anti-Müllerian Hormone Expression Research Articles

Endometrial expression of anti-Müllerian hormone and its type II receptor in women with polycystic ovary syndrome

Is endometrial expression of anti-Müllerian hormone (AMH) and its receptor II (AMH-R) altered in women with polycystic ovary syndrome (PCOS) and affected by lifestyle intervention? Endometrial immunostaining of AMH and AMH-R was evaluated in obese women with PCOS (OB-PCOS, n = 18) before and after 3 months of lifestyle intervention, as well as in BMI-matched controls (OB-C, n = 10), normal-weight women with PCOS (n = 11) and healthy normal-weight controls (n = 11). Before lifestyle modification, serum concentrations of AMH were higher in women with PCOS compared with BMI-matched controls, but there were no differences in endometrial immunostaining of AMH or AMH-R between the groups. Following lifestyle modification, a subgroup of OB-PCOS women started to ovulate. Still, there were no differences in endometrial immunostaining of AMH between ovulatory and anovulatory women with PCOS and controls, and no variation within the menstrual cycle. However, immunostaining of stromal AMH-R increased from cycle days 6-8 to 21-23 in all three groups. Furthermore, endometrial immunostaining of AMH-R correlated positively with oestrogen receptor alpha on cycle days 21-23 in the groups of women with PCOS, as well as in the controls (r = 0.66, P = 0.007 and r = 0.85, P < 0.001, respectively). Although PCOS is associated with increased serum concentrations of AMH, protein expression of AMH and its receptor in the endometrium was no different to controls, and moreover not affected by lifestyle modification. These results imply that circulating AMH is not affecting expression of AMH and its receptor in the endometrium.

Reparative effects of lycium barbarum polysaccharide on mouse ovarian injuries induced by repeated superovulation

To explore the repair effect of lycium barbarum polysaccharide (LBP) on ovarian injuries induced by repeated superovulation in mice, a model of ovarian injury was established, and ovarian repair was assessed after intragastric administration of LBP. The oocyte quality and blastocyst rates of pronuclear embryos in vitro were observed. The levels of 8-hydroxydeoxyguanosine (8-OHdG) and lipid peroxide (LPO) in ovarian tissue were measured, and ovarian damage was assessed in paraffin sections. The groups with significant injury were selected according to the above observation, mice in the significant injury group were intragastrically administered with LBP (low dose, 25 mg/kg; medium dose, 35 mg/kg; and high dose, 45 mg/kg) for 30 days. The above measurements and anti-Müllerian hormone (AMH) expression were detected in the mouse ovaries and the breeding verification was carried out. Our results showed that repeated superovulation could cause mouse oocyte quality to drop, significant differences started from 4 superovulation events (P < 0.05). The levels of 8-OHdG and LPO in the ovary increased gradually as the number of superovulation events increased, and significant differences were observed after 4–6 superovulations (P < 0.05). The ratios of primordial follicles, primary, tertiary and mature follicles decreased and the ratio of atresia follicles increased as the number of superovulation events increased, especially in 4–6 superovulation groups. Thus, the groups of superovulation 4–6 events were considered as significant injury groups. LBP-medium dose groups significantly improved the number and quantity of oocytes and embryo blastocyst rate (P < 0.05), significantly decreased 8-OHdG and LPO levels in mice ovary (P < 0.05), also improved the ratios of all stages follicles and reduced the rate of atresia follicles, increased the numbers of litter size, live birth, weaning survival, and repaired the expression of AMH in ovary significantly (P < 0.05). In conclusion, the degree of ovarian injury was affected by the number of superovulation. LBP repaired ovarian injuries most likely through scavenging oxidative products 8-OHdG and LPO and increasing AMH protein expression.

