Anti-Müllerian Hormone Signaling Research Articles

Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a condition characterized by absent puberty and infertility due to gonadotropin releasing hormone (GnRH) deficiency, which is often associated with anosmia (Kallmann syndrome, KS). We identified loss-of-function heterozygous mutations in anti-Müllerian hormone (AMH) and its receptor, AMHR2, in 3% of CHH probands using whole-exome sequencing. We showed that during embryonic development, AMH is expressed in migratory GnRH neurons in both mouse and human fetuses and unconvered a novel function of AMH as a pro-motility factor for GnRH neurons. Pathohistological analysis of Amhr2-deficient mice showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in reduced fertility in adults. Our findings highlight a novel role for AMH in the development and function of GnRH neurons and indicate that AMH signaling insufficiency contributes to the pathogenesis of CHH in humans.

Association of gene polymorphisms in the anti-Müllerian hormone signalling pathway with ovarian function: a systematic review and meta-analysis.

This systematic review evaluated whether single nucleotide polymorphisms of AMH and AMHRII genes are associated with ovarian function. A literature search of PubMed and Embase was complemented by hand searches in the reference lists. Eight studies involving 3155 participants were included in a meta-analysis and 10 studies included for description. For AMH c.146T>G polymorphism, no significant difference in serum anti-Müllerian hormone (AMH) levels was found between participants with different genotypes (weighted mean difference [WMD] 0.42, 95% confidence interval [CI] -0.16 to 0.99). Subgroup analyses showed similar results for the European region and in healthy and infertile populations. Regarding AMHRII -482 A>G, there was no significant difference in serum AMH levels between participants with A/A genotype and G/A or G/G genotype carriers (WMD -0.04, 95% CI -0.31 to 0.23). In subgroup analysis, an interesting trend was confirmed in healthy women and polycystic ovary syndrome (PCOS) patients (WMD -0.36, 95% CI -0.63 to -0.09, P=0.009; WMD 0.46, 95% CI 0.15 to 0.77, P=0.004). The review suggests that AMH c.146T>G is not associated with AMH levels, while AMHRII -482 A>G may be related to AMH levels in PCOS and healthy subgroups. However, the impact of polymorphisms in the AMH signalling pathway on ovarian function still requires further investigation.

Functional Genetic Variation in the Anti-Müllerian Hormone Pathway in Women With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a highly heritable, common endocrine disorder characterized by hyperandrogenism, irregular menses, and polycystic ovaries. PCOS is often accompanied by elevated levels of anti-Müllerian hormone (AMH). AMH inhibits follicle maturation. AMH also inhibits steroidogenesis through transcriptional repression of CYP17A1. We recently identified 16 rare PCOS-specific pathogenic variants in AMH. To test whether additional members of the AMH signaling pathway also contribute to the etiology of PCOS. Targeted resequencing of coding and regulatory regions of AMH and its specific type 2 receptor, AMHR2, was performed on 608 women affected with PCOS and 142 reproductively normal control women. Prediction tools of deleteriousness and in silico evidence of epigenetic modification were used to prioritize variants for functional evaluation. Dual-luciferase reporter assays and splicing assays were used to measure the impact of genetic variants on function. We identified 20 additional variants in/near AMH and AMHR2 with significantly reduced signaling activity in in vitro assays. Collectively, from our previous study and as reported herein, we have identified a total of 37 variants with impaired activity in/near AMH and AMHR2 in 41 women affected with PCOS, or 6.7% of our PCOS cohort. Furthermore, no functional variants were observed in the 142 phenotyped controls. The functional variants were significantly associated with PCOS in our cohort of 608 women with PCOS and 142 controls (P = 2.3 × 10-5) and very strongly associated with PCOS relative to a larger non-Finnish European (gnomAD) population-based control cohort (P < 1 × 10-9). The AMH signaling cascade plays an important role in PCOS etiology.

GnRH Transactivates Human AMH Receptor Gene via Egr1 and FOXO1 in Gonadotrope Cells

Background/Objectives: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. Method/Results: Here, we demonstrated in perifused murine LβT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LβT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (–53/–37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and β-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. Conclusions: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.

