anti-Müllerian Hormone Production Research Articles

Reduced anti-Müllerian hormone action in cumulus-oocyte complexes is beneficial for oocyte maturation without affecting oocyte competence.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.

Omaveloxolone Prevents Polystyrene Microplastic-Induced Ovarian Granulosa Cell Apoptosis via the Keap1/Nrf2/HO-1 Pathway in Rats.

Microplastics (MPs) are persistent environmental pollutants that enter the circulatory system and subsequently reduce sperm quantity and quality. However, the influence of polystyrene MPs (PS-MPs) on the ovary and relevant mechanisms remain elusive. Herein, we aimed to examine the impact of PS-MPs on oxidative disorders in ovarian tissues and elucidate the underlying mechanisms. Healthy female rats were treated with different concentrations of 0.5µm PS-MPs (diluted in deionized H2O) for 90 days. Upon examination of hematoxylin-eosin-stained ovarian tissue sections, the number of growing follicles was reduced in PS-MP-treated rats when compared with that in control rats. Enzyme-linked immunosorbent assays revealed that PS-MP exposure markedly reduced anti-Müllerian hormone (AMH) levels. Treatment with PS-MPs downregulated superoxide dismutase, glutathione, and catalase activities in ovarian tissues while upregulating malondialdehyde levels. Furthermore, exposure to PS-MP blocked the Keap1/Nrf2/HO-1 signal transduction pathway. PS-MPs also triggered apoptosis in the ovarian tissue, as evidenced by increased TUNEL staining and expression levels of cleaved caspase-9, Bax, and Bcl-2. To reactivate the Keap1/Nrf2/HO-1 pathway, rats were co-administered PS-MPs and omaveloxolone (Oma), an Nrf2 activator, for 1week. We found that Oma could counteract the PS-MP-mediated effects on oxidative disorder, apoptosis, AMH production, and follicle number in rat ovarian tissues. To develop an in vitro model, granulosa cells (GCs) were treated with 10μM H2O2 for 12h to induce oxidative stress. H2O2-stimulated GCs exhibited attenuated cell growth and upregulated apoptosis and oxidative stress. Oma administration could ameliorate the H2O2-induced effects in terms of regulating cell viability, apoptosis, and oxidative stress in GCs. In summary, PS-MPs could induce apoptosis and oxidative stress via the Keap1/Nrf2/HO-1 signaling pathway in both rats and GCs.

Neurotrophin-4 promotes in vitro development and maturation of human secondary follicles yielding metaphase II oocytes and successful blastocyst formation.

