Persistent Müllerian duct syndrome associated with genetic defects in the regulatory subunit of myosin phosphatase.

STUDY QUESTION: Can mutations of genes other than AMH or AMHR2, namely PPP1R12A coding myosin phosphatase, lead to persistent Müllerian duct syndrome (PMDS)? SUMMARY ANSWER: The detection of PPP1R12A truncation mutations in five cases of PMDS suggests that myosin phosphatase is involved in Müllerian regression, independently of the anti-Müllerian hormone (AMH) signaling cascade. WHAT IS KNOWN ALREADY: Mutations of AMH and AMHR2 are detectable in an overwhelming majority of PMDS patients but in 10% of cases, both genes are apparently normal, suggesting that other genes may be involved. STUDY DESIGN, SIZE, DURATION: DNA samples from 39 PMDS patients collected from 1990 to present, in which Sanger sequencing had failed to detect biallelic AMH or AMHR2 mutations, were screened by massive parallel sequencing. PARTICIPANTS/MATERIALS, SETTING, METHODS: To rule out the possibility that AMH or AMHR2 mutations could have been missed, all DNA samples of good quality were analyzed by targeted next-generation sequencing. Twenty-four samples in which the absence of AMH or AMHR2 biallelic mutations was confirmed were subjected to whole-exome sequencing with the aim of detecting variants of other genes potentially involved in PMDS. MAIN RESULTS AND THE ROLE OF CHANCE: Five patients out of 24 (21%) harbored deleterious truncation mutations of PP1R12A, the gene coding for the regulatory subunit of myosin phosphatase, were detected. In addition to PMDS, three of these patients presented with ileal and one with esophageal atresia. The congenital abnormalities associated with PMDS in our patients are consistent with those described in the literature for PPP1R12A variants and have never been described in cases of AMH or AMHR2 mutations. The role of chance is therefore extremely unlikely. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study is the lack of experimental validation of the role of PPP1R12A in Müllerian regression. Only circumstantial evidence is available, myosin phosphatase is required for cell mobility, which plays a major role in Müllerian regression. Alternatively, PPP1R12A mutations could affect the AMH transduction pathway. WIDER IMPLICATIONS OF THE FINDINGS: The study supports the conclusion that failure of Müllerian regression in males is not necessarily associated with a defect in AMH signaling. Extending the scope of molecular analysis should shed light upon the mechanism of the initial steps of male sex differentiation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by la Fondation Maladies Rares, GenOmics 2021_0404 and la Fondation pour la Recherche Médicale, grant EQU201903007868. The authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Genetics of anti-Müllerian hormone and its signaling pathway.

Anti-Müllerian hormone (AMH) is a member of the TGF-ß family produced essentially by the supporting somatic cells of the testis. Initially known for its inhibiting role upon the development of female internal organs, AMH has been shown to exert many other effects namely upon germ cells. Circulating AMH reflects the ovarian reserve of young developing follicles and is used to evaluate the fertility potential in assisted reproduction. The signaling pathway of AMH is both similar and different from that of other members of the TGF-ß family. Like these, it signals through two distinct serine/threonine receptors, type 1 and type 2, that phosphorylate cytoplasmic effectors, the Smads. It also shares type 1 receptors and Smads with other members of the family. However, AMH is the only family member with its own, dedicated, ligand-specific type 2 receptor, AMHR2. The monogamic relationship between AMH and AMHR2 is supported by molecular studies of the Persistent Müllerian Duct Syndrome, characterized by the presence of Müllerian derivatives in otherwise normally virilized males: mutations of AMH or AMHR2 are clinically indistinguishable.

What Does AMH Tell Us in Pediatric Disorders of Sex Development?