Anti-Mullerian Hormone Gene Expression and Serum Protein Concentration in Females Suffered from Diabetes Mellitus: An Animal Study

This study aimed to evaluate the effect of diabetes on fertility on diabetic women especially. Sixty adult female patients aged (18 – 40) years are divided equally into three groups; G1; control healthy group, G2; type one diabetic women, G3; non fertile women. Blood samples of all individuals were analyzed for measurement of glucose, estradiol, progestrone, anti-mullerian hormone (AMH), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), superoxide dismutase (SOD) and nitric oxide (NO), serum insulin and glycated hemoglobin (HbA1c) showed a significant increase in diabetic fertile group. Also plasma level of FSH, LH and SOD activity were significantly lowered in diabetic infertile group than healthy group and diabetic fertile group. NO was higher in diabetic fertile group. In AMH mRNA expression in ovarian tissues, 40 adult female Sprague-Dawley rats were divided equally into four groups; G1; normal rats, G2; diabetic 30 days group rat, G3; diabetic 45 days group rat, G4; diabetic 60 days group rat. the expression of AMH in ovaries was significantly increased in diabetic rats at day 30 compared to healthy rats and significantly decreased at days 45 and 60 compared to healthy rats. In conclusion, diabetes mellitus can affect the health status and fertility markers in females that was also observed in expression of AMH in ovarian tissues in rats

Anti-Müllerian hormone is expressed and secreted by bovine oviductal and endometrial epithelial cells.

In this study, we investigated whether bovine oviducts and endometria produce anti-Müllerian hormone (AMH) (for paracrine and autocrine signaling). Reverse transcription-polymerase chain reaction and western blotting detected AMH expression in oviductal and endometrial specimens. Immunohistochemistry revealed robust AMH expression in the ampulla and isthmus epithelia, and the glandular and luminal endometrial epithelia (caruncular endometria). AMH mRNA (measured by real-time PCR) and protein expression in these layers did not significantly differ among estrous phases in adult Japanese Black (JB) heifers (p>.1). Furthermore, the expression in these layers also did not differ among Holstein cows (93.8±5.8months old), JB heifers (25.5±0.4months old), and JB cows (97.9±7.9months old). We also compared AMH concentrations in the oviduct and uterine horn fluids among the three groups (measured by immunoassays). Interestingly, the AMH concentration in the oviduct fluid, but not in the uterine horn fluid, of Holstein cows was lower than those in JB heifers and cows (p<.05). Therefore, bovine oviducts and endometria express AMH and likely secrete it into the oviduct and uterine fluids.

Ovarian Growth Factor Protein Expression in Pediatric Patients before and after Cytotoxic Therapy

The improved survival of young cancer patients results often in follicle destruction. The infertility level varies by age, with alkylating agents considered the most gonadotoxic. One option for fertility preservation is ovarian cryostorage, often conducted post-chemotherapy. Large number of follicles remain in ovaries of pediatric patients post-chemotherapy. Anti-Mullerian hormone (AMH), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are ovarian markers. Their ovarian expression in post-chemotherapy girls might provide an indication of follicle normality, and if it is worthwhile to cryopreserve such tissue. AMH, GDF9 and BMP15 protein expression in human ovarian samples from 49 girls pre-and-post-chemotherapy was evaluated by regular immunohistochemistry (IHC) and immunofluorescence (IF) for confocal laser microscopy. Protein expression for the three growth factors in follicles of pediatric patients pre-and-post-chemotherapy (including alkylating agent exposure) was identified by both methods from primordial stages onwards in oocytes and granulosa cells, and in irregularly shaped follicles. In the pre-chemotherapy group, IHC AMH staining seemed strongest at age ≤ 8 years, and was more abundant in pubertal-than-pre-pubertal patients by IF. Post-chemotherapy patients had significantly more immunostaining for AMH and BMP15 in atretic follicles after exposure to alkylating agents. IHC immunostaining for all three proteins seemed fuller before than after chemotherapy; there were more follicles containing growth factor-expressing granulosa cells post-chemotherapy; BMP15 staining seemed stronger pre-than-post-chemotherapy. The growth factor distribution post-chemotherapy as well as the high numbers of remaining follicles, suggests that it is worthwhile to cryostore ovarian tissue from pediatric patients even after chemotherapy initiation, including alkylating agent exposure.

Neonatal Exposure to Agonists and Antagonists of Sex Steroid Receptors Affects AMH and FSH Plasma Level and Their Receptors Expression in the Adult Pig Ovary.