Vitamin D attenuates the effect of advanced glycation end products on anti-Mullerian hormone signaling

Vitamin D3 (1,25-dihydroxyvitamin D3, VD3) in vitro attenuates the effect of the pro-inflammatory advanced glycation end products (AGEs) on steroidogenesis in human granulosa cells (GCs) by downregulating the receptor for AGEs (RAGE). It has been shown that VD3 alone downregulates anti-Mullerian hormone (AMH) type 2 receptor (AMHR-2) gene expression and suppresses AMH-induced SMAD 1/5/8 phosphorylation in granulosa cells. However, the effect of AGEs, in the absence or presence of VD3, on AMH action in GCs has not been studied. Using human GCs, this study showed that human glycated albumin (HGA), an in vitro representative for AGEs, upregulated AMHR-2 mRNA but did not alter AMH mRNA expression levels. VD3 inhibited the HGA-induced increase in AMHR-2 mRNA expression levels. In KGN granulosa cell line, recombinant AMH induced SMAD 1/5/8 phosphorylation. HGA augmented the recombinant AMH-induced SMAD 1/5/8 phosphorylation while the addition of VD3 to HGA attenuated the recombinant AMH-induced SMAD 1/5/8 phosphorylation. Thus, AGEs could potentially affect folliculogenesis as reflected by changes in AMH signaling. These findings have significant implications for women with polycystic ovary syndrome who have significantly elevated serum and ovarian AGEs.

Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression

In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct, and upper vagina. It is well established that transforming growth factor-β family member anti-Müllerian hormone (AMH) secreted from the fetal testis and its type 1 and 2 receptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global nonbiased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identified Osterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a male-specific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern of Osx expression. Together, these results indicate that AMH signaling is necessary and sufficient for Osx expression in the MD mesenchyme. In addition, MD regression was delayed in Osx-null males, identifying Osx as a factor that regulates MD regression.

Anti-Müllerian hormone overexpression restricts preantral ovarian follicle survival.

Anti-Müllerian hormone (AMH) is an ovarian regulator that affects folliculogenesis. AMH inhibits the developmental activation of the dormant primordial follicles and the oocyte within. In more mature follicles, AMH reduces granulosa cell sensitivity to follicle-stimulating hormone (FSH). We examined the effects of AMH overexpression on the stages of ovarian folliculogenesis, and the development of embryos, with a transgenic mouse that overexpresses human AMH in central nervous system neurons under the control of the mouse Thy1.2 promoter (Thy1.2-AMHTg mice). These mice are severely sub-fertile, despite relatively normal ovulation rates. The embryos of Thy1.2-AMHTg females exhibited delayed preimplantation development and extensive mid-gestation fetal resorption. Young Thy1.2-AMHTg mouse ovaries exhibited only a slight reduction in the rate of primordial follicle activation but large declines in the number of developing follicles surviving past the primary stage. It was expected that Thy1.2-AMHTg mice would retain more primordial follicles as they aged, but at 5 months, their number was significantly reduced relative to wild-type females. These data indicate that moderate elevations in AMH levels can severely restrict reproductive output and the number of developing follicles in the ovary. This evidence suggests that early antral follicles are a target for AMH signaling, which may regulate early follicle survival.

The cell biology and molecular genetics of Müllerian duct development.

The Müllerian ducts are part of the embryonic urogenital system. They give rise to mature structures that serve a critical function in the transport and development of the oocyte and/or embryo. In most vertebrates, both sexes initially develop Müllerian ducts during embryogenesis, but they regress in males under the influence of testis-derived Anti-Müllerian Hormone (AMH). A number of regulatory factors have been shown to be essential for proper duct development, including Bmp and Wnt signaling molecules, together with homeodomain transcription factors such as PAX2 and LIM1. Later in development, the fate of the ducts diverges between males and females and is regulated by AMH and Wnt signaling molecules (duct regression in males) and Hox genes (duct patterning in females). Most of the genes and molecular pathways known to be involved in Müllerian duct development have been elucidated through animal models, namely, the mouse and chicken. In addition, genetic analysis of humans with reproductive tract disorders has further defined molecular mechanisms of duct formation and differentiation. However, despite our current understanding of Müllerian duct development, some questions remain to be answered at the molecular genetic level. This article is categorized under: Early Embryonic Development > Development to the Basic Body Plan.

The mechanisms underlying the effects of AMH on Müllerian duct regression in male mice.

