Persistent Müllerian duct syndrome due to anti-Müllerian hormone receptor 2 microdeletions: a diagnostic challenge.

The persistent Müllerian duct syndrome (PMDS) is defined by the persistence of Müllerian derivatives in an otherwise normally virilized 46,XY male. It is usually caused by mutations in either the anti-Müllerian hormone (AMH) or AMH receptor type 2 (AMHR2) genes. We report the first cases of PMDS resulting from a microdeletion of the chromosomal region 12q13.13, the locus of the gene for AMHR2. 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. Diagnostic methods are discussed, with an emphasis on comparative genomic hybridization and targeted massive parallel sequencing.

Insensitivity to anti-müllerian hormone due to a mutation in the human anti-müllerian hormone receptor.

Anti-Müllerian hormone (AMH) and its receptor are involved in the regression of Müllerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Müllerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor.

Serine/threonine kinase receptors and ligands.

Serine/threonine receptors transduce signals for the TGF-beta family, several members of which, such as decapentaplegic and bone morphogenetic proteins, are involved in early patterning of the embryo. The gene encoding the anti-Müllerian hormone (AMH) receptor has recently been cloned; gene targeting produces the same effects as targeting of the AMH gene itself. Another divergent member of the TGF-beta family, GDNF, signals through Ret, a tyrosine kinase receptor; binding to Ret requires the cooperation of GDNFR-alpha. The signal transduction pathway of serine/threonine receptors is now being intensively studied; the immunophilin FKBP-12 and MAD proteins are known to be involved.

Hormonal and cellular regulation of Sertoli cell anti-Müllerian hormone production in the postnatal mouse.

Anti-Müllerian hormone (AMH) is secreted by immature testicular Sertoli cells. Clinical studies have demonstrated a negative correlation between serum AMH and testosterone in puberty but not in the neonatal period. We investigated AMH regulation using mouse models mimicking physiopathological situations observed in humans. In normal mice, intratesticular, not serum, testosterone repressed AMH synthesis, explaining why AMH is downregulated in early puberty when serum testosterone is still low. In neonatal mice, AMH was not inhibited by intratesticular testosterone, due to the lack of expression of the androgen receptor in Sertoli cells. We had shown previously that androgen-insensitive patients exhibit elevated AMH in coincidence with gonadotropin activation. In immature normal and in androgen-insensitive Tfm mice, follicle stimulating hormone (FSH) administration resulted in elevation of AMH levels, indicating that AMH secretion is stimulated by FSH in the absence of the negative effect of androgens. The role of meiosis on AMH expression was investigated in Tfm and in pubertal XXSxrb mice, in which germ cells degenerate before meiosis. We show that meiotic entry acts in synergy with androgens to inhibit AMH. We conclude that AMH represents a useful marker of androgen and FSH action within the testis, as well as of the onset of meiosis.

Anti-Müllerian hormone and intersex states.

Anti-Müllerian hormone (AMH), alias Müllerian-inhibiting substance or factor, plays a key role in fetal sex differentiation. The cloning of the human gene, a member of the transforming growth-factor-beta family and the development of immunochemical reagents recognizing circulating human AMH have opened new perspectives for clinical research. AMH assays and genetic studies now provide meaningful information regarding testicular function in infancy and the molecular basis of a rare form of male pseudohermaphroditism, the persistent Müllerian duct syndrome.

Cloning, expression, and alternative splicing of the receptor for anti-Müllerian hormone.