Simple SummaryThe ovarian development and the establishment of ovarian reserve during fetal and/or neonatal life is critical for future reproductive success. Many environmental chemicals are known to negatively affect development and physiology of human and animal ovaries by interfering with endocrine systems, resulting in aberrant reproductive functions. The present study shows the long-term impact of neonatal exposure to agonists and antagonists of sex steroid receptors on AMH and FSH signalling in the ovary of adult pigs. Our findings suggest alteration in ovarian follicle recruitment from ovarian reserve arising from neonatal disruption of androgen/estrogen signalling induced by environmental endocrine active compounds. Everyday use of many endocrine disruptors is already prohibited after their harmful impacts on normal physiology have become known. Nevertheless, market introduction of new chemicals with potential deleterious influence on reproductive physiology has continued. Our outcomes confirm that a neonatal window plays an essential role in the physiological programming of ovarian function in adult pigs. The influence of environmental chemicals on this critical neonatal window needs to be investigated in order to gain a comprehensive view of deleterious interactions between endocrine disrupting chemicals and ovarian function.In this study piglets were injected with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen) or corn oil (controls) between postnatal days 1 and 10 (N = 5/group). Then plasma anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) concentration and the expression of their receptors were examined in the adult pig ovary. TP and FLU decreased plasma AMH and FSH concentration. In preantral follicles, TP resulted in upregulation of AMHR2 and FSHR expression, but decreased AMH protein abundance. FLU upregulated AMHR2 expression, while OP increased FSHR mRNA. In small antral follicles, OP upregulated ACVR1 and BMPR1A expression, while FLU increased BMPR1A mRNA. FLU and ICI resulted in upregulation of AMHR2 expression. TP and FLU upregulated AMH expression, while it was downregulated in response to OP or ICI. Moreover, OP and ICI resulted in downregulation of FSHR expression, while FLU decreased FSHR protein abundance. In conclusion, neonatal exposure to either agonist or antagonist of androgen receptor affected AMH and FSH signalling systems in preantral follicles. In small antral follicles these systems were influenced by compounds with estrogenic, antiestrogenic, and antiandrogenic activity. Consequently, these hormonal agents may cause an accelerated recruitment of primordial follicles and affect the cycling recruitment of small antral follicles in pigs.

Regulation of AMH, AMHR-II, and BMPs (2,6) Genes of Bovine Granulosa Cells Treated with Exogenous FSH and Their Association with Protein Hormones.

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3–8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3–8 mm), medium (9–12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.

Expression of the anti-Mullerian hormone, kisspeptin 1, and kisspeptin 1 receptor in polycystic ovary syndrome and controlled ovarian stimulation rat models.

Polycystic ovary syndrome represents a significant cause of female infertility. The aim of this study was to investigate the expression of anti-Mullerian hormone (AMH), kisspeptin 1 (KISS-1), and kisspeptin 1 receptor (KISS1r) in rat models of polycystic ovary syndrome (PCOS) and controlled ovarian stimulation (COS). For this purpose, 28 rats were assigned into four groups. Estrus and Diestrus groups consisted of rats in estrus and diestrus phases, respectively, while COS and PCOS groups consisted of rats with induced COS and PCOS, respectively. The serum AMH, KISS-1, and estradiol levels, and ovarian KISS1r levels were analyzed by enzyme-linked immunosorbent assay. Furthermore, histopathological analysis of the ovary tissue was done and ovarian KISS-1 expression was determined by immunohistochemical assay. The results revealed that ovarian KISS1r levels were higher in the Estrus (1271.43±51.98 pg/mL) and COS (1191.43±85.67 pg/mL) groups, compared to Diestrus and PCOS groups. The highest level of AMH was found in the Estrus group (16.91±2.12 ng/mL). The results indicate that AMH had no effect on the development of COS and PCOS, while KISS-1 was found to affect the development of COS in rats.

The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome.

What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries? Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS. In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women. Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor β (ERβ) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERβ. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability. AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERβ was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis. Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7M (P< 0.05; P< 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. 5α-DHT produced a significant dose-related increase in AMH protein secreted into the media (P= 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERβ produced a corresponding increase in AMH mRNA expression (P= 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P< 0.05, one-way ANOVA) and FSHR (P< 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n= 7, PCOM n= 5, PCOS n= 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P< 0.05, normal n= 6, PCOS n= 3). AMH at 20 and 50ng/ml decreased KGN-GC cell proliferation but not viability after 8