Anti-Müllerian hormone (AMH) produced in the developing testis induces the regression of the Müllerian duct, which develops into the oviducts, uterus and upper vagina. In our true hermaphrodite mouse with an ovary on one side and a testis on the other (O/T), the oviduct and uterus are present only on the ovary side, and nothing derived from the Müllerian duct is present on the testis side. Here, we investigate the mechanism underlying the unilateral Müllerian duct regression and the mode of AMH signaling, by performing immunohistology, Western blotting, and organ culture analyses. The histological analysis revealed that during the start of the Müllerian duct regression, the duct in the O/T mice was clearly regressed on the AMH-positive testis side compared to the AMH-negative ovary side. The immunohistochemistry showed a diffuse immunoreaction of AMH in the interstitium surrounding the testis cord and boundary region between the testis and mesonephros, especially in the cranial portion. Western blotting revealed that the amount of AMH in the cranial half of the mesonephros was larger than that in the caudal half. AMH injected into the gonads in organ culture induced the regression of the Müllerian duct via the interstitium of the organ. These results suggest that AMH acts on the Müllerian duct in male mice by exuding into the interstitium surrounding the testis cord and infiltrating through the cranial region from the testis to the mesonephros.

Efficacy of predictive models for polycystic ovary syndrome using serum levels of two antimüllerian hormone isoforms (proAMH and AMHN,C)

To compare total antimüllerian hormone (AMH), proAMH, AMHN,C, and the ratio of the two forms in predictive models for polycystic ovary syndrome (PCOS) diagnosis. Total AMH consists of proAMH (inactive precursor) and AMHN,C (receptor-competent), but neither isoform has been tested individually for their ability to predict PCOS diagnosis. Cross-sectional study using biobanked samples collected between July 2008 and January2010. Not applicable. Overweight, premenopausal women aged 18-45years with PCOS (n = 45, with 21 fulfilling National Institutes of Health diagnostic criteria and 24 fulfilling European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE) criteria, but not National Institutes of Health criteria) and without PCOS (n = 23 controls). None. Serum concentrations of proAMH and total AMH (proAMH and AMHN,C combined) were determined by immunoassay. The AMHN,C concentrations were calculated by subtraction ([AMHN,C] = [total AMH] - [proAMH]). Relative levels of proAMH were expressed as the AMH prohormone index (API = [ProAMH]/[Total AMH] × 100). In women with PCOS, total AMH, proAMH, and AMHN,C levels were higher, and the API was lower (P=.010), than in controls indicating increased conversion of proAMH to AMHN,C. Receiver-operating characteristic analysis for proAMH (area under the curve [AUC] = 0.82), AMHN,C (AUC = 0.86), and API (AUC = 0.70) did not improve the prediction for PCOS when compared with total AMH (AUC = 0.86). The proAMH and AMHN,C do not appear to improve the ability to predict a diagnosis of PCOS beyond total AMH assays. However, the ratio of inactive proAMH precursor to receptor-competent AMHN,C (API) differs in women with PCOS relative to unaffected controls indicating that AMH signaling mechanisms may be altered in women with PCOS.

A Long Noncoding RNA, lncRNA-Amhr2, Plays a Role in Amhr2 Gene Activation in Mouse Ovarian Granulosa Cells.

Anti-Müllerian hormone (AMH) is critical to the regression of Müllerian ducts during mammalian male differentiation and targets ovarian granulosa cells and testicular Sertoli and Leydig cells of adults. Specific effects of AMH are exerted via its receptor, AMH type II receptor (Amhr2), but the mechanism by which the Amhr2 gene is specifically activated is not fully understood. To see whether a proximal promoter was sufficient for Amhr2 gene activation, we generated transgenic mice that bore the enhanced green fluorescent protein (EGFP) gene driven by a 500-bp mouse Amhr2 gene promoter. None of the established 10 lines, however, showed appropriate EGFP expression, indicating that the 500-bp promoter was insufficient for Amhr2 gene activation. As a regulatory element, we found a long noncoding RNA, lncRNA-Amhr2, transcribed from upstream of the Amhr2 gene in ovarian granulosa cells and testicular Sertoli cells. In primary granulosa cells, knockdown of lncRNA-Amhr2 resulted in a decrease of Amhr2 messnger RNA level, and a transient reporter gene assay showed that lncRNA-Amhr2 activation increased Amhr2 promoter activity. The activity was correlated with lncRNA-Amhr2 transcription in stably transfected OV3121 cells derived from mouse granulosa cells. Moreover, by the Tet-on system, the induction of lncRNA-Amhr2 transcription dramatically increased Amhr2 promoter activity in OV3121 cells. These results indicate that lncRNA-Amhr2 plays a role in Amhr2 gene activation in ovarian granulosa cells by enhancing promoter activity, providing insight into Amhr2 gene regulation underlying the AMH signaling in the female reproductive system.