Anti-Müllerian hormone, also called Müllerian-inhibiting substance or factor, is a glycoprotein dimer belonging to the transforming growth factor-beta superfamily and synthesized by immature Sertoli cells and postnatal granulosa cells. Anti-Müllerian hormone plays a key role in sex differentiation by inducing the regression of Müllerian ducts in the male fetus. It is also responsible for the stunting and masculinization of fetal ovaries in bovine freemartin fetuses and may be involved in the control of follicular maturation in the postnatal ovary. Using a degenerate probe for a consensus region of the transforming growth factor-beta receptor superfamily to screen a complementary DNA library from rabbit fetal ovaries, we cloned a complementary DNA coding for a transmembrane serine/threonine kinase, which is expressed around the fetal Müllerian duct, in fetal and adult granulosa cells, and in immature Sertoli cells. Two transcripts, generated by alternative splicing of an exon coding for an N-terminal 61-amino acid domain, are strongly expressed in anti-Müllerian hormone target organs and Sertoli cells. The longer, 569-amino acid, isoform binds anti-Müllerian hormone when transiently expressed in COS cells and is believed to encode its functional receptor.

Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily.

Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.

A monoclonal antibody against bovine anti-Müllerian hormone.

Anti-Müllerian hormone (AMH) was partially purified from incubation medium of calf fetal testes and injected into a BALB/c mouse, whose splenocytes were fused with Sp2/Ag8 myeloma cells. Hybridomas were screened for specific antibody production by double antibody precipitation of labeled AMH, which was obtained by incubating fetal calf testes in the presence of tritiated fucose and submitting the medium to the standard procedure of purification. In spite of the extremely low concentration of AMH in the preparation used for immunization, three hybridomas gave positive results in the screening assay. One was cloned and grown in mice. The monoclonal antibody purified from ascites fluid abolished anti-Müllerian activity of partially purified AMH, whether or not the immune complex was removed from solution by a second, antimouse immunoglobulin antibody. The monoclonal antibody also blocked anti-Müllerian activity of calf but not rat fetal testes. Our results indicate that the monoclonal antibody is species specific and is directed towards the antigenic determinant responsible for biological activity.

Production of anti-Müllerian hormone: another homology between Sertoli and granulosa cells.

Anti-Müllerian hormone (AMH) has been detected by RIA in the follicular fluid of mature bovine ovaries and in incubation medium of bovine granulosa cells. Purification of AMH from two independent batches of follicular fluid was achieved with a yield of 11% and 15% respectively. Both ovarian and control testicular AMH produced near-complete regression of fetal rat Müllerian ducts exposed to it in culture at a final concentration of 200-300 mU/ml and were recognized by the same monoclonal and polyclonal antibodies. These findings indicate that adult mammalian granulosa cells are capable of producing immunoreactive and bioactive AMH at a rate apparently similar to that already demonstrated for mature Sertoli cells and add yet another item to the homologies reported between male and female somatic gonadal cells.

A sensitive radioimmunoassay for bovine anti-Müllerian hormone, allowing its detection in male and freemartin fetal serum.

Two monoclonal antibodies have been used to set up a solid-phase RIA for bovine anti-Müllerian hormone (bAMH). One AMH unit is defined as the amount released by 1 g of bovine fetal testicular tissue during a 4 h incubation period. Calibration curves were prepared using aliquots of a standard 500 ml pool of incubation medium, containing 200 AMH mU/ml, diluted either in 50% pig testes incubation medium, 5% horse serum, 10% female calf fetal serum or pure female calf fetal serum. Linearization of the calibration curves was achieved through "logit-log" transformation, all four lines were parallel. Within and between-assay variability were less than 5%. The RIA is at least 600 times more sensitive than the bioassay for anti-Müllerian activity and can detect AMH in male and freemartin fetal serum.

Anti-Müllerian hormone is not cytotoxic to human endometrial cancer in tissue culture.

