Genome-wide association study of anti-Müllerian hormone levels in pre-menopausal women of late reproductive age and relationship with genetic determinants of reproductive lifespan.

Anti-Müllerian hormone (AMH) is required for sexual differentiation in the fetus, and in adult females AMH is produced by growing ovarian follicles. Consequently, AMH levels are correlated with ovarian reserve, declining towards menopause when the oocyte pool is exhausted. A previous genome-wide association study identified three genetic variants in and around the AMH gene that explained 25% of variation in AMH levels in adolescent males but did not identify any genetic associations reaching genome-wide significance in adolescent females. To explore the role of genetic variation in determining AMH levels in women of late reproductive age, we carried out a genome-wide meta-analysis in 3344 pre-menopausal women from five cohorts (median age 44-48 years at blood draw). A single genetic variant, rs16991615, previously associated with age at menopause, reached genome-wide significance at P = 3.48 × 10-10, with a per allele difference in age-adjusted inverse normal AMH of 0.26 standard deviations (SD) (95% confidence interval (CI) [0.18,0.34]). We investigated whether genetic determinants of female reproductive lifespan were more generally associated with pre-menopausal AMH levels. Genetically-predicted age at menarche had no robust association but genetically-predicted age at menopause was associated with lower AMH levels by 0.18 SD (95% CI [0.14,0.21]) in age-adjusted inverse normal AMH per one-year earlier age at menopause. Our findings provide genetic support for the well-established use of AMH as a marker of ovarian reserve.

Anti-Müllerian Hormone Accelerates Pathological Process of Insulin Resistance in Polycystic Ovary Syndrome Patients.

Insulin resistance (IR) is one of the most common features of polycystic ovary syndrome (PCOS), which is related to obesity. Whether increased anti-Müllerian hormone (AMH) levels in PCOS are involved in the pathogenesis of insulin resistance remains unclear. We investigated serum levels of leptin and AMH along with basic clinical and metabolic parameters in 114 PCOS patients and 181 non-PCOS women. PCOS patients presented higher fasting blood glucose, insulin concentrations and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) in addition to body mass index (BMI), lipids profiles and hormone levels. HOMA-IR showed a positive correlation with BMI, AMH, leptin, and low-density lipoprotein-cholesterol (LDL-c) levels. Interestingly, AMH is strongly positively correlated with HOMA-IR and insulin concentrations for 1st and 2nd hours of glucose treatment after fasting. Among PCOS women with BMI≥25 kg/m2, high AMH level group showed an increased HOMA-IR when compared to normal AMH level. However, among PCOS women with normal BMI, women with high AMH presented an elevated fasting insulin levels but not HOMA-IR when compared to normal AMH group. In vitro treatment of isolated islet cells with high concentration of leptin (200 ng/ml) or high leptin plus high concentration of AMH (1 ng/ml) significantly enhanced insulin secretion. Importantly, co-treatment of AMH plus leptin upregulates the expression of pro-apoptotic proteins, such as Bax, caspase-3, and caspase-8 after incubating with a high level of glucose. These results suggest that AMH may involve in the pathological process of pancreatic ß-cells in obese PCOS women.

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-ß 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.

AMH prevents primordial ovarian follicle loss and fertility alteration in cyclophosphamide-treated mice.

The follicular ovarian reserve, constituted by primordial follicles (PMFs), is established early in life, then keeps declining regularly along reproductive life. The maintenance of a normal female reproductive function implies the presence of a vast amount of dormant PMFs. This process involves a continuous repression of PMF activation into early growing follicle through the balance between factors activating the initiation of follicular growth, mainly actors of the PI3K signaling pathway, and inhibiting factors such as anti-Müllerian hormone (AMH). Any disruption of this balance may induce follicle depletion and subsequent infertility. It has been recently proposed that cyclophosphamide (Cy), an alkylating agent commonly used for treating breast cancer, triggers PMF activation, further leading to premature ovarian insufficiency. Preventing chemotherapy-induced ovarian dysfunction might represent an interesting option for preserving optimal chances of natural or medically assisted conceptions after healing. The aim of the present study was to evaluate, in a model of Cy-treated pubertal mice, whether AMH administration might restrain PMF depletion. The counting of the total PMF number within mouse ovaries showed that recombinant AMH prevented Cy-induced PMF loss. Western blot analysis revealed activation of PI3K signaling pathway after Cy administration. After AMH injection, FOXO3A phosphorylation, a main actor of PMF activation, was significantly decreased. Taken together, these results support a protective role of AMH against Cy-induced follicular loss. We also provide evidence for a possible role of autophagy in the preservation of follicular pool reserve. Therefore, concomitant recombinant AMH administration during chemotherapy might offer a new option for preserving young patients' fertility.-Sonigo, C., Beau, I., Grynberg, M., Binart, N. AMH prevents primordial ovarian follicle loss and fertility alteration in cyclophosphamide-treated mice.

