development
Complete androgen insensitivity syndrome
Constitutional delay of puberty
Disorders of sex development
Insulin-like factor 3
Müllerian inhibiting substance
Protein kinase A
Persistent Müllerian duct syndrome
Transforming growth factor β
In the adult male, the appraisal of the endocrine function of the gonadal axis usually relies on the assessment of serum levels of gonadotropins, testosterone and inhibin B. In pediatric ages, basal testosterone and gonadotropin levels may be largely uninformative. In fact, gonadotropin and testosterone secretion is active only during 3 to 6 months after birth in the male; thereafter, their serum levels remain very low or undetectable until the onset of puberty . However, the use of non-classical biomarkers, like anti-Müllerian hormone (AMH), has become increasingly widespread since it not only informs about the activity of the male gonad without the need for dynamic tests but also reflects the action of FSH and androgens within the gonad . This review will address the usefulness of AMH as a biomarker of testicular function in prepubertal and adolescent males, based on the knowledge of the endocrine regulation of testicular AMH secretion during pre- and post-natal development.
Developmental physiology of the hypothalamic-pituitary-testicular axis
Testicular function is mainly regulated by the pituitary gonadotropins LH and FSH, which in turn depend on gonadotropin-releasing hormone (GnRH) action, from the hypothalamus. This hypothalamic-pituitary-gonadal axis evolves throughout development, from fetal life through adulthood. Specific maturational changes take place both in these organs as a whole and in the different cell types that make them up.
While sperm production has classically been the focus of adult reproductive function, somatic cells are crucial for the maintenance of spermatogenesis and gamete production. In the interstitial tissue, Leydig cells synthesize androgens and the insulin-like factor 3 (INSL3) , whereas in the seminiferous tubules, Sertoli cells regulate the nutrients and factors that reach the germ cells by means of the blood-testis barrier. Sertoli cells not only regulate the inflow of external substances, but also produce several substances which are critical to the proper progression of spermatogenesis . Therefore, it appears evident that the assessment of gonadal function and the definition of male hypogonadism should rely on the understanding of normal testicular physiology resulting from the integrated function of the tubular and interstitial compartments, and its developmental changes from fetal life through maturity .
Sertoli cells as the most active population in the developing testis
Unlike the adult testis, where germ cells represent most of the gonadal size and Leydig cells are the most active endocrine cell population, in the prepubertal testis, Sertoli cells are the most numerous  and active testicular cell population [7, 8]. Even though Sertoli cells remain active during infancy and childhood, the testes have been erroneously considered as quiescent due to the reduced activity of the hypothalamic-gonadotrope axis. This activity is clearly reflected on the high levels of serum AMH and inhibin B.
It is around the onset of puberty that Sertoli cells undergo major morphological and physiological changes, leading to the switch from a proliferative, immature state, to a quiescent, mature one. Morphologically, there are changes in the nucleus and nucleolus . The blood-testis barrier becomes distinct, creating two separate compartments within the tubules. Germ cells in the adluminal compartment become dependent on the function of the, now mature, Sertoli cells . These maturational changes observed in Sertoli cells are induced essentially by an increase of intratesticular testosterone concentration early in pubertal development [14, 16]. Interestingly, Sertoli cells do not show maturational changes, in spite of the active androgen testicular production, during fetal and neonatal periods of life. This is due to the fact that before the age of 1 yr in humans [17, 18], the androgen receptor is not expressed in Sertoli cells (Fig. 1b), as experimentally confirmed in mice [19, 20].
AMH as a marker of prepubertal Sertoli cells: physiological concepts
AMH, also known as Müllerian Inhibiting Substance (MIS), is a glycoprotein dimer belonging to the transforming growth factor β (TGF-β) family [21, 22], which plays a major role in fetal sex differentiation by inducing the regression of the Müllerian ducts.
Serum AMH levels in normal boys
15 days – 6 months
6 months – 2 years
Because AMH is exclusively secreted into the circulation by Sertoli cells [33, 34], it has become one of the most useful markers to study testicular function during the prepubertal period in the male [35, 36, 37]. In the female, AMH is produced by ovarian granulosa cells of primary and small growing follicles up until transition to menopause [38, 39, 40, 41].
AMH as a marker of FSH action in the testis
AMH as a marker of androgen action in the testis
Androgen-mediated AMH down-regulation is also not observed in fetal life and during the first year of postnatal life (Fig. 3), even in patients with precocious puberty, owing to the above-mentioned physiological androgen insensitivity of Sertoli cells, which is consequence of the lack of androgen receptor expression in Sertoli cells in those periods of life [17, 18, 54, 55].
Ever since Alfred Jost postulated the existence of AMH , it has been referred to as the fetal testicular hormone guiding the regression of the Müllerian ducts in the male fetus. The biological reasons for ongoing expression of AMH throughout childhood have been the source of many debates. Nonetheless, AMH detection in serum has become a very powerful tool in pediatrics. In the following part of this review, we aim to summarize the main conditions in which AMH can be used as a proper marker of Sertoli cell function in boys.
