Serum Anti-Müllerian Hormone Is an Effective Indicator of Antral Follicle Counts but Not Primordial Follicle Counts.

Serum anti-Müllerian hormone (AMH) is a biomarker for predicting antral follicle counts but there is no clear consensus on whether AMH is indicative of primordial follicle counts in humans. Mice were used as a model species in this study to obtain accurate follicle counts across the reproductive phase of life. Serum AMH was measured in 62 female C57Bl6/J mice aged 25 to 401 days. Primordial and primary follicles were determined by stereological counts and all secondary and antral follicles were counted in serial histological sections. Serum AMH was most strongly correlated with small- and medium-sized antral follicles. Immunohistochemistry and stepwise multiple regression confirmed that these follicle development stages are the key determinants of serum AMH, with little contribution from other stages. Primordial follicles were not found to have strong correlations with serum AMH or antral follicle counts, particularly in younger females, but the strength of the association appeared to increase with age. This result is likely attributed to high interindividual variation in primordial follicle activation and preantral follicle survival rates. Recent large studies in human populations have shown similar results but the primary limitation of these studies was that primordial follicle counts were determined from ovarian cortical biopsies, where regional variation in follicle distribution may affect the quality of the data. In the present study, whole ovaries were surveyed, eliminating this limitation. The findings indicate that primordial follicle counts are not closely related with either serum AMH or antral follicle counts in females in the early phase of the reproductive phase of life.

Key signalling pathways underlying the aetiology of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine condition characterised by a range of reproductive, endocrine, metabolic and psychological abnormalities. Reports estimate that around 10% of women of reproductive age are affected by PCOS, representing a significant prevalence worldwide, which poses a high economic health burden. As the origin of PCOS remains largely unknown, there is neither a cure nor mechanism-based treatments leaving patient management suboptimal and focused solely on symptomatic treatment. However, if the underlying mechanisms underpinning the development of PCOS were uncovered then this would pave the way for the development of new interventions for PCOS. Recently, there have been significant advances in our understanding of the underlying pathways likely involved in PCOS pathogenesis. Key insights include the potential involvement of androgens, insulin, anti-Müllerian hormone and transforming growth factor beta in the development of PCOS. This review will summarise the significant scientific discoveries on these factors that have enhanced our knowledge of the mechanisms involved in the development of PCOS and discuss the impact these insights may have in shaping the future development of effective strategies for women with PCOS.

Fetal resorption coincides with dysregulated LH secretion in AMH-overexpressing mice.

Female anti-Müllerian hormone (AMH) overexpressing (Thy1.2-AMHTg/0) mice experience fetal resorption (miscarriage) by mid-gestation. This study examined whether the ovary, uterine implantation sites and hypothalamus are potential sites of AMH action, as AMH type-2 receptor (AMHR2) expression is reported in each tissue. Pregnancy in Thy1.2-AMHTg/0 mice was compared to wild-type (WT) mice via histological examination of implantation sites, hormone assays, embryo culture and embryo transfer. Uterine AMH and AMHR2 expression was examined by RT-qPCR and immunohistochemistry. The first signs of fetal resorption in the Thy1.2-AMHTg/0 dams occurred at embryonic day 9.5 (E9.5) with 100% of fetuses resorbing by E13.5. Cultured embryos from Thy1.2-AMHTg/0 dams had largely normal developmental rates but a small proportion experienced a minor developmental delay relative to embryos from WT dams. However, embryos transferred from WT donor females always failed to survive to term when transferred into Thy1.2-AMHTg/0 dams. Amh and Amhr2 mRNA was detected in the gravid uterus but at very low levels relative to expression in the ovaries. Progesterone and estradiol levels were not significantly different between WT and Thy1.2-AMHTg/0 dams during pregnancy but luteinizing hormone (LH) levels were significantly elevated in Thy1.2-AMHTg/0 dams at E9.5 and E13.5 relative to WT dams. Collectively, these experiments suggest that AMH overexpression does not cause fetal resorption through an effect on oocytes or preimplantation embryo development. The Thy1.2-AMHTg/0 fetal resorption phenotype is nearly identical to that of transgenic LH overexpression models, suggesting that neuroendocrine mechanisms may be involved in the cause of the miscarriage.

Proteolytic activation of anti-Müllerian hormone is suppressed in adolescent girls.