Anti-Müllerian hormone as a qualitative marker - or just quantity?

PURPOSE OF REVIEW: In this review, we will summarize research looking into anti-Müllerian hormone (AMH) as a measure of oocyte quality. RECENT FINDINGS: AMH is a key factor involved in embryogenesis but also in the development of early follicles. Owing to its relatively small inter and intracycle variability, it has become a widely used method of ovarian reserve testing. In the realm of assisted reproductive technology, it has demonstrated a reliable ability to gauge the number of oocytes obtained during an in-vitro fertilization cycle. For these purposes, AMH is a readily measured quantitative tool. However, its qualitative role is as yet undefined. SUMMARY: Although levels of this hormone have been associated with fertilization, blastulation, implantation, and clinical pregnancy rates, there is no clear link with live-birth rates. Furthermore, AMH levels do not appear to correspond with risk of fetal trisomy. AMH does show significant predictive value for the risk of premature ovarian insufficiency and time to onset of menopause.

Age effect on in vitro fertilization pregnancy mediated by anti-Mullerian hormone (AMH) and modified by follicle stimulating hormone (FSH).

BACKGROUND: Both follicle stimulating hormone (FSH) and anti-mullerian hormone (AMH) are widely used to assess the ovarian reserve in women for in vitro fertilization (IVF). However, studies also showed that both AMH and FSH are significantly associated with age: as age increases, AMH decreases and FSH increases. This study aims to investigate the mechanism of age effect on IVF live birth rate, particularly through mediation and interaction by AMH and FSH. METHODS: We conducted a retrospective cohort study of 13970 IVF cycles collected by eIVF from 2010 to 2016. A series of logistic mixed models were used to estimate the association of live birth and AMH (or FSH). The mediation effects and proportion mediated, were quantified by our previously proposed mediation analyses. We further investigated the FSH-modified mediation effects on live birth rate through AMH, accounting for the nonlinear age effect. RESULTS: Our analyses showed that age effect on live birth was mediated more by AMH than by FSH (18 vs. 6%). The mediation effect through AMH can be further modified by FSH level regardless of women's age. CONCLUSIONS: In summary, mediation model provides a new perspective elucidating the mechanism of IVF successful rate by age. The majority of the age effect on live birth rate remained unexplained by AMH and FSH, suggesting its importance and independent role in IVF.

Endometriosis reduces ovarian response in controlled ovarian hyperstimulation independent of AMH, AFC, and women's age measured by follicular output rate (FORT) and number of oocytes retrieved.

PURPOSE: To determine the influence of endometriosis on the ovarian response during controlled ovarian hyperstimulation measured by number of oocytes retrieved and the follicular output rate (FORT). METHODS: A retrospective, single center study included 96 women, who underwent ICSI treatments for male factor infertility according to World Health Organisation between 2016 until 2018. A total of 96 patients were included in the study with 205 fresh ICSI cycles. The study group included 26 patients with endometriosis after surgical and medical treatment; the control group included 70 patients without endometriosis. The women with endometriosis underwent 47 and the control group 158 ICSI cycles. Women underwent fresh intracytoplasmatic sperm injection cycles after controlled ovarian hyperstimulation following a GnRH-antagonist protocol. The FORT was calculated as the ratio of pre-ovulatory follicle count × 100/small antral follicle count at baseline. RESULTS: A lower number of retrieved oocytes (5.89 vs. 7.25, p = 0.045), lower FORT (75.67 vs. 94.63, p = 0.046), lower number of metaphase II oocytes (4.87 vs. 6.04, p = 0.046), and lower fertilization rate after intracytoplasmatic sperm injection (40.61 vs. 57.76, p = 0.003) were found in women with endometriosis compared to women without endometriosis. The number of oocyctes retrieved was 0.71 lower in the group with endometriosis than in the group without (p = 0.026). The FORT was 24.55% lower in the group with endometriosis (p = 0.025). CONCLUSIONS: Endometriosis reduces the FORT and the number of metaphase-II oocytes after controlled ovarian hyperstimulation independly of women's age, antral follicle count and anti-Müllerian hormone.