Serum AMH in the diagnosis of conditions affecting testicular function
Serum AMH levels according to clinical presentation
Anorchidism (Testicular regression, bilateral gonadectomy)
PMDS - AMH mutation
Primary hypogonadism (testicular dysgenesis syndrome)
Rules out testicular dysgenesis
PMDS - AMHR mutation
Fetal testicular regression
Absence of puberty
Constitutional delay of puberty
Precocious pubertal signs
Central Precocious Puberty
Leydig cell tumor
Congenital adrenal hyperplasia
Adrenal androgen-secreting tumors
Exogenous androgen exposure
Sex-cord stromal tumors
46,XY Complete gonadal dysgenesis
46,XY Partial gonadal dysgenesis
Sex-chromosome gonadal dysgenesis
Androgen synthesis defects
46,XY Malformative DSD
46,XX male (Testicular DSD)
Androgen synthesis defects
In patients with non-palpable gonads, it is necessary to determine whether there is intraabdominal functional testicular tissue. The utility of gonadotropins, as indirect markers, is limited since they may be normal even in anorchid children . Conversely, in boys with non-palpable gonads detectable serum AMH levels are highly predictive of the existence of testicular tissue while an undetectable AMH value is indicative of anorchidism [33, 34] (Table 2). An extremely rare exception is the Persistent Müllerian Duct Syndrome caused by AMH gene mutations, which may explain the finding of undetectable serum AMH in a boy with abdominal testes . Vanishing or regression of testicular tissue occurring in the second half of fetal life does not preclude virilization, but micropenis and hypoplastic scrotum occur (Table 2). Serum AMH is low or undetectable, according to the amount of remaining functional testicular tissue [33, 34].
Monorchidism is the presence of a solitary testis, which may undergo a compensatory volume increase. There is a dissociated capacity of the remaining testis to compensate for the absence of the other gonad: while Leydig cell function is largely compensated, lower AMH and higher FSH in monorchid boys indicate that Sertoli cell proliferation and function is insufficient to fully compensate the function of the absent one .
No overt signs of hypogonadism are evident before puberty in Klinefelter syndrome, a sex-chromosome aneuploidy with late-onset testicular dysgenesis. Serum AMH is normal during childhood and early puberty, in correlation with normal inhibin B and FSH, indicating that Sertoli cell function is preserved until advanced stages of puberty [64, 65]. At the onset of puberty, like in normal boys, androgens provoke a physiological decrease in serum AMH also in patients with Klinefelter syndrome. However, in the latter, Sertoli cell function deteriorates progressively from mid-puberty, resulting in extremely low or undetectable AMH, in coincidence with undetectable inhibin B, very high FSH levels and small testis volume. Germ cell degeneration has been described already in early fetal development with a clear progression during postnatal life, mainly after pubertal onset .
Cryptorchidism and micropenis: suspicion of central hypogonadism
During the neonatal period and infancy, some clinical features associated with cryptorchidism, like micropenis and microorchidism, or the coexistence of anosmia or other pituitary hormone deficiencies are suggestive of central hypogonadism. Serum AMH is below the normal range in most cases of isolated central hypogonadism and of multiple pituitary hormone deficiency [42, 43, 49] (Table 2 and Fig. 3), although normal AMH levels do not rule out the diagnosis . The lack of FSH stimulation during fetal and neonatal life is responsible for the decreased Sertoli cell numbers and low AMH expression in patients with congenital hypogonadotropic hypogonadism.[44, 45] The increase in serum AMH in those patients receiving FSH may be useful to monitor treatment efficacy [42, 43, 49].
Sertoli cells markers have been assessed to distinguish between constitutional delay of puberty and central hypogonadism. AMH is within normal prepubertal levels in boys with constitutional delay of puberty, reflecting a eugonadal state in these patients . In untreated patients of pubertal age with congenital central hypogonadism, serum AMH levels are above those expected for age –reflecting that intratesticular testosterone is too low to inhibit AMH– but below those expected for Tanner stage 1, indicating that Sertoli cells have not been exposed to FSH [49, 68] (Table 2 and Fig. 3). Treatment with recombinant FSH provokes an increase in serum AMH, whereas further administration of hCG results in an elevation of intratesticular androgen levels and a decline in AMH [42, 49]. Conversely, down-regulation of AMH is less notorious when patients receive exogenous testosterone, probably due to the lower intratesticular androgen levels obtained with this treatment .
Serum AMH determination may be particularly helpful in the diagnostic workup of boys with incipient signs of precocious puberty, e.g. testis volume increase from 2 to 3 ml with or without penile enlargement, in whom basal gonadotropin and testosterone levels are not yet informative. As already mentioned, the decline in serum AMH is an early biochemical sign of the increase in intratesticular testosterone concentration . In infants below the age of 1 yr, AMH may not be useful (Figs. 3 and 4): serum levels are normal, owing to the lack of androgen receptor expression in Sertoli cells at that age, which makes this particular cell population of the testis transiently insensitive to androgens .
AMH may also be useful to monitor effectiveness during treatment with GnRH analogues, ketoconazole or anti-androgens. The decrease in testosterone production or action is reflected in the recovery of prepubertal AMH levels [18, 46]. Interestingly, lack of adherence to treatment resulting in intermittent inhibition of testosterone production can be suspected when AMH does not normalize .