Does a matrix-free culture system supplemented with neurotrophic factor 4 (NT4) improve human in vitro follicular development and meiotic maturation, ultimately resulting in fertilizable oocytes? NT4 supplementation of in vitro culture significantly enhances the growth, steroid hormone production, and maturity potential of human secondary follicles derived from fresh ovarian medulla (from post- and pre-pubertal patients), thereby yielding fertilizable oocytes. Reconstituting folliculogenesis in vitro is of paramount importance in the realms of fertility preservation, reproductive biology research, and reproductive toxicity assessments. However, the efficiency of in vitro culture systems remains suboptimal, as the attainment of fertilizable oocytes from in vitro growth (IVG) of human follicles remains unachieved, with the data being particularly scant regarding follicles from prepubertal girls. We have previously found that mouse oocytes from secondary follicles derived from IVG are deficient in neuroendocrine regulation. NT4 and its corresponding receptor have been identified in human follicles. Significantly, the addition of NT4 during the IVG process markedly enhances both follicle growth and oocyte maturation rates in mice. Fresh medulla tissue obtained during tissue preparation for ovarian tissue cryopreservation (OTC) were collected from 10 patients aged from 6 to 21 years old, all of whom had undergone unilateral oophorectomy as a means of fertility preservation. Isolated secondary follicles were individually cultured in vitro with or without NT4 in a matrix-free system. Secondary follicles, extracted via enzymatic digestion and mechanical disruption from each patient, were randomly allocated to either a control group or an NT4-supplemented group (100 ng/ml), followed by individual culture on an ultra-low attachment plate. Follicle growth and viability were assessed by microscopy. Levels of anti-Müllerian hormone (AMH), estradiol, and progesterone in the medium were quantified. An oocyte-specific marker was identified using confocal fluorescence microscopy following DEAD box polypeptide 4 (DDX4) staining. The competence of individual oocytes for maturation and fertilization were assessed after IVM and ICSI with donated sperm samples. Overall, isolated follicles from both groups survived up to 6 weeks with increasing diameters over the duration (P < 0.05), reaching terminal diameters of almost 1 mm with confirmed steroidogenesis and expression of oocyte marker (DDX4), and producing morphologically normal MII oocytes. When compared with the control group, the NT4 group had a similar initial follicular diameter (206 ± 61.3 vs 184 ± 93.4 μm) but exhibited a significant increase in follicular diameter from the ninth day of culture onwards (P < 0.05). From Week 3, estradiol and progesterone production were significantly increased in the NT4 group, while no significant difference was observed in AMH production between groups. The proportion of 'fast-growth' follicles in the NT4 group was significantly higher than that in the control group (13/23 vs 6/24, P < 0.05). An increased efficiency of MII oocyte maturation per live follicle in the NT4 group was also observed (control group vs NT4 group, 4/24 vs 10/23, P < 0.05). It is noteworthy that an MII oocyte obtained from the control group exhibited abnormal fertilization after ICSI. In contrast, an MII oocyte acquired from the NT4 group progressed to the blastocyst stage and showed potential for transfer. N/A. The cohort examined in this study was all patients diagnosed with beta-thalassemia major. Whether this culture system is effective for patients with other diseases remains unknown. Since the chosen dose of NT4 was established based on dose finding in mice, the optimal dose for use in a human IVG system needs further confirmation. The oocytes and embryos procured from this study have not been quantified for ploidy status or epigenetic signatures. Fresh medulla tissue obtained during tissue preparation for OTC may serve as a precious source of fertilizable oocytes for female fertility preservation, even for pre-pubertal girls, without the threat of tumour reintroduction. After further characterization and optimization of the system, this culture system holds the potential to provide a powerful future research tool, for the comprehensive exploration of human follicular development mechanisms and for conducting reproductive toxicity evaluations. This work was supported by the National Key R&D Program of China (grant number 2022YFC2703000) and National Natural Science Foundation of China (grant numbers 82271651 and 81871214). The medium used in human follicle in vitro culture in this study has been applied for a national invention patent in China (No. 202211330660.7). The inventors of the patent, in order, are: Y.G., C.F., and X.L.

Anti-Müllerian hormone: a new approach to fertility assessment in cattle – a review

The anti-Müllerian hormone (AMH) enhances fertility in cattle and hence used as a fertility marker. AMH is produced by granulosa cells of all primordial, primary, secondary follicles, and antral follicles up to 4-5 mm diameter, whereas AMH production decreases once antral stage follicles reach the 4-5 mm stage. There is an individual variation in antral follicle count (AFC) and AMH concentration. One-time determination of AMH concentration will help predict the animals’ reproductive performance at the heifer stage. In turn, it will help to cull those animals with low reproductive potential. There is no significant fluctuation in the AMH concentration during the estrous cycle, and hence it can be determined at any stage of the estrous cycle. This review explores the potential of AMH as a fertility marker and its association with the antral follicle population (AFP) as AMH can be an effective segregative tool to screen low, average or high fertility future performance in cattle.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 μM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. N/A.

Value of Assessing AMH in the Management of Polycystic Ovarian Syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in reproductive age affecting 5 “ 10% of women and is the leading cause of ovulatory dysfunction. Anti-mullerian hormone (AMH) is a dimeric glycoprotein releases from the granulosa cells of small preantral and antral follicles and these concentrations have shown to be proportional to the number of developing follicles in the ovaries. In women with PCOS serum levels are elevated 2- to 3- fold in comparison with normo-ovulatory women, consistent with the increased number of small antral follicles in these women. AMH production per granulosa cell is increased on average 75-fold in anovulatory PCOS compared with normal ovaries. AMH is a marker for ovarian reserve of the women. It also can be used for diagnosis of PCOS. The high level of PCOS has negative effect on reproduction. It inhibits the function of FSH and causes follicular arrest and anovulation. In presence of high AMH there is poor response to oral ovulation inducing agent, gonadotropin and laparoscopic ovarian drilling. Quality of embryo, implantation rate and live birth rate are significantly reduced in presence of high AMH. High LH, hyperandrogenism, hyperinsulinemia have association with high AMH. Androgen is responsible for increasing the number of preantral follicles, hence AMH. Adjuvant therapy like metformin can correct hyperandrogenism, reduce the serum level of AMH and improve ovulation, pregnancy and live birth rate. Pre-treatment AMH level 7.77ng/ml is a cutoff level to predict the response to fertility treatment in PCOS. Ovulation, pregnancy and live birth rate are significantly lower when AMH level is &gt;7.77ng/ml. So, to predict response to treatment and to fix the treatment plan value of assessment of AMH is important in case of PCOS. Bangladesh J Obstet Gynaecol, 2022; Vol. 37(1): 47-62

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100-130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E2), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E2 and FSH to the culture medium.