Disorders of sex development (DSD) are conditions where genetic, gonadal, and/or internal/external genital sexes are discordant. In many cases, serum testosterone determination is insufficient for the differential diagnosis. Anti-Müllerian hormone (AMH), a glycoprotein hormone produced in large amounts by immature testicular Sertoli cells, may be an extremely helpful parameter. In undervirilized 46,XY DSD, AMH is low in gonadal dysgenesis while it is normal or high in androgen insensitivity and androgen synthesis defects. Virilization of a 46,XX newborn indicates androgen action during fetal development, either from testicular tissue or from the adrenals or placenta. Recognizing congenital adrenal hyperplasia is usually quite easy, but other conditions may be more difficult to identify. In 46,XX newborns, serum AMH measurement can easily detect the existence of testicular tissue, leading to the diagnosis of ovotesticular DSD. In sex chromosomal DSD, where the gonads are more or less dysgenetic, AMH levels are indicative of the amount of functioning testicular tissue. Finally, in boys with a persistent Müllerian duct syndrome, undetectable or very low serum AMH suggests a mutation of the AMH gene, whereas normal AMH levels orient toward a mutation of the AMH receptor.

A mutation inactivating the distal SF1 binding site on the human anti-Müllerian hormone promoter causes persistent Müllerian duct syndrome.

The persistent Müllerian duct syndrome (PMDS) is a 46,XY disorder of sexual development characterized by the persistence of Müllerian duct derivatives, uterus and tubes, in otherwise normally masculinized males. The condition, transmitted as a recessive autosomal trait, is usually due to mutations in either the anti-Müllerian hormone (AMH) gene or its main receptor. Many variants of these genes have been described, all targeting the coding sequences. We report the first case of PMDS due to a regulatory mutation. The AMH promoter contains two binding sites for steroidogenic factor 1 (SF1), one at -102 and the other at -228. Our patient carries a single base deletion at -225, significantly decreasing its capacity for binding SF1, as measured by the electrophoresis mobility shift assay. Furthermore, by linking the AMH promoter to the luciferase gene, we show that the transactivation capacity of the promoter is significantly decreased by the mutation, in contrast to the disruption of the -102 binding site. To explain the difference in impact we hypothesize that SF1 could partially overcome the lack of binding to the -102 binding site by interacting with a GATA4 molecule linked to a nearby response element. We show that disruption of both the -102 SF1 and the -84 GATA response elements significantly decreases the transactivation capacity of the promoter. In conclusion, we suggest that the distance between mutated SF1 sites and potentially rescuing GATA binding motifs might play a role in the development of PMDS.

WOMEN IN REPRODUCTIVE SCIENCE: Anti-Müllerian hormone: a look back and ahead.

Anti-Müllerian hormone (AMH) is a member of the TGF-ß family secreted by immature Sertoli cells and by granulosa cells of growing ovarian follicles. In males, it induces the regression of fetal Müllerian ducts and represses androgen synthesis through receptors located on the Leydig cell membrane. In female mice, AMH inhibits primary follicle recruitment and sensitivity to FSH. Measurement of circulating AMH is of value to pediatric endocrinologists allowing them to detect the presence and functional activity of testicular tissue without resorting to stimulation by human chorionic gonadotropin. In women, AMH levels are correlated with the size of the ovarian follicle pool and provide information on the likelihood of spontaneous or induced pregnancy.

Persistent Müllerian duct syndrome: an update.

Male sex differentiation is driven by two hormones, testosterone and anti-Müllerian hormone (AMH), responsible for regression of Müllerian ducts in male fetuses. Mutations inactivating AMH or AMH receptor type 2 (AMHR2) are responsible for persistent Müllerian duct syndrome (PMDS) in otherwise normally virilised 46,XY males. This review is based on published cases, including 157 personal ones. PMDS can present in one of three ways: bilateral cryptorchidism, unilateral cryptorchidism with contralateral hernia and transverse testicular ectopia. Abnormalities of male excretory ducts are frequent. Testicular malignant degeneration occurs in 33% of adults with PMDS. Cancer of Müllerian derivatives is less frequent. Fertility is rare but possible if at least one testis is scrotal and its excretory ducts are intact. Up to January 2019, 81 families with 65 different mutations of the AMH gene, mostly in exons 1, 2 and 5, have been identified. AMHR2 gene mutations comprising 64 different alleles have been discovered in 79 families. The most common mutation, a 27-bp deletion in the kinase domain, was found in 30 patients of mostly Northern European origin. In 12% of cases, no mutation of AMH or AMHR2 has been detected, suggesting a disruption of other pathways involved in Müllerian regression.

The Persistent Müllerian Duct Syndrome: An Update Based Upon a Personal Experience of 157 Cases.