Anti-Müllerian hormone: a critical factor for female fertility and reproductive health.

Anti-Müllerian hormone (AMH) is a glycoprotein produced by the granulosa cells of preantral and small antral follicles. AMH concentrations reflect ovarian physiology with high precision, thus serving as a more sensitive marker of the ovarian re-serve than chronological age. This hormone plays a role in the pathogenesis of menstrual disorders and fertility in both obesity and polycystic ovary syndrome. The evaluation of AMH may also be useful in diagnosing or monitoring therapy of granulosa cell ovarian tumors.

Hyperproliferation of mitotically active germ cells due to defective anti-Müllerian hormone signaling mediates sex reversal in medaka.

The function of AMH (Anti-Müllerian hormone), a phylogenetically ancient member of the TGFß family of proteins, in lower vertebrates is largely unknown. Previously, we have shown that the gene encoding the type II anti-Müllerian hormone receptor, amhrII, is responsible for excessive germ cell proliferation and male-to-female sex reversal in the medaka hotei mutant. In this study, functional analyses in cultured cells and of other amhrII mutant alleles indicate that lack of AMH signaling causes the hotei phenotype. BrdU incorporation experiments identified the existence of both quiescent and mitotically active germ cells among the self-renewing, type I population of germ cells in the developing gonad. AMH signaling acts in supporting cells to promote the proliferation of mitotically active germ cells but does not trigger quiescent germ cells to proliferate in the developing gonad. Furthermore, we show that the male-to-female sex reversal phenotype in hotei mutants is not a direct consequence of AMH signaling in supporting cells, but is instead mediated by germ cells. Our data demonstrate that interfollicular AMH signaling regulates proliferation at a specific stage of germ cell development, and that this regulation is crucial for the proper manifestation of gonadal sex directed by sex determination genes.

Role of Anti-Müllerian Hormone in pathophysiology, diagnosis and treatment of Polycystic Ovary Syndrome: a review.

Polycystic ovary syndrome (PCOS) is the most common cause of chronic anovulation and hyperandrogenism in young women. Excessive ovarian production of Anti-Müllerian Hormone, secreted by growing follicles in excess, is now considered as an important feature of PCOS. The aim of this review is first to update the current knowledge about the role of AMH in the pathophysiology of PCOS. Then, this review will discuss the improvement that serum AMH assay brings in the diagnosis of PCOS. Last, this review will explain the utility of serum AMH assay in the management of infertility in women with PCOS and its utility as a marker of treatment efficiency on PCOS symptoms. It must be emphasized however that the lack of an international standard for the serum AMH assay, mainly because of technical issues, makes it difficult to define consensual thresholds, and thus impairs the widespread use of this new ovarian marker. Hopefully, this should soon improve.

Unifying theory of adult resting follicle recruitment and fetal oocyte arrest.

One of the biggest mysteries of ovarian physiology is what controls the emergence of adult primordial follicles from the resting stage, and their steady depletion over the woman's lifetime. A related mystery is why do early oogonia begin meiosis in the fetus and then suddenly arrest for most of fetal and adult life. If fetal oocyte arrest did not occur after meiotic activation, there would be no oocytes left in the female baby by the time she is born. Similarly, without a steady controlled release in the adult ovary of resting follicles, the adult woman would run out of her eggs prematurely and have an early menopause. Could there be a similarity between what causes fetal oocyte arrest and what causes adult oocyte recruitment? The answer begins with the observation of a sudden massive recruitment of primordial follicles after human ovarian transplantation, and the embryologic discoveries about oocyte activation and the time of differentiation of cortex and medulla. The unifying theory is that ovarian cortical tissue pressure controls both fetal oocyte arrest and adult oocyte recruitment.

Ovarian response biomarkers: physiology and performance.

PURPOSE OF REVIEW: This review summarizes recent technological developments in the measurement of anti-Müllerian hormone (AMH) and presents an update of the relative performance characteristics of both AMH and antral follicle count (AFC) in predicting the ovarian response to controlled stimulation. RECENT FINDINGS: The introduction of two automated AMH immunoassays appears to have resolved the majority of preanalytical and analytical limitations of the manual assays thereby facilitating the delivery of consistent and accurate results. However, as they exhibit different calibration from preexisting assays, derivation of new reference ranges and clinical thresholds for prediction of ovarian response categories will be required. Randomized controlled trials have highlighted the superiority of AMH to AFC in ovarian response prediction and provide a sound basis for its ongoing assessment for stratification and personalization of treatment. SUMMARY: Trial evidence combined with full automation of AMH assay measurement suggests that the future for ovarian response prediction will be AMH focused. Sonography will continue to be invaluable for the identification of tubal, ovarian and endometrial pathologies, but the days of counting follicles are numbered.

