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.

Early onset of primary hypogonadism revealed by serum anti-Müllerian hormone determination during infancy and childhood in trisomy 21.

Male patients with an extra sex chromosome or autosome are expected to present primary hypogonadism at puberty owing to meiotic germ-cell failure. Scarce information is available on trisomy 21, a frequent autosomal aneuploidy. Our objective was to assess whether trisomy 21 presents with pubertal-onset, germ-cell specific, primary hypogonadism in males, or whether the hypogonadism is established earlier and affects other testicular cell populations. We assessed the functional status of the pituitary-testicular axis, especially Sertoli cell function, in 117 boys with trisomy 21 (ages: 2months-20year). To compare with an adequate control population, we established reference levels for serum anti-Müllerian hormone (AMH) in 421 normal males, from birth to adulthood, using a recently developed ultrasensitive assay. In trisomy 21, AMH was lower than normal, indicating Sertoli cell dysfunction, from early infancy, independently of the existence of cryptorchidism. The overall prevalence rate of AMH below the 3rd percentile was 64.3% in infants with trisomy 21. Follicle-stimulating hormone was elevated in patients <6months and after pubertal onset. Testosterone was within the normal range, but luteinizing hormone was elevated in most patients <6months and after pubertal onset, indicating a mild Leydig cell dysfunction. We conclude that in trisomy 21, primary hypogonadism involves a combined dysfunction of Sertoli and Leydig cells, which can be observed independently of cryptorchidism soon after birth, thus prompting the search for new hypotheses to explain the pathophysiology of gonadal dysfunction in autosomal trisomy.

[Two immunoassays for antimullerian hormone measurement: analytical and clinical performances]. / Deux dosages de l'hormone antimüllérienne : performances analytiques et cliniques.

Today, serum antimullerian hormone (AMH) is considered as an interesting marker of fertility potential in women to determine follicular status and in men to evaluate testicular function. We study analytical and clinical characteristics of two AMH commercialized immunoassays: Immunotech and DSL methods. The detection limits (close to 0.13 ng/mL), functional sensitivities (close to 0.30 ng/mL) are equivalent, and imprecision results are acceptable for entirely manual assays. The Immunotech method is linear within the calibration range (up to 21 ng/mL) and the DSL method presents a lack of linearity making it accurate only up to 11 ng/mL (and not up to 14 ng/mL as it is indicated by the manufacturer). The two methods allow to measure human AMH, don't cross react with TGF-beta superfamily proteins and the DSL immunoassay recognize mouse (25%), rat (68%) and calf (100%) AMH. The comparison between the two methods (from 0.3 to 200 ng/mL) shows a good correlation (r = 0.979) with not statistically different results (p = 0.31). From a clinical point of view, the two methods allow the evaluation of follicular status in normo-ovulatory women and in women with polycystic ovary syndrome. Results are in agreement with studies showing that AMH serum concentration is strongly correlated with the number of antral follicles. In conclusion, the Immunotech method seems to be more efficient than the DSL method even if the two methods are suitable for clinical applications needing AMH measurements.

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.

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.

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.

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.

Ovarian granulosa cell tumors express a functional membrane receptor for anti-Müllerian hormone in transgenic mice.

Anti-Müllerian hormone inhibits granulosa cell growth and function. Both anti-Müllerian hormone and its type II receptor are expressed in normal granulosa cells. We show by histologic and molecular analyses that ovarian tumors developing in transgenic mice, obtained by targeted oncogenesis using an anti-Müllerian hormone promoter-SV40 oncogene construct, are of granulosa-cell origin. Because tissue-specific, cell-surface molecules are of particular interest for the analysis and treatment of tumors, we examined the expression of anti-Müllerian hormone type II receptor in the ovaries of these transgenic mice. We demonstrate that the anti-Müllerian hormone type II receptor is expressed not only in normal ovarian follicles, but also in granulosa cell tumors. Using a cell line derived from one of these tumors, we show that the anti-Müllerian hormone type II receptor protein is present on the surface of tumor cells and binds anti-Müllerian hormone. Furthermore, we show that the anti-Müllerian hormone receptor is functional in the granulosa tumor cell line, with anti-Müllerian hormone treatment inducing selective activation of Smad1. In conclusion, in this study we present a new murine transgenic model of granulosa cell tumors of the ovary and, using this model, we demonstrate for the first time cell-surface expression of a highly tissue-specific molecule, anti-Müllerian hormone type II receptor, as well as the selective activation of Smad proteins by anti-Müllerian hormone, in granulosa tumor cells.

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.

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.

Detection of minimal levels of serum anti-Müllerian hormone during follow-up of patients with ovarian granulosa cell tumor by means of a highly sensitive enzyme-linked immunosorbent assay.

Granulosa cell tumors (GCT) are ovarian neoplasms that tend to recur and spread in the pelvis and the abdomen several years after the initial treatment. Anti-Mülerian hormone (AMH) is a reliable serum marker of these tumors. To enhance the availability and the sensitivity of serum AMH determination, we developed an ultrasensitive enzyme-linked immunosorbent assay. In this work we compare the results of serum AMH levels, obtained using the ultrasensitive and the traditional assays, in 31 patients with ovarian GCT followed up for up to 7 yr. The ultrasensitive enzyme-linked immunosorbent assay has a significantly higher sensitivity than the traditional one. This resulted in the detection of low serum AMH levels, which were undetectable with the traditional assay, in several cases including one patient in whom a recurrence of a GCT had developed and two patients in whom the treatment had not been completely successful. These cases highlight the importance of the availability of a highly sensitive assay allowing evaluation with high precision of the results of treatment and to detect the recurrences of GCT at an early, preclinical stage.