To determine whether anti-Müllerian hormone (AMH) is the factor responsible for the cytotoxic effect of testicular preparations towards endometrial cancer, homogenates and incubation media of bovine fetal testes were fractionated by lectin affinity chromatography on wheat germ agglutinin and their cytotoxic effect tested on an endometrial cancer cell line, HEC-1, and on a control cell line of renal origin. Homogenate-derived testicular proteins proved more cytotoxic than incubation medium-derived ones, and the cytotoxic factor did not bind to the lectin of wheat germ, in contrast to AMH which did. Bioactive AMH purified by monoclonal antibody chromatography, at a concentration, determined by RIA, more than 20 times that present in the cytotoxic testicular preparations, had no effect upon the growth of the malignant endometrial cells. These results indicate that the testicular agent cytotoxic to malignant endometrial cells is a substance other than AMH.

Antimüllerian hormone in patients with hypogonadotropic hypogonadism.

Antimullerian hormone (AMH) is produced by immature Sertoli cells until pubertal maturation. At puberty, elevation of serum testosterone correlates with a decrease in serum AMH. To further investigate the hormonal control of AMH secretion, serum AMH levels were measured in 20 normal men (20-60 yr), in 12 patients (19-30 yr) with congenital hypogonadotropic hypogonadism (CHH), and in 18 patients (19-65 yr) with acquired hypogonadotropic hypogonadism (AHH) either untreated or during testosterone or human chorionic gonadotropin (hCG) therapy. Mean serum AMH levels in normal adult men were low (20+/-4.9 pmol/L). In untreated CHH patients, mean serum AMH levels were significantly higher than in normal men (292+/-86 pmol/L, P < 0.001) and were similar to those previously reported in prepubertal boys. In men with AHH, mean serum AMH levels were also significantly increased (107+/-50 pmol/L; P < 0.01) when compared with healthy men but were less than in men with CHH. In addition, in 10 patients treated for prostate cancer, a modest but significant increase of serum AMH (from 11.4 +/-5.7 pmol/L to 49+/-9.9 pmol/L; P < 0.01) was observed 12 months after suppression of the gonadal axis with the GnRH agonist Triptorelin (3.75 mg IM once a month). Plasma testosterone (T) and serum AMH levels were measured at baseline and at 3 and 6 months in 10 HH patients (6 CHH and 4 AHH) treated with hCG (1500 IU/twice weekly for 6 months) and in 8 HH (4 CHH and 4 AHH) patients treated with T (T enanthate 250 mg/3 weeks for 6 months). hCG treatment induced an increase of plasma T (from 1.0+/-0.7 to 11+/-2.4 and 19+/-4.8 nmol/L, at 3 and 6 months respectively) associated with a dramatic decrease of serum AMH (from 314+/-93 to 56+/-30 and 17+/-4.3 pmol/L). The similar increase in plasma T levels (from 1.4+/-1.0 to 15.6+/-4.2 and 23+/-6.2 ng/mL) obtained with exogenous T induced a lesser decrease of serum AMH (from 221+/-107 pmol/L to 114+/-50 and 66+/-17 pmol/L, at 3 and 6 months respectively). In conclusion, high plasma AMH levels in CHH patients are related to the absence of pubertal maturation of Sertoli cells. The high AMH levels in AHH and its increase after Triptorelin-induced gonadotropin deficiency suggest that the suppression of AMH is a reversible phenomenon. Finally, the inhibition of AMH production by Sertoli cells is induced by intratesticular T.

An enzyme linked immunoassay for anti-müllerian hormone: a new tool for the evaluation of testicular function in infants and children.

Anti-Müllerian hormone (AMH), also called Müllerian-inhibiting substance or factor was measured by an interspecific enzyme-linked immunoassay in the serum of 218 normal children and adults of both sexes and in 110 boys with various developmental disorders. AMH levels were high [81.67 ng/ml +/- 7.44(SEM)] in normal males under 2 yr of age, fell progressively in older boys and, decreased sharply at puberty. Serum AMH was not detectable in adults or in females at any age, with very rare exceptions. AMH serum concentrations were significantly decreased in infants with disorders of sex differentiation, particularly testicular dysgenesis, and increased in patients with delayed puberty. In contrast, levels were not significantly affected by either cryptorchidism or chorionic gonadotropin stimulation. AMH shows promise as a marker of testicular function in infancy.