ß-Catenin is essential for Müllerian duct regression during male sexual differentiation.

During male sexual differentiation, the transforming growth factor-ß (TGF-ß) signaling molecule anti-Müllerian hormone (AMH; also known as Müllerian inhibiting substance, MIS) is secreted by the fetal testes and induces regression of the Müllerian ducts, the primordia of the female reproductive tract organs. Currently, the molecular identity of downstream events regulated by the AMH signaling pathway remains unclear. We found that male-specific Wnt4 expression in mouse Müllerian duct mesenchyme depends upon AMH signaling, implicating the WNT pathway as a downstream mediator of Müllerian duct regression. Inactivation of ß-catenin, a mediator of the canonical WNT pathway, did not affect AMH signaling activation in the Müllerian duct mesenchyme, but did block Müllerian duct regression. These data suggest that ß-catenin mediates AMH signaling for Müllerian duct regression during male sexual differentiation.

The ovarian reserve of primordial follicles and the dynamic reserve of antral growing follicles: what is the link?

The growing follicles develop from a reserve of primordial follicles constituted early in life. From this pre-established reserve, a second ovarian reserve is formed, which consists of gonadotropin-responsive small antral growing follicles and is a dynamic reserve for ovulation. Its size, evaluated by direct antral follicular count or endocrine markers, determines the success of assisted reproductive technologies in humans and embryo production biotechnologies in animals. Strong evidence indicates that these two reserves are functionally related. The size of both reserves appears to be highly variable between individuals of similar age, but the equilibrium size of the dynamic reserve in adults seems to be specific to each individual. The dynamics of both follicular reserves appears to result from the fine tuning of regulations involving two main pathways, the phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDPK1)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) and the bone morphogenetic protein (BMP)/anti-Müllerian hormone (AMH)/SMAD signaling pathways. Mutations in genes encoding the ligands, receptors, or signaling effectors of these pathways can accelerate or modulate the exhaustion rate of the ovarian reserves, causing premature ovarian insufficiency (POI) or increase in reproductive longevity, respectively. With female aging, the decline in primordial follicle numbers parallels the decrease in the size of the dynamic reserve of small antral follicles and the deterioration of oocyte quality. Recent progress in our knowledge of signaling pathways and their environmental and hormonal control during adult and fetal life opens new perspectives to improve the management of the ovarian reserves.

Is AMH a regulator of follicular atresia?

We discuss the hypothesis that AMH is an intraovarian regulator that inhibits follicular atresia within the human ovary. Several indirect lines of evidence derived from clinical and basic science studies in a variety of different patient populations and model systems collectively support this hypothesis. Evidence presented herein include 1) timing of onset of menopause in women with polycystic ovary syndrome, 2) site of cellular origin and timing of AMH production, 3) AMH's influence on other critical growth factors and enzymes involved in folliculogenesis, and 4) AMH's inhibition of granulosa apoptosis. If this hypothesis is true, it may provide insight for treatment strategies for prevention and treatment of premature ovarian insufficiency, slowing natural ovarian aging, and/or delaying eventual ovarian failure. Such findings may lead to the development of 1) AMH agonists for retarding the onset of menopause and/or as a chemoprotectant prior to cancer therapy and 2) AMH antagonists for the treatment of PCOS.

The Role of Anti-Müllerian Hormone in Ovarian Function.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor ß (TGFß) superfamily, whose actions are restricted to the endocrine-reproductive system. Initially known for its role in male sex differentiation, AMH plays a role in the ovary, acting as a gatekeeper in folliculogenesis by regulating the rate of recruitment and growth of follicles. In the ovary, AMH is predominantly expressed by granulosa cells of preantral and antral follicles (i.e., post primordial follicle recruitment and prior to follicle-stimulating hormone (FSH) selection). AMH signals through a BMP-like signaling pathway in a manner distinct from other TGFß family members. In this review, the latest insights in AMH processing, signaling, its regulation of spatial and temporal expression pattern, and functioning in folliculogenesis are summarized. In addition, effects of AMH variants on ovarian function are reviewed.