PURPOSE: The ratio of the anti-Müllerian hormone (AMH) precursor (proAMH) to active AMH (AMHN,C) is higher in childhood than in adulthood but has never been quantified during adolescence. The ratio of proAMH to total AMH (AMH prohormone index, API) was examined during the puberty in healthy girls. The API was also compared between girls with and without polycystic ovary syndrome (PCOS) to determine if there were differences that could assist in PCOS diagnosis during adolescence. METHODS: Total AMH and proAMH were measured by immunoassay in a single-centre, cross-sectional observational study; 61 controls and 29 girls with PCOS were included in the study (age range 8-21 years). The API was calculated as proAMH as a percentage of total AMH. Differences in API between control and PCOS subjects and across age-groups were examined by Welch's ANOVA. The relationship between API and a range of metabolic parameters was examined by Pearson correlation. RESULTS: The API in healthy females increased between the ages of 10~15 years and declined from 15~20 years (p < 0.001). The API was negatively correlated with body mass index in the control (p = 0.04) and PCOS groups (p = 0.007). The API was associated with factors related to adiposity and lipid metabolism. The API was not significantly different in control girls and girls with PCOS. CONCLUSIONS: Higher API during adolescence suggests that proteolytic activation of proAMH is suppressed during this life stage. API was not different between control girls and girls with PCOS indicating that it is not useful in diagnosis of PCOS during adolescence.

Hyperactivation of dormant primordial follicles in ovarian endometrioma patients.

Serum anti-Müllerian hormone (AMH) levels decrease after surgical treatment of ovarian endometrioma. This is the main reason that surgery for ovarian endometrioma endometriosis is not recommended before in vitro fertilization, unless the patient has severe pain or suspected malignant cysts. Furthermore, it has been suggested that ovarian endometrioma itself damages ovarian reserve. This raises two important challenges: (1) determining how to prevent surgical damage to the ovarian reserve in women with ovarian endometrioma and severe pain requiring surgical treatment and (2) deciding the best treatment for women with ovarian endometrioma without pain, who do not wish to conceive immediately. The mechanisms underlying the decline in ovarian reserve are potentially induced by both ovarian endometrioma and surgical injury but the relative contribution of each process has not been determined. Data obtained from various animal models and human studies suggest that hyperactivation of dormant primordial follicles caused by the local microenvironment of ovarian endometrioma (mechanical and/or chemical cues) is the main factor responsible for the decreased primordial follicle numbers in women with ovarian endometrioma. However, surgical injury also induces hyperactivation of dormant primordial follicles, which may further reduce ovarian reserve after removal of the endometriosis. Although further studies are required to elucidate the mechanisms underlying diminished ovarian reserve in women with ovarian endometrioma, the available data strongly suggests the need to prevent/minimize hyperactivation of dormant primordial follicles, regardless of whether surgery is performed, for better clinical management of ovarian endometrioma.

The proportion of cleaved anti-Müllerian hormone is higher in serum but not follicular fluid of obese women independently of polycystic ovary syndrome.

RESEARCH QUESTION: Does the relative distribution of anti-Müllerian hormone (AMH) isoforms differ between patients depending on their body mass index (BMI) and polycystic ovary syndrome (PCOS) status in serum and follicular fluid? DESIGN: Obese and normal weight patients (PCOS [n = 70]; non-PCOS [n = 37]) were selected for this case-control study in the serum. Between 2018 and 2019, obese (n = 19) and normal weight (n = 20) women with or without PCOS who were receiving IVF treatment were included in the follicular fluid study. The bio-banked serums and follicular fluid were tested for total AMH (proAMH and AMHN,C combined) and proAMH using an automatic analyzer. The AMH prohormone index (API = [proAMH]/[total AMH]x 100) was calculated as an inverse marker of conversion of proAMH to AMHN,C, with only the latter isoform that could bind to the AMH receptor complex. RESULTS: The API was not significantly different between controls and women with PCOS, whereas obese women had a lower API compared with their normal weight counterparts. Grouping PCOS and controls, a lower API was found in obese versus normal weight women, suggesting a greater conversion of proAMH to AMHN,C. The API in the serum was significantly correlated with metabolic parameters. In the follicular fluid, API is not different between obese and normal weight women independently of PCOS and is higher than in the concomitant serum. CONCLUSIONS: The proportion of inactive form of AMH in the serum is higher in normal weight versus obese women but not in the follicular fluid, independently of PCOS. The conversion of proAMH into the cleaved isoform is likely to occur in extra-ovarian tissues and to exacerbate in obese individuals.

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

OBJECTIVE: 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. DESIGN: Cross-sectional study using biobanked samples collected between July 2008 and January 2010. SETTING: Not applicable. PATIENT(S): Overweight, premenopausal women aged 18-45 years 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). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 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). RESULT(S): 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). CONCLUSION(S): 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.

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 (TGFß) 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 TGFß 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, TGFß 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.

Increased circulating leukocyte numbers and altered macrophage phenotype correlate with the altered immune response to brain injury in metallothionein (MT)-I/II null mutant mice.

BACKGROUND: Metallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system. METHODS: Wild type and MT-I/II(-/-) mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay. RESULTS: Comparison of MT-I/II(-/-) mice with wild type controls following cryolesion brain injury revealed that the MT-I/II(-/-) mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II(-/-) mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II(-/-) mice that was independent of brain injury. CONCLUSIONS: These results contribute to the evidence that MT-I/II(-/-) mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II(-/-) mice after brain injury relative to wild type mice.