The persistent Müllerian duct syndrome: a molecular approach.

A rare form of male pseudohermaphroditism is characterized by the persistence of Müllerian derivatives in phenotypic males. To determine the etiology of this syndrome, we studied the expression of anti-Müllerian hormone (AMH) in six boys, including three brothers, with the persistent Müllerian duct syndrome. All except one presented with an inguinal hernia containing the Müllerian derivatives, and in two boys the hernial sac contained the contralateral testis. AMH was normally expressed in the testicular tissue of two patients, as shown by bioassay of anti-Müllerian activity and immunocytochemistry. The testicular tissue of the other patients had no detectable bioactive or immunoreactive AMH, yet they expressed AMH mRNA with a normal transcription initiation site and in the amount expected for their age. These results prove the heterogeneity of the persistent Müllerian duct syndrome and suggest that it may sometimes involve peripheral insensitivity to AMH.

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.

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.

Purification of testicular anti-Müllerian hormone allowing direct visualization of the pure glycoprotein and determination of yield and purification factor.

An improved method is described for the purification of anti-Müllerian hormone from incubation medium of bovine fetal testes, using RIA to optimize the yield at different steps. Proteins present in incubation medium are precipitated by ammonium sulphate at 30-45% saturation and subjected to ion-exchange chromatography on DEAE-Sepharose. The bulk of the hormone is eluted from the ion-exchanger by a 0.12 M concentration of NaCl. Purification is achieved by immunochromatography on a monoclonal antibody: usually, addition of extraneous protein to the eluting buffer is required, but can be dispensed with if a second, cumulative, immunochromatography is performed by pooling the eluates from the first procedure. AMH obtained by this procedure has been studied using Coomassie blue and immunoblotting, and compared with fucose-labelled AMH, which is recognized by the same monoclonal antibodies as the carrier of the anti-Müllerian biological activity. In the absence of reducing conditions, several multimers, from 145 000 to 235 000 in molecular weight, are present. Reduction of disulphide bond linkages results in the disappearance of the multimeric forms, and the appearance of a 72 000 monomer. Equivalence between RIA and weight units has been established in one experiment: one AMH unit corresponds to 15 micrograms of protein. Pure AMH isolated by this procedure is highly bioactive at a concentration of 200 mU/ml.

Biosynthesis of labelled anti-müllerian hormone by fetal testes: evidence for the glycoprotein nature of the hormone and for its disulfide-bonded structure.

Tritiated fucose incorporated into proteins released by fetal calf tests into incubation medium proved to be a marker for anti-müllerian hormone (AMH) once non-specific glycoproteins had been eliminated by partial purification. When partially purified incubation medium from fetal calf teste s was fractionated by gel filtration, sucrose density gradient sedimentation or preparative electrofocusing, a single radioactive protein peak co-purified with anti-müllerian activity. Partially purified medium from bull testes--which are devoid of anti-müllerian activity--has a much lower fucose content than that derived from fetal testes. Antisera directed against 'fetal'--but not 'bull'--partially purified incubation medium, and capable of blocking anti-müllerian activity, precipitated the radioactive protein peak. The molecular weight of labelled AMH was 215,000 when determined by gel filtration and 124,000 when determined by density gradient sedimentation. By SDS-PAGE the molecular weight of labelled AMH was 123,000 and dissociation into a 72,000 subunit was demonstrated under conditions which reduce disulfide bonds.

A mouse Sertoli cell line expressing anti-Müllerian hormone and its type II receptor.

Anti-Müllerian hormone (AMH) induces the regression of Müllerian ducts in the male foetus; it is secreted by prepubertal testicular Sertoli cells and repressed at puberty. Using an AMH promoter/Simian virus 40 (SV40) oncogene fusion gene, we generated transgenic mouse lines exhibiting heritable Sertoli cell tumorigenesis. One cell line, derived from an adult male, expressed mRNAs characteristic of mature Sertoli cells, but no AMH. Two other cell lines were obtained from pretumoral testes at 6.5 days. One was cloned to yield SMAT1, whose expression pattern was characteristic of prepubertal Sertoli cells, namely no transferrin and high SF-I and AMH expression. SMAT1 also secretes AMH protein into the culture medium and expresses the AMH receptor. To the best of our knowledge, this is the first Sertoli cell line stably expressing AMH and its receptor. Our results show that, in targeted oncogenesis, the timing of cell line derivation plays a critical role even when using a developmentally regulated promoter.

Secretion of anti-Müllerian hormone by immature bovine Sertoli cells in primary culture, studied by a competition-type radioimmunoassay: lack of modulation by either FSH or testosterone.

Secretion of anti-Müllerian hormone (AMH) by immature bovine Sertoli cells in primary culture was studied through a competition-type RIA employing a polyclonal antibody and 125I-labelled purified AMH. This RIA is approximately 10 times more sensitive than the solid-phase two-site monoclonal antibody-based RIA described previously. Biosynthesis and secretion of AMH by cultured Sertoli cells require approximately 48 h, are not influenced by FSH or testosterone and steadily decrease over a one-week period of culture. Cyclic AMP response to FSH stimulation is normal in cultured cells. Whether the factors responsible for the extinction of AMH production in vitro are in any way related to those operating during normal maturation, which lead to repression of AMH biosynthesis in adult Sertoli cells, is not known at the present time and deserves further study.