Anti-müllerian hormone and testosterone serum levels are inversely during normal and precocious pubertal development.

Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance or factor, is produced by Sertoli cells from fetal life until puberty. In the present study, AMH, testosterone (T), LH, and FSH were measured by immunochemical methods in the serum of 50 boys with normal or delayed pubertal development, 4 patients with suspected androgen insensitivity, and 11 patients with either central (CPP) or gonadotropin-independent (GIPP) precocious puberty to investigate the hormonal regulatory mechanisms of AMH secretion at puberty. An inverse relationship between AMH and T levels was demonstrated. In boys with normal or delayed puberty with T concentrations below 6.7 nmol/L, AMH values were elevated (mean +/- SEM, 22.4 +/- 3.1 micrograms/L) and widely dispersed. In subjects with T levels over 6.7 nmol/L, AMH levels were uniformly low (3.4 +/- 0.5 micrograms/L), except in patients with suspected androgen insensitivity. No significant relationship was found between AMH and gonadotropin levels. Similar results were obtained in patients with either CPP or GIPP. Longitudinal studies were performed on four boys with CPP and two with GIPP before and after treatment. At the time of diagnosis, the T concentration was high, and AMH levels were usually low in CPP and GIPP patients alike. When appropriate treatment was initiated, the T concentration was normalized within 2-4 weeks, but restoration of prepubertal AMH levels required several months. Mature Sertoli cells were observed in testicular biopsies performed in three patients with untreated GIPP. Our results suggest that gonadotropins are not directly implicated in repression of AMH synthesis at puberty, but, rather, that the decrease in AMH production is the consequence of an androgen-mediated, long term, reversible chain of events leading to morphological and functional maturation of the Sertoli cells. Thus, the fall in serum AMH levels appears to be an excellent marker of Sertoli cell pubertal development.

Anti-müllerian hormone in children with androgen insensitivity.

Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance or factor, is secreted in high amounts by the immature Sertoli cell; it is negatively regulated by testosterone at puberty. In the present study, we measured serum AMH in 20 patients with defects of androgen synthesis or action: 9 with complete androgen insensitivity syndrome, 9 with a partial form, 1 patient with 3 beta-hydroxysteroid dehydrogenase deficiency, and 1 with Leydig cell agenesis. AMH was also determined in 15 control patients with idiopathic male pseudohermaphroditism. The serum AMH concentration was elevated in all testosterone-insensitive or -deficient patients compared with control levels during the first year of life. From 1 yr of age to the onset of puberty, serum AMH levels in patients with androgen insensitivity returned to normal values, but after pubertal development began, AMH levels again rose to extremely high levels in the complete androgen insensitivity syndrome. These results suggest that AMH is negatively regulated by testosterone not only at puberty, but also during the postnatal period. An elevation of serum AMH appears to be an interesting marker of androgen resistance or defect of androgen production in sexually ambiguous male infants.

Autosomal recessive segregation of a truncating mutation of anti-Müllerian type II receptor in a family affected by the persistent Müllerian duct syndrome contrasts with its dominant negative activity in vitro.

Anti-Müllerian hormone belongs to the TGFbeta family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Müllerian hormone type II receptor occur naturally, causing the persistent Müllerian duct syndrome. In a family with two members with persistent Müllerian duct syndrome and one normal sibling, we detected two novel mutations of the anti-Müllerian hormone type II receptor gene. One, transmitted by the mother to her three sons, is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain. The other, a missense mutation in the substrate-binding site of the kinase domain, is transmitted by the father to the two sons affected by persistent Müllerian duct syndrome, indicating a recessive autosomal transmission as in other cases of persistent Müllerian duct syndrome. Truncating mutations in receptors of the TGFbeta family exert dominant negative activity, which was seen only when each of the mutant anti-Müllerian hormone receptors was overexpressed in an anti-Müllerian hormone-responsive cell line. We conclude that assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis.

Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement.

Fetal male sexual differentiation is driven by two testicular hormones: testosterone (synthesized by interstitial Leydig cells) and antimüllerian hormone (AMH; produced by Sertoli cells present in the seminiferous tubules). Intersex states result either from gonadal dysgenesis, in which both Leydig and Sertoli cell populations are affected, or from impaired secretion or action of either testosterone or AMH. Until now, only Leydig cell function has been assessed in children with ambiguous genitalia, by means of testosterone assay. To determine whether serum AMH would help in the diagnosis of intersex conditions, we assayed serum AMH levels in 107 patients with ambiguous genitalia of various etiologies. In XY patients, AMH was low when the intersex condition was caused by abnormal testicular determination (including pure and partial gonadal dysgenesis) but was normal or elevated in patients with impaired testosterone secretion, whereas serum testosterone was low in both groups. AMH was also elevated during the first year of life and at puberty in intersex states caused by androgen insensitivity. In 46,XX patients with a normal male phenotype or ambiguous genitalia, in whom the diagnosis of female pseudohermaphroditism had been excluded, serum AMH levels higher than 75 pmol/L were indicative of the presence of testicular tissue and correlated with the mass of functional testicular parenchyma. In conclusion, serum AMH determination is a powerful tool to assess Sertoli cell function in children with intersex states, and it helps to distinguish between defects of male sexual differentiation caused by abnormal testicular determination and those resulting from isolated impairment of testosterone secretion or action.

Biochemical analysis of bovine testicular anti-Müllerian hormone.

Direct biochemical analysis has been applied to bovine testicular anti-Müllerian hormone (AMH), purified from incubation medium of bovine fetal testes by immunochromatography on a monoclonal antibody. The hormone contains a high proportion of hydrophobic amino acids and 13.5% carbohydrate. The oligosaccharide composition suggests that both N- and O-glycosidically linked chains are present. The molecular extinction coefficient is 3.27 +/- 0.06. One RIA unit, defined as the amount of hormone released by 1 g fetal bovine testicular tissue incubated during 4 h, corresponds to 3.06 +/- 0.17 microgram protein.

Disorders of gonadal differentiation and congenital adrenal hyperplasia.

Ambiguity of the external genitalia may be based on disordered steroid biosynthesis or may arise from fundamental genetic and chromosomal abnormalities that produce failure of gonadogenesis. This article first discusses congenital adrenal hyperplasia and then considers the diverse abnormalities that cause disordered gonadal differentiation.

Immunocytochemical detection of anti-müllerian hormone in Sertoli cells of various mammalian species including human.

An immunocytochemical method, based on the use of a polyclonal antibody raised against purified bovine anti-Müllerian hormone (AMH), was used to detect AMH in Sertoli cell cytoplasm of various mammalian species, including human. Immunopurification of antiserum by AMH-affinity chromatography, although not mandatory, leads to better results and increased sensitivity. In human testicular tissue, AMH is detectable up to 6 years of age. In rats, AMH production is initiated at 13 days post coitum, peaks between 15 and 17 days, and is no longer detectable 1 week after birth. The reaction is strongest in Sertoli cells of calves, sheep, goats, and pigs, species characterized by a high degree of development of the rough endoplasmic reticulum. It is fainter in human, rat, rabbit, and cat Sertoli cells, in which the rough endoplasmic reticulum is not as abundant. This correlation is not unexpected, in view of the localization of reaction product in this cytoplasmic organelle. Preliminary results indicate that there may be a relationship between the amount of immunoreactive AMH present in testicular biopsies of intersex patients and the degree of regression of the Müllerian duct on the ipsilateral side. This may help to elucidate whether persistence of Müllerian ducts results from lack of testicular production of AMH or from peripheral resistance of the Müllerian primordia to the hormone.