AMH in Males: Effects of Body Size and Composition on Serum AMH Levels.

Serum concentrations of anti-Müllerian hormone (AMH) have been found to decrease with increasing body mass index (BMI) in many studies. It is not yet clear whether this stems from an adverse effect of adiposity on AMH production, or from dilution due to the greater blood volume that accompanies a larger body size. To investigate a possible hemodilution effect, we explored the relationships between serum AMH levels and different parameters of body composition using linear regression models in a cohort of adult males. Body weight, lean mass (LM), and body surface area (BSA) were found to be better predictors of AMH than measures of adiposity, such as BMI or fat mass. Since both LM and BSA correlate with plasma volume better than adipose tissue, we conclude that hemodilution of AMH does occur in adult males and should be considered for normalization in future studies.

Threshold value of Anti Mullerian Hormone (AMH) and its correlation with other related hormones in Polycystic Ovarian Disease

Background: Polycystic ovarian disease (PCOD) is the commonest endocrine disorder affecting women at reproductive age. As a complex multisystem disorder, its background can be confusing to understand. The key feature is an increased production of AMH by antral follicles. Aim: The aim of the present study was to assess the threshold value of serum AMH in PCOD and its correlation with other related hormones such as FSH, LH and Prolactin. Materials and Methods: Serum AMH, FSH, LH and Prolactin in all subject (PCOD and Non PCOD groups) were estimated using Cobas-e-411analyser. The results analyzed statistically by Using Z-test (p value &lt;0.05 considered as significant), Correlation test and ROC analysis. Result: Serum AMH and LH shown significant increase level in PCOD groups compared to Non PCOD groups (p value=0.00, 0.001 respectively). Serum FSH and Prolactin have shown no much significant variation between the two groups (p value=0.75, 0.8 respectively). Threshold values of serum AMH and LH was observed as 8.47 ng/ml and 15.57 mIU/ml respectively. Additionally, serum AMH and LH has shown the strong positive correlation in PCOD group; no other parameters shown any correlation in both groups. Conclusion: Serum AMH level estimation can be considered as a better choice for diagnosis of PCOD especially in the situation wherever ultrasonography examination is not convenient to conduct

AMH Regulation by Steroids in the Mammalian Testis: Underlying Mechanisms and Clinical Implications.

Anti-Müllerian hormone (AMH) is a distinctive biomarker of the immature Sertoli cell. AMH expression, triggered by specific transcription factors upon fetal Sertoli cells differentiation independently of gonadotropins or sex steroids, drives Müllerian duct regression in the male, preventing the development of the uterus and Fallopian tubes. AMH continues to be highly expressed by Sertoli until the onset of puberty, when it is downregulated to low adult levels. FSH increases testicular AMH output by promoting immature Sertoli cell proliferation and individual cell expression. AMH secretion also showcases a differential regulation exerted by intratesticular levels of androgens and estrogens. In the fetus and the newborn, Sertoli cells do not express the androgen receptor, and the high androgen concentrations do not affect AMH expression. Conversely, estrogens can stimulate AMH production because estrogen receptors are present in Sertoli cells and aromatase is stimulated by FSH. During childhood, sex steroids levels are very low and do not play a physiological role on AMH production. However, hyperestrogenic states upregulate AMH expression. During puberty, testosterone inhibition of AMH expression overrides stimulation by estrogens and FSH. The direct effects of sex steroids on AMH transcription are mediated by androgen receptor and estrogen receptor α action on AMH promoter sequences. A modest estrogen action is also mediated by the membrane G-coupled estrogen receptor GPER. The understanding of these complex regulatory mechanisms helps in the interpretation of serum AMH levels found in physiological or pathological conditions, which underscores the importance of serum AMH as a biomarker of intratesticular steroid concentrations.

Association between an AMH promoter polymorphism and serum AMH levels in PCOS patients.