Although precocious puberty is one of the classic components of McCune-Albright syndrome, macro-orchidism in the absence of androgen-dependent secondary sexual characteristics has also been reported in boys [70, 71]. In some cases, increased Sertoli cell proliferation was detected by the presence of small hyperechogenic foci in ultrasound imaging . The typical somatic activating mutation in the GNAS1 gene, encoding for the Gsα protein involved in the FSH receptor transduction pathway, was present only in Sertoli cells, thus resulting in isolated Sertoli cell hyperfunction with Sertoli cell hyperplasia and increased AMH (Table 2), without activating Leydig cells .
Sex-cord stromal tumors
AMH immunohistochemistry is useful to identify the sex-cord stromal origin in testicular tumors. AMH expression has been shown in overt Sertoli cell tumors [73, 74], in large cell calcifying Sertoli cell tumors frequently associated with Peutz-Jeghers syndrome , in primary or metastatic granulosa cell tumors of the testis , and in intratubular Sertoli cell proliferations, which has been suggested to represent an “in situ” or early stage of Sertoli cell tumors . Although a single determination of serum AMH may not be helpful to establish the initial diagnosis of the tumors in most of pediatric cases, because high AMH is normally found in the prepubertal boy, increasing AMH levels may be suggestive of a progressive lesion.
Chemotherapy and radiotherapy affect primarily germ cells of the testis, while steroid secreting Leydig cells are less affected. Sertoli cell function has not been extensively studied in cancer survivors. Two reports including few patients treated with poly-chemotherapy or hematopoietic cell transplantation for medulloblastoma or posterior fossa ependymoma have shown AMH below normal range for age [75, 76], whereas our group could not demonstrate any decrease in serum AMH in a large series of patients with Acute Lymphoblastic Leukemia or Lymphoblastic Lymphoma who received poly-chemotherapy .
When a 46,XY newborn is born with ambiguous or female genitalia, i.e. a 46,XY disorder of sex development (DSD), causes of insufficient virilization should be investigated . 46,XY DSD may result from disorders affecting both tubular and interstitial testicular compartments, like gonadal dysgenesis, or from a condition affecting only the interstitial compartment, like Leydig cell aplasia or steroidogenic enzyme defects. While testosterone is low in both situations, serum AMH is helpful to establish a differential diagnosis since it is low in patients with gonadal dysgenesis but normal or high in patients with isolated androgen deficiency [53, 62, 78, 79, 80] (Table 2 and Fig. 3).
Alternatively, the action of androgen in target tissue may be affected in the androgen insensitivity syndrome. In these patients, both Sertoli and Leydig cell activity is preserved, as reflected by normal to elevated serum AMH and androgen levels [53, 62, 78, 79, 80] (Table 2 and Fig. 3).
In boys with isolated hypospadias, AMH and testosterone are usually normal, indicating that there is no testicular dysfunction, and a malformative DSD should be suspected. When hypospadias is associated with other clinical manifestations of undervirilization like cryptorchidism, a higher risk of abnormal hormone secretion by the gonads or androgen end-organ defects exists [81, 82].
The Persistent Müllerian Duct Syndrome is a rare form of 46,XY DSD usually diagnosed by the unexpected finding of Müllerian duct remnants during a surgical procedure for cryptorchidism. Serum AMH levels are useful to differentiate its etiology, with normal serum AMH in patients with AMH receptor mutations and extremely low or undetectable AMH levels in patients with AMH gene mutations  (Table 2).
In 46,XX DSD patients with ambiguous external genitalia, AMH levels above the normal female range exclude the diagnosis of congenital adrenal hyperplasia, aromatase defects or virilizing tumors, and are highly suggestive of an Ovotesticular DSD [53, 78, 83].
In fully virilized 46,XX DSD patients (XX males), AMH and testosterone are in the normal male range (Table 2), indicating that Leydig and Sertoli cells are not primarily affected [53, 78]. However, germ cells fail to progress through meiosis and undergo apoptosis at puberty, associated with low testicular volume .
Serum AMH is an extremely helpful marker for assessing testicular function in pediatric patients. In 46,XY patients with non-palpable gonads and in newborns with DSD, serum AMH is informative about the existence and functional capacity of testicular tissue. Serum AMH levels are commensurate with the amount of functional Sertoli cells present in prepubertal patients, including those with micro- or macro-orchidism, or ovotesticular DSD. Serum AMH is also a reliable marker of FSH action in the prepubertal testis, both in basal conditions to diagnose central hypogonadism and to monitor FSH treatment. Finally, declining serum AMH is indicative of effective androgen action within the seminiferous tubules, and therefore a useful marker in the diagnosis and follow-up of patients with precocious or delayed puberty.
This work was partially supported by grants PIP-11220120100279 of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, to RAR and HFS, and PICT 2014-2490 of the Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT), Argentina to RPG.
Availability of data and material
All authors participated in the conception and writing of the manuscript. Its final version was approved by all the authors.
RPG and RAR have received honoraria from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, for technology services using the AMH ELISA. NYE and HFS declare that they have no competing interests.
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