Analysis of androgen receptor and anti-Müllerian hormone pathways in human granulosa cells under luteinizing hormone treatment.

BACKGROUND: The objective of this study was to determine the gene expression profiles of the androgen/androgen receptor (AR) and anti-Müllerian hormone (AMH)/ Sry-related high-mobility group box 9 (SOX9) pathways in granulosa-luteal cells from patients undergoing standard in vitro fertilization (IVF) with or without recombinant luteinizing hormone (rLH) therapy. METHODS: Levels of reproductive hormones in the pre-ovulatory follicular fluid and the expression levels of LHR (luteinizing hormone receptor), AR, SOX9, AMH, AR-associated protein 54(ARA54)and ARA70 were determined in granulosa-luteal cells by real-time reverse-transcription PCR. The effects of androgen and rLH treatments on AR and AMH expression levels were also tested in vitro using HO23 cells. RESULTS: We collected 35 an 70 granulosa cell samples from patients cycled with and without rLH supplementation, respectively. The clinical outcomes were similar in patients who received rLH therapy and those who did not, though the pre-ovulatory follicular fluid levels of androstenedione, testosterone, and estradiol were significantly higher and progesterone was lower in the rLH supplementation group. Moreover, granulosa-luteal cell mRNA levels of LHR, AR, AMH, and SOX9 were significantly higher in the rLH supplementation group relative to the group that did not receive rLH supplementation. In addition, we observed significant correlations between LHR and AR mRNA expression and among AR, AMH, and SOX9 mRNA expression in granulosa-luteal cells from patients undergoing standard IVF treatment. CONCLUSIONS: Increased expression of LHR, AR, AMH, and SOX9 is characteristic of granulosa-luteal cells from IVF/ intracytoplasmic sperm injection (ICSI) patients receiving rLH supplementation.

The bed nucleus of the stria terminalis has developmental and adult forms in mice, with the male bias in the developmental form being dependent on testicular AMH.

Canonically, the sexual dimorphism in the brain develops perinatally, with adult sexuality emerging due to the activating effects of pubescent sexual hormones. This concept does not readily explain why children have a gender identity and exhibit sex-stereotypic behaviours. These phenomena could be explained if some aspects of the sexual brain networks have childhood forms, which are transformed at puberty to generate adult sexuality. The bed nucleus of stria terminalis (BNST) is a dimorphic nucleus that is sex-reversed in transsexuals but not homosexuals. We report here that the principal nucleus of the BNST (BNSTp) of mice has developmental and adult forms that are differentially regulated. In 20-day-old prepubescent mice, the male bias in the principal nucleus of the BNST (BNSTp) was moderate (360 ± 6 vs 288 ± 12 calbindin(+ve) neurons, p < 0.0001), and absent in mice that lacked a gonadal hormone, AMH. After 20 days, the number of BNSTp neurons increased in the male mice by 25% (p < 0.0001) and decreased in female mice by 15% (p = 0.0012), independent of AMH. Adult male AMH-deficient mice had a normal preference for sniffing female pheromones (soiled bedding), but exhibited a relative disinterest in both male and female pheromones. This suggests that male mice require AMH to undergo normal social development. The reported observations provide a rationale for examining AMH levels in children with gender identity disorders and disorders of socialization that involve a male bias.

Presence of anti-müllerian hormone in the squirrel monkey (Saimiri boliviensis): gender and seasonal differences.

BACKGROUND: Anti-mullerian hormone (AMH) is a glycoprotein of the transforming growth factor-ß superfamily secreted by male Sertoli cells and female ovarian granulosa cells. This study determined squirrel monkey AMH levels as influenced by gender and seasonality. METHODS: Squirrel monkey sera AMH were measured by an enzymatically amplified two-site immunoassay. RESULTS: A significant difference (P<0.001) was found in AMH levels between male (mean=3.46 ng/ml) and female squirrel monkeys (mean=22.12 ng/ml). A significant difference (P<0.05) was found in male AMH levels between breeding (mean=4.21 ng/ml) and non-breeding seasons (mean=2.78 ng/ml). No significant differences were found between female groups. CONCLUSIONS: Anti-mullerian hormone levels in female squirrel monkeys are the highest in any primate species reported, whereas in the male, levels are within reported ranges. The AMH assay may allow us soon to assess the squirrel monkey fertility potential as a function of various factors.