STUDY QUESTIONDo polymorphisms in the anti-Müllerian hormone (AMH) promoter have an effect on AMH levels in patients with polycystic ovary syndrome (PCOS)?SUMMARY ANSWERWe have identified a novel AMH promoter polymorphism rs10406324 that is associated with lower serum AMH levels and is suggested to play a role in the mechanism of regulation of AMH gene expression in women.WHAT IS KNOWN ALREADYFollicle number is positively correlated with serum AMH levels, reflected by elevated AMH levels in women with PCOS. In addition, it is suggested that AMH production per follicle is higher in women with PCOS than in normo-ovulatory women, implying an altered regulation of AMH in PCOS.STUDY DESIGN, SIZE, DURATIONA discovery cohort of 655 PCOS women of Northern European ancestry and both an internal and external validation PCOS cohort (n = 458 and n = 321, respectively) were included in this study. Summary-level data of an AMH genome-wide association study meta-analysis including 7049 normo-ovulatory women was included as a control cohort. A genetic approach was taken through association analysis and in silico analysis of the associated variants in the AMH promoter. In vitro analysis was performed to investigate the functional mechanisms.PARTICIPANTS/MATERIALS, SETTING, METHODSAll common two-allelic single-nucleotide polymorphisms (SNPs) in the region Chr19:2 245 353–2 250 827 bp (Build 37) were selected for the analysis. Linear regression analyses were performed to determine the association between SNPs in the AMH promoter region and serum AMH levels. For the in silico analysis, the webtools ‘HaploReg’ v4.1 for ENCODE prediction weight matrices and ‘atSNP’ were used. In vitro analysis was performed using KK1 cells, a mouse granulosa cell line and COV434 cells, a human granulosa tumor cell line. Cells were transfected with the reference or the variant human AMH promoter reporter construct together with several transcription factors (TFs). Dual-Glo® Luciferase Assay was performed to measure the luciferase activity.MAIN RESULTS AND THE ROLE OF CHANCEPolymorphism rs10406324 was significantly associated with serum AMH levels in all three PCOS cohorts. Carriers of the minor allele G had significantly lower log-transformed serum AMH levels compared to non-carriers (P = 8.58 × 10−8, P = 1.35 × 10−3 and P = 1.24 × 10−3, respectively). This result was validated in a subsequent meta-analysis (P = 3.24 × 10−12). Interestingly, rs10406324 was not associated with follicle count, nor with other clinical traits. Also, in normo-ovulatory women, the minor allele of this variant was associated with lower serum AMH levels (P = 1.04 × 10−5). These findings suggest that polymorphism rs10406324 plays a role in the regulation of AMH expression, irrespective of clinical background. In silico analysis suggested a decreased binding affinity of the TFs steroidogenenic factor 1, estrogen-related receptor alpha and glucocorticoid receptor to the minor allele G variant, however in vitro analysis did not show a difference in promoter activity between the A and G allele.LIMITATIONS, REASONS FOR CAUTIONFunctional analyses were performed in a mouse and a human granulosa cell line using an AMH promoter reporter construct. This may have limited assessment of the impact of the polymorphism on higher order chromatin structures. Human granulosa cells generated from induced pluripotent stem cells, combined with gene editing, may provide a method to elucidate the exact mechanism behind the decrease in serum AMH levels in carriers of the −210 G allele. We acknowledge that the lack of follicle number in the external validation and the control cohort is a limitation of the paper. Although we observed that the association between rs10406324 and AMH levels was independent of follicle number in our discovery and internal validation PCOS cohorts, we cannot fully rule out that the observed effects on serum AMH levels are, in part, caused by differences in follicle number.WIDER IMPLICATIONS OF THE FINDINGSThese results suggest that variations in serum AMH levels are not only caused by differences in follicle number but also by genetic factors. Therefore, the genetic context should be taken into consideration when assessing serum AMH levels in women. This may have clinical consequences when serum AMH levels are used as a marker for the polycystic ovarian morphology phenotype.STUDY FUNDING/COMPETING INTEREST(S)No external funding was used. J.S.E.L. has received consultancy fees from the following companies: Ferring, Roche Diagnostics and Ansh Labs and has received travel reimbursement from Ferring. J.A.V. has received royalties from AMH assays, paid to the institute/lab with no personal financial gain. The other authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A.

Testosterone and androstenedione are positively associated with anti-Müllerian hormone in premenopausal women.