Male sex differentiation is driven by 2 hormones, testosterone and anti-müllerian hormone (AMH), responsible for the regression of müllerian ducts in male fetuses. Mutations inactivating AMH or its receptor AMHRII lead to the persistent müllerian duct syndrome (PMDS) in otherwise normally virilized 46,XY males. Our objective was to review the clinical, anatomical, and molecular features of PMDS based upon a review of the literature and upon 157 personal cases. Three clinical presentations exist: bilateral cryptorchidism, unilateral cryptorchidism with contralateral hernia, and transverse testicular ectopia. Abnormalities of male excretory ducts are frequent. Testicular malignant degeneration occurs in 33% of adults with the disorder, while cancer of müllerian derivatives is less frequent. Fertility is rare but possible if at least one testis is scrotal and its excretory ducts are intact. Eighty families with 64 different mutations of the AMH gene have been identified, mostly in exons 1, 2, and 5. AMHRII gene mutations representing 58 different alleles have been discovered in 75 families. The most common mutation, a 27-bp deletion in the kinase domain, was found in 30 patients of mostly Northern European origin. In 12% of cases, no mutation of AMH or AMHRII has been detected, suggesting a disruption of other pathways involved in müllerian regression.

The BOC ELISA, a ruminant-specific AMH immunoassay, improves the determination of plasma AMH concentration and its correlation with embryo production in cattle.

Plasma anti-Müllerian hormone (AMH) concentrations have been recently found to be predictive of the number of embryos recovered after FSH superovulatory treatment in the cow. However, the sensitivity of the Active Müllerian-inhibiting substance/AMH ELISA (ref. 10-14400; DSL-Beckman-Coulter) used to make these measurements in bovine plasma samples is low because it was developed to measure human AMH levels. To overcome this limitation, we developed an immunoassay specific for the bovine (B), ovine (O), and caprine (C) species, the bovine-ovine-caprine (BOC) ELISA. For this purpose, we produced recombinant bovine AMH for standardization, and we used monoclonal antibodies raised against bovine AMH, previously prepared by our laboratory. We evaluated the precision, accuracy, specificity, limit of detection, and functional sensitivity of the assay. The intra-assay coefficient of variation ranged between 3.4% and 11.3% for AMH concentrations between 23.68 and 1.74 ng/mL, and the interassay coefficient of variation ranged between 4.8% and 20.5% for concentrations between 25.53 and 1.42 ng/mL, respectively. The assay displayed a good linearity, had a detection limit of 0.4 ng/mL and a functional sensitivity of 1.4 ng/mL. It also cross-reacted with ovine and caprine AMHs. Both the mean and median AMH levels measured in 40 cow plasma samples using the BOC ELISA were approximately 44 fold higher than the mean and median AMH levels measured with the Active Müllerian-inhibiting substance/AMH ELISA. Moreover, a higher correlation was observed between the average number of embryos recovered from each cow after superovulatory treatment and AMH concentrations measured with the BOC ELISA. This BOC ELISA provides a very efficient tool for evaluating the ovarian follicular reserve of cows and predicting their embryo production capacity.

Anti-müllerian hormone: a valuable addition to the toolbox of the pediatric endocrinologist.

Anti-Müllerian hormone (AMH), secreted by immature Sertoli cells, provokes the regression of male fetal Müllerian ducts. FSH stimulates AMH production; during puberty, AMH is downregulated by intratesticular testosterone and meiotic germ cells. In boys, AMH determination is useful in the clinical setting. Serum AMH, which is low in infants with congenital central hypogonadism, increases with FSH treatment. AMH is also low in patients with primary hypogonadism, for instance in Down syndrome, from early postnatal life and in Klinefelter syndrome from midpuberty. In boys with nonpalpable gonads, AMH determination, without the need for a stimulation test, is useful to distinguish between bilaterally abdominal gonads and anorchism. In patients with disorders of sex development (DSD), serum AMH determination helps as a first line test to orientate the etiologic diagnosis: low AMH is indicative of dysgenetic DSD whereas normal AMH is suggestive of androgen synthesis or action defects. Finally, in patients with persistent Müllerian duct syndrome (PMDS), undetectable serum AMH drives the genetic search to mutations in the AMH gene, whereas normal or high AMH is indicative of an end organ defect due to AMH receptor gene defects.