Anti-Müllerian hormone signaling is influenced by Follistatin 288, but not 14 other transforming growth factor beta superfamily regulators.

The hypothesis that, in contrast to other transforming growth factor-beta (TGFβ) superfamily ligands, the dose-response curve of Anti-Müllerian hormone (AMH) is unmodulated was tested by examining whether known TGFB superfamily modulators affect AMH signaling, using a P19/BRE luciferase reporter assay. AMHC and AMHN,C activated the reporter with an EC50 of approximately 0.5 nM. Follistatins (FS) produced concentration-dependent increases in AMHC - and AMHN,C -initiated reporter activity, with FS288 being more potent than FS315; however, the maximum bioactivity of AMH was not altered by either follistatin. Thirteen other TGFβ regulators (Chordin, Chordin-like 1, Chordin-like 2, Differential screening-selected gene aberrative in neuroblastoma [DAN], Decorin, Endoglin, Follistatin-like 1, Follistatin-like 3, Follistatin-like 4, Noggin, α2 macroglobulin, TGFβ receptor 3, Von Willebrand factor C domain-containing 2) had little or no effect. Surface plasmon resonance analysis showed no significant association between FS288 and AMHC , suggesting that FS288 indirectly regulates AMH signaling. Activin A, a direct target of FS288, did not itself induce reporter activity in P19 cells, but did prevent the FS288-induced increase in AMH signaling. Hence, local concentrations of FS288 and Activin A may influence the response of some cell types to AMH.

Pathogenic Anti-Müllerian Hormone Variants in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS), a common endocrine condition, is the leading cause of anovulatory infertility. Given that common disease-susceptibility variants account for only a small percentage of the estimated PCOS heritability, we tested the hypothesis that rare variants contribute to this deficit in heritability. Unbiased whole-genome sequencing (WGS) of 80 patients with PCOS and 24 reproductively normal control subjects identified potentially deleterious variants in AMH, the gene encoding anti-Müllerian hormone (AMH). Targeted sequencing of AMH of 643 patients with PCOS and 153 control patients was used to replicate WGS findings. Dual luciferase reporter assays measured the impact of the variants on downstream AMH signaling. We found 24 rare (minor allele frequency < 0.01) AMH variants in patients with PCOS and control subjects; 18 variants were specific to women with PCOS. Seventeen of 18 (94%) PCOS-specific variants had significantly reduced AMH signaling, whereas none of 6 variants observed in control subjects showed significant defects in signaling. Thus, we identified rare AMH coding variants that reduced AMH-mediated signaling in a subset of patients with PCOS. To our knowledge, this study is the first to identify rare genetic variants associated with a common PCOS phenotype. Our findings suggest decreased AMH signaling as a mechanism for the pathogenesis of PCOS. AMH decreases androgen biosynthesis by inhibiting CYP17 activity; a potential mechanism of action for AMH variants in PCOS, therefore, is to increase androgen biosynthesis due to decreased AMH-mediated inhibition of CYP17 activity.

Expressional changes of AMH signaling system in the quail testis induced by photoperiod.

Gonadal sex differentiation proceeds by the interplay of various genes including the transcription factors and secretory factors in a complex network. The sex-differentiating genes are expressed not only during early sex differentiation but also throughout the gonadal development and even in the adult gonads. In addition, the evidence that they actually function in the adult gonads have been accumulated from the studies using the conditional knockout mice. However, many previous studies were focused on one single gene though those genes function in a network. In this study, the expressions of various sex-differentiating genes were analyzed simultaneously in the adult testis of the Japanese quail (Coturnix japonica), whose testicular functions are dramatically changed by altering the photoperiod, to elucidate the roles of them in the adult gonad. Anti-Müllerian hormone (AMH) was significantly upregulated in the regressed testis induced by the short-day condition. The expressions of the transcription factors that promote AMH expression in mammals (SF1, SOX9, WT1 and GATA4) were also increased in the regressed testis. Moreover, AMH receptor (AMHR2) showed similar expression pattern to its ligand. We also analyzed the expressions of other transforming growth factor beta (TGFB) superfamily members and their receptors. The expressions of the ligands and receptors of TGFB family, and follistatin and betaglycan in addition to inhibin subunits were increased in the regressed testis. These results suggest that AMH is involved in the adult testicular functions of the Japanese quail together with other TGFB superfamily members.