To document associations between anti-Müllerian hormone (AMH) and circulating androgens in nonhealthcare-seeking premenopausal women. Community-based, cross-sectional study. Eastern states of Australia. Women aged 18-39 years not using systemic hormones, not pregnant or breastfeeding within 3 months, and not postmenopausal. AMH, measured by the Beckman Access 2, 2 site immunometric assay from fresh samples, and testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 11-oxygenated C19 steroids, measured by liquid chromatography-tandem mass spectrometry. Data were available for 794 women, median age of 33 years (range: 18-39). 76.1% were of European ancestry and 48.2% were parous. Serum AMH was positively associated with testosterone (rho = .29, p < .001) androstenedione (rho = .39, p < .001) and DHEA (rho = .10, p = .005) but not 11-ketoandrostenedione or 11-ketotestosterone. When adjusted for age, body mass index and smoking, using quantile regression, independent positive associations remained between AMH and testosterone (β coefficient: 20.90, 95% confidence interval [CI]: 13.79-28.03; p < .001) and androstenedione (β coefficient: 5.90, 95% CI: 3.76-8.03; p < .001). The serum concentration of testosterone was greater at the top AMH quintile than other quintiles (0.56 nmol/L [range: 0.21-1.90] vs. 0.36 nmol/L [range: 0.13-0.87]; p = .001) in women with self-reported polycystic ovary syndrome. The positive associations between serum testosterone and androstenedione and AMH in premenopausal women is consistent with androgens directly or indirectly influencing AMH production during follicular development. As the highest AMH concentrations are most likely to be seen in women with multifollicular ovaries, it would be expected that women with multifollicular ovaries would have higher serum testosterone. Therefore, whether hyperandrogenemia and multifollicular ovaries should be considered independent characteristics of polycystic ovary syndrome warrants review.

Impact of Obesity on Anti-Mullerian Hormone (AMH) Levels in Women of Reproductive Age.

Obesity negatively impacts reproductive health, including ovarian function. Obesity has been posited to alter Anti-Müllerian hormone (AMH) production. Understanding biological factors that could impact AMH levels is necessary given the increasing use of AMH for predicting reproductive health outcomes in response to controlled ovarian stimulation, diagnosing ovulatory disorders, onset of menopause, and natural conception. In this narrative review, we evaluated the impact of obesity on AMH levels in healthy, regularly cycling reproductive-age women (18–48 years). Thirteen studies (n = 1214 women; (811, non-obese (body mass index; BMI < 30 kg/m2); 403, obese (BMI > 30 kg/m2))) were included, of which five reported decreased AMH levels with obesity, whereas eight showed comparable AMH levels between groups. Inclusion of women with higher obesity classes (Class 3 versus Class 1) may have been a factor in studies reporting lower AMH levels. Together, studies reporting AMH levels in otherwise healthy women remain limited by small sample sizes, cross-sectional designs, and lack of representation across the entire adiposity spectrum. Ultimately, the degree to which obesity may negatively impact AMH levels, and possibly ovarian reserve, in otherwise healthy women with regular menstrual cycles should be deemed uncertain at this time. This conclusion is prudent considering that the biological basis for an impact of obesity on AMH production is unknown.

Could hormonal and follicular rearrangements explain timely menopause in unilaterally oophorectomized women?

Does unilateral oophorectomy modify the relationship between serum anti-Müllerian hormone (AMH) levels and antral follicle count (AFC)? No altered 'per-ovary' and 'per-follicle' AMH production and antral follicle distribution was evident in unilaterally oophorectomized women compared to matched controls. The age of menopause onset is relatively unchanged in patients having undergone unilateral oophorectomy. Mechanisms that occur to preserve and maintain ovarian function in this context remain to be elucidated. Forty-one infertile women, with no polycystic ovary syndrome (PCOS) and no endometriosis, aged 19-42 years old, having undergone unilateral oophorectomy (One Ovary group; average time since surgery: 23.8 ± 2.2 months) were retrospectively age-matched (±1 year) with 205 infertile women having two intact ovaries and similar clinical features (Control group). Serum AMH levels, 3-4 mm AFC, 5-12 mm AFC, and total AFC (3-12 mm) were assessed on cycle Day 3 in both groups. Hormonal and ultrasonographic measurements obtained from patients in the Control group (i.e. having two ovaries) were divided by two to be compared with measurements obtained from patients of the One Ovary group (i.e. having one single remaining ovary). To estimate per-follicle AMH production, we calculated the ratio between serum AMH levels over 3-4 mm AFC, 5-12 mm AFC, and total AFC (3-12 mm), and the strength of the correlation between serum AMH levels and total AFC. The main outcome measure was to assess Day 3 AMH/Day 3 AFC ratio and hormonal-follicular correlation. As expected, before correction, mean serum AMH levels (1.46 ± 0.2 vs 2.77 ± 0.1 ng/ml, P < 0.001) and total AFC (7.3 ± 0.6 vs 15.1 ± 0.4 follicles, P < 0.0001) were lower in the One Ovary group compared to the Control group, respectively. Yet, after correction, per-ovary AMH levels (1.46 ± 0.2 vs 1.39 ± 0.1 ng/ml) and total AFC (7.3 ± 0.6 vs 7.5 ± 0.2 follicles) values were comparable between the two groups. Consistently, per-follicle AMH levels (3-4 mm, 5-12 mm, and total) were not significantly different between the two groups (0.39 ± 0.05 vs 0.37 ± 0.02 ng/ml/follicle; 0.69 ± 0.12 vs 0.59 ± 0.05 ng/ml/follicle, and 0.23 ± 0.03 vs 0.19 ± 0.01 ng/ml/follicle; respectively). In addition, the prevalence of 3-4 mm follicles was comparable between the two groups (66.7% for One Ovary group vs 58.8% for Control group, respectively). Finally, the correlation between serum AMH levels and total AFC was similar for patients in the One Ovary group (r = 0.70; P < 0.0001) compared to those in the Control group (r = 0.68; P < 0.0001). The retrospective character of the analysis might lead to potential bias. The present investigation did not provide evidence of altered 'per-ovary' and 'per-follicle' AMH production and antral follicle distribution in unilaterally oophorectomized women compared to matched controls. Further studies are warranted to support the hypothesis that follicle-sparing mechanisms are clearly at stake in remaining ovaries after unilateral oophorectomy to explain their long-lasting function and timely menopausal onset. The authors have no funding or competing interests to declare. N/A.

PCOCheck AMH ELISA: A Clinical Case Report to Resolve Miss-Matched Antral Follicle Counts (AFC) to Serum AMH

Introduction: At times clinicians wonder why a subject’s Anti-Mullerian Hormone (AMH) does not correlate to the antral follicle count (AFC). AMH is secreted as a full-length protein and undergoes proteolytic cleavage at amino acid 451 to become biologically active. Additional proteolytic processing takes place at aa229. This processing, which may differ between individuals with different clinical conditions, exposes new antigenic sites which affect AMH measurements. Moreover, AMH epitopes might be masked by protein interaction in the circulation. Clinical Case: A 24-year-old woman gravida 0 presented with metrorrhagia for several months. The bleeding pattern was unresponsive to a trial of oral contraceptive pills, and they were discontinued. The patient’s history is remarkable for a microprolactinoma, managed with dopamine agonist. She has facial acne, but no hirsutism. Her BMI is 24 kg/m2. Laboratory studies revealed normal testosterone (27 ng/mL) and DHEAS (64 mcg/dL). Her prolactin was 1.1 ng/mL Thyroid function tests were in the normal range. hCG was negative. A random progesterone was 12 ng/mL, consistent with an ovulatory cycle. Her ovarian assessment showed FSH of 6.6 IU/L, LH of 6.7 IU/L, estradiol of 51 pg/mL, Inhibin B of 139 pg/mL, all in the normal range. Transvaginal pelvic ultrasound determined the ovarian volumes to be 8 and 6 mL, respectively, with a basal antral follicle count of 15 per side. This would have predicted a normal AMH level in this patient. However, AMH levels were repeatedly found to be low (0.05 to 0.1 ng/mL) using the pro-Mature AMH assay. As this patient was interested in future fertility, additional studies to further investigate her AMH production were carried out. AMH Measurement: Novel AMH ELISAs with antibody epitopes specific to Pro-Mature, Mature-Mature and Pro-Pro were used to analyze the sample. The observed AMH concentration by Pro-AMH ELISA, Mature-Mature ELISA and PCOCheck ELISAs were 0.1 ng/mL, 0.14 ng/mL, and 4.99 ng/mL, respectively. AMH Pro-Pro assay (PCOCheck) uses a linear epitope two-sided antibody that is not impacted by post-translational modifications, known AMH mutations or conformational changes due to thermal instability. Conclusions: The AMH measurements indicate that the patient’s serum may not contain pro-mature associated form of AMH or the C-fragmented AMH in circulation. This may be due to mutated AMH in the C-terminus and can result in C-truncation. This subject’s AMH is mostly fragmented and contains enriched pro-region AMH, which was measured by PCOCheck ELISA. The Teaching: Tests such as PCOCheck ELISA, targeted to specific regions of AMH will provide clinicians better tools for patient management. Such tests will also help resolve the observed AMH discrepancies with AFC using legacy AMH tests. PCOCheck ELISA can be a valuable tool for assessment of ovarian reserve or polycystic ovary syndrome (PCOS).