A dominant negative mutation at the ATP binding domain of AMHR2 is associated with a defective anti-Müllerian hormone signaling pathway.

Does a heterozygous mutation in AMHR2, identified in whole-exome sequencings (WES) of patients with primary ovarian insufficiency (POI), cause a defect in anti-Müllerian hormone (AMH) signaling? The I209N mutation at the adenosine triphosphatebinding domain of AMHR2 exerts dominant negative defects in the AMH signaling pathway. Previous studies have demonstrated the associations of several sequence variants in AMH or AMHR2 with POI, but no functional assay has been performed to verify whether there was any defect on AMH signaling. Ninety-six unrelated female Chinese Han patients were diagnosed with idiopathic POI and subjected to WES. In silico analysis was done for the sequence variants followed by molecular assays to examine the functional effects of the sequence variants in human granulosa cells. In silico analysis, immunostaining, Western analysis, genome-wide expression analysis, quantitatively polymerase chain reaction were applied to the characterization of the sequence variants. We identified one novel heterozygous missense variant, p.Ala17Glu (A17E), in AMHR2. Subsequently, A17E and two independently reported missense variants, p.Ile209Asn (I209N) and p.Leu354Phe (L354F), were evaluated for effects on the AMH signaling pathway. In silico analysis predicted that all three variants may be deleterious. However, only one variant, I209N, showed severe defects in transducing the AMH signal as well as impaired SMAD1/5/8 phosphorylation. Furthermore, using genome-wide gene expression analysis, we identified genes whose expression was affected by the mutation, these included genes previously reported to participate in AMH signaling as well as newly identified genes. They are EMILIN2, FAM155A, GATA2, HES5, ID1, ID2, RLTPR, SMAD7, CBL, MALAT1 and SMARCA2. None. Although the in vitro assays demonstrated the causative effect of I209N on AMH signaling, further studies need to validate its long-term effects on folliculogenesis and POI. These results will aid both researchers and clinicians in understanding the molecular pathology of AMH signaling and POI to develop diagnostic assays or therapeutics approaches. Research funding is provided by the Ministry of Science and Technology of China [2012CB944704; 2012CB966702], and the National Natural Science Foundation of China [Grant number: 31171429]. The authors declare no conflict of interest.

Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independentinduction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.

Anti-Müllerian hormone (AMH) receptor type II expression and AMH activity in bovine granulosa cells

Anti-Müllerian hormone (AMH) produced by granulosa cells has previously been proposed to play a role in regulating granulosa cell differentiation and follicle selection. Although AMH receptor type II (AMHR2) dimerizes with a type I receptor to initiate AMH signaling, little is known about the regulation of AMHR2 expression in bovine granulosa cells and the role of AMH in follicle development. The primary objectives of this study were to: (1) characterize AMHR2 expression in granulosa cells during follicle development; (2) identify factors that regulate AMHR2 mRNA expression in granulosa cells; and (3) examine the role of AMH signaling in granulosa cell differentiation and proliferation. Bovine granulosa cells were isolated from 5- to 8-mm follicles before selection and deviation, as well as from 9- to 12-mm and 13- to 24-mm follicles after selection. Analyses revealed that expression of AMHR2 was greater in 5- to 8-mm follicles compared with 13- to 24-mm follicles (P < 0.05). Granulosa cells treated with bone morphogenetic protein 6 (BMP6) or BMP15, but not BMP2, significantly increased AMHR2 expression when compared with control cultured cells (P < 0.05). In addition, expression of AMH was greater in granulosa cells cultured with BMP2, BMP6, or BMP15 when compared with controls (P < 0.05). Finally, treatment with recombinant human AMH, in vitro, inhibited CYP19A1 expression in a dose-related (10–100 ng/mL) fashion, and reduced granulosa cell proliferation at 48 and 72 hours (P < 0.05). Results from these studies indicate that AMH signaling plays a role in both regulating granulosa cell proliferation and preventing granulosa cells from 5- to 8-mm follicles from undergoing premature differentiation before follicle selection.

The Anti-Müllerian Hormone Precursor (proAMH) Is Not Converted to the Receptor-Competent Form (AMHN,C) in the Circulating Blood of Mice.