After cyclophosphamide exposure, granulosa cells recover their anti-müllerian hormone-producing ability but not their numbers.

Anti‐müllerian hormone (AMH) produced by granulosa cells (GCs), reserves the ovarian follicle pool for future recruitment and ovulation. However, women who have undergone cyclophosphamide (Cy) treatment have decreased AMH levels due to damaged GCs. This study establishes flow cytometry protocols for identification of GCs and investigates the cause of the Cy‐induced AMH decrease by analyzing the number of GCs and their AMH production at the single cell level. Over 2 weeks, C57BL/6 mice were intraperitoneally injected 6 times with 100 mg/kg Cy and sacrificed either immediately or 4 weeks after Cy treatment. Twenty‐four hours post‐Cy exposure, a decrease in serum AMH levels was seen due to a reduction in the number of follicle‐stimulating hormone receptor (FSHR)+AMH+ GCs and their ability to produce AMH. However, 4 weeks after Cy treatment, serum AMH levels were still decreased due to the decreased number of FSHR+AMH+ GCs, however, their AMH‐producing ability was unaltered. Consistently, in vitro, Cy‐induced low AMH production in FSHR+AMH+ hGL5 cells (immortalized human GCs) was restored 24 h after Cy treatment, although their numbers remained decreased. Thus, the surviving GCs after Cy exposure had intact AMH‐producing ability. In future, an effort to minimize GC death by Cy treatment is required, while maintaining its therapeutic effects.

The association between in utero exposure to maternal psychological stress and female reproductive function in adolescence: A prospective cohort study

BackgroundExperimental studies suggest that prenatal stress affects reproductive function in female offspring, but human evidence is sparse and inconsistent. In this present study, we aim to investigate whether maternal psychological stress, quantified as stressful life events during pregnancy, affect reproductive function in the female offspring. MethodIn a large population-based pregnancy cohort study (The Raine Study) continuously followed from prenatal life through to adolescence we examined the association between the number of maternal stressful life events in both early and late gestation and subsequent ovarian and uterine function in 228 female adolescent offspring. Mothers prospectively reported stressful life events during pregnancy at 18 and 34 weeks using a standardized 10-point questionnaire. Female offspring (n ​= ​228) age 14–16 years underwent gynecological examination including transabdominal abdominal ultrasound (TAUS) to measure uterine volume and ovarian AFC. Plasma samples on day 2–6 of the spontaneous menstrual cycle measured circulating AMH and inhibin B. Multivariate linear regression analysis was used to examine the associations between maternal stressful life events and reproductive function in female offspring. Adolescents taking hormonal contraception were excluded. ResultsMost adolescents (145/228, 64%) were exposed to at least one stressful life event in early gestation and around half (125/228, 55%) were exposed to at least one in later gestation. Exposure to one or more maternal stressful life events in late gestation was associated with a greater uterine volume (β ​= ​0.13, 95% CI 0.04; 0.23) and higher ovarian AFC (β ​= ​0.19, 95% CI 0.02; 0.35) at age 14–16 years. No associations between maternal stressful events in late gestation and reproductive function were identified. No associations between stressful life events in early or late gestation and circulating AMH or Inhibin B were observed. ConclusionMaternal psychological stress in late, but not early gestation was associated with a significantly greater uterine volume and ovarian antral follicle count (AFC) in adolescent offspring but did not affect ovarian production of antimullerian hormone (AMH) or Inhibin B. These findings suggest that female reproductive function is influenced by prenatal exposure to stress.

Impact of imatinib or dasatinib coadministration on in vitro preantral follicle development and oocyte acquisition in cyclophosphamide-treated mice.

ObjectiveWe investigated the impact of tyrosine kinase inhibitor (imatinib or dasatinib) coadministration with cyclophosphamide (Cp) on preantral follicle development in an in vitro mouse model.MethodsSeventy-three female BDF1 mice were allocated into four experimental groups: group A, saline; group B, Cp (25 mg/kg); group C, Cp (25 mg/kg) and imatinib (7.5 mg/kg); and group D, Cp (25 mg/kg) and dasatinib (7.5 mg/kg). Preantral follicles were isolated and cultured in vitro up to 12 days. Final oocyte acquisition and spindle integrity of metaphase II (MII) oocytes were assessed. Levels of 17β-estradiol and anti-Müllerian hormone (AMH) in the final spent media were measured by enzyme-linked immunosorbent assays, and the mRNA levels of Star, Sod1, Mapk3, and Casp3 in the final follicular cells were quantified by real-time polymerase chain reaction.ResultsThe percentage of MII oocytes per initiated follicle, the proportion of MII oocytes with normal spindles, and the 17β-estradiol level were similar in all four groups. The median AMH level in group B (7.74 ng/mL) was significantly lower than that in group A (10.84 ng/mL). However, the median AMH levels in group C (9.96 ng/mL) and group D (9.71 ng/mL) were similar to that in group A. The mRNA expression levels of Star, Sod1, Mapk3, and Casp3 were similar in all four groups.ConclusionCoadministration of imatinib or dasatinib with Cp could preserve AMH production capacity in this in vitro mice preantral follicle culture model, and it did not affect MII oocyte acquisition.

Resveratrol (3,4ʹ,5-trihydroxy-trans-stilbene) or caffeic acid phenethyl ester supplementation prevents premature ovarian insufficiency due to chemotherapy

IntroductionThis study was planned to investigate the protective effect of resveratrol (RSV), caffeic acid phenethyl ester (CAPE), and coenzyme Q10 (CoQ10) on chemotherapy-induced premature ovarian insufficiency (POI).Material and methodsTwenty-eight female rats were divided into four groups with 7 rats in each group. The groups consisted of a vehicle-treated control group (group 1), rats treated with chemotherapy followed by intraperitoneal RSV injection (group 2) or rats treated with chemotherapy followed by CoQ10 (group 3), or rats treated with chemotherapy followed by intraperitoneal CAPE injection (group 4). Cisplatin was administered intraperitoneally once daily at doses of 2.0 mg/kg for 10 days. Rats in groups 2, 3, and 4 were given RSV (100 mg/kg, i.p.), CoQ10 (20 mg/kg, oral), and CAPE (10 mg/kg, i.p.) respectively. Five animals in the sham group underwent laparotomy for ovarian weight measurements on the first day of cisplatin injection. Animals in the treatment and control groups were sacrificed two weeks later, and their ovaries were excised for histopathological analysis. Serum levels of anti-Mullerian hormone (AMH) were also analyzed.ResultsRats in groups 2 and 4 showed vaginal smears under estrogenic effect. At the end of the second week, the total body weight and ovarian weight of the animals in group 1 increased. Significantly higher mean follicle count was detected in groups 2 and group 4 compared to group 3. The ovaries of the rats in the control group showed follicles in all stages of development. While the ovaries of the rats in the RSV or CAPE showed significantly decreased numbers of primordial follicles they showed increased numbers of early growing follicles.ConclusionsBoth RSV and CAPE administration might improve follicle survival and AMH production in rats with chemotherapy-induced POI but further research is necessary.

Molecular mechanisms underlying AMH elevation in hyperoestrogenic states in males

Anti-Müllerian hormone (AMH) is secreted by Sertoli cells of the testes from early fetal life until puberty, when it is downregulated by androgens. In conditions like complete androgen insensitivity syndrome (CAIS), AMH downregulation does not occur and AMH increases at puberty, due in part to follicle-stimulating hormone (FSH) effect. However, other conditions like Peutz-Jeghers syndrome (PJS), characterised by low FSH, also have increased AMH. Because both CAIS and PJS may present as hyperoestrogenic states, we tested the hypothesis that oestradiol (E2) upregulates AMH expression in peripubertal Sertoli cells and explored the molecular mechanisms potentially involved. The results showed that E2 is capable of inducing an upregulation of endogenous AMH and of the AMH promoter activity in the prepubertal Sertoli cell line SMAT1, signalling through ERα binding to a specific ERE sequence present on the hAMH promoter. A modest action was also mediated through the membrane oestrogen receptor GPER. Additionally, the existence of ERα expression in Sertoli cells in patients with CAIS was confirmed by immunohistochemistry. The evidence presented here provides biological plausibility to the hypothesis that testicular AMH production increases in clinical conditions in response to elevated oestrogen levels.