Anti-Müllerian hormone (AMH) is a gonadal hormone that regulates aspects of male sexual differentiation and ovarian function. AMH is synthesized as the AMH proprotein precursor (proAMH), which is converted to a receptor-binding form (AMHN,C) by proteolytic cleavage. ProAMH appears to be the predominant species in the ovary, whereas AMHN,C is the prevalent form in circulation. The aim of this study was to determine whether cleavage of proAMH occurs before it is released from the gonad or while in circulation. The individual half-lives of the proAMH and AMHN,C were also determined, as this has important implications for understanding the mechanisms of AMH signaling. Recombinant human (rh)-proAMH or rh-AMHN,C was injected iv into mice. AMH levels were analyzed in a series of repeated blood samples using an assay that detects human, but not murine, AMH. The degree of cleavage of injected proAMH was assessed by immunoprecipitation and Western blotting. The elimination half-life curves were biphasic. The fast-phase elimination was estimated at 6 and 11 minutes for rh-proAMH and rh-AMHN,C, respectively. The slow-phase half-life estimates were 2.4 and 3.8 hours for rh-proAMH and rh-AMHN,C, respectively. Immunoprecipitation of rh-proAMH 1 hour after injection determined that no detectable conversion of proAMH to AMHN,C was occurring in circulation. The data suggest that the ratio of proAMH to AMHN,C in the circulation is not altered after it is released from the gonads and that the levels of these 2 circulating forms are likely to reflect AMH activity in the gonad.

A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone

The utility of serum anti-Müllerian hormone (AMH) assays in assessment of female fertility have been investigated extensively but little is known about the biological activity of the hormone being studied. ProAMH is the proprotein precursor and is incapable of binding to the AMH-specific type II receptor. Proteolytic cleavage generates receptor-competent AMHN,C which is a non-covalent complex of the N- and C-terminal cleavage fragments. Commercially available AMH assays do not differentiate the two forms of AMH. Techniques were developed to dissociate the AMHN,C complex and abolish its two-site immunoassay immunoreactivity. This allowed specific quantification of proAMH. The surfactant sodium deoxycholate (DOC) dissociated AMHN,C without disrupting binding of proAMH to the capture-antibody with an optimal concentration of 0.1–0.2%w/v. The incorporation of a DOC incubation step into the AMH Gen II ELISA detected proAMH, with AMHN,C cross-detection conservatively estimated at 6.0% ± 2.5% (mean ± S.D.). The intra-assay and inter-assay variability were estimated at 8.0%CV and 13.0%CV respectively. The levels of proAMH and total AMH were assessed in 5 boys and 5 men and the proportion of proAMH was found to be significantly higher in boys (p = 0.005). This study will facilitate further investigation of the role of proteolytic cleavage in AMH signalling.

Role of vitamin D in ovarian physiology and its implication in reproduction: a systematic review

To report an update on the role of vitamin D (VD) in ovarian physiology with a focus on genes involved in steroidogenesis, follicular development, and ovarian reserve, as well as ovulatory dysfunction associated with polycystic ovary syndrome (PCOS), and ovarian response to assisted reproductive technology (ART). Systematic review. Not applicable. Human, animal, and cell culture models. Pubmed literature search. Granulosa cell function, serum antimüllerian hormone (AMH), AMH and its receptor gene expression, soluble receptor for advanced glycation end-products (sRAGE), PCOS parameters, and ART outcome. In human granulosa cells, VD alters AMH signaling, FSH sensitivity, and progesterone production and release, indicating a possible physiologic role for VD in ovarian follicular development and luteinization. In the serum, 25-hydroxyvitamin D (25OH-D) is positively correlated with AMH, and appropriate VD supplementation in VD-depleted women can suppress the seasonal changes that occur in serum AMH. In VD-deficient women with PCOS, VD supplementation lowers the abnormally elevated serum AMH levels, possibly indicating a mechanism by which VD improves folliculogenesis. The antiinflammatory sRAGE serum levels significantly increase in women with PCOS after VD replacement. Although follicular fluid 25OH-D correlates with IVF outcomes, there is a lack of data pertaining to the impact of VD supplementation on pregnancy rates following IVF. This review underscores the need for understanding the mechanistic actions of VD in ovarian physiology and the critical need for randomized trials to elucidate the impact of VD supplementation on controlled ovarian hyperstimulation/IVF outcome and ovulatory dysfunction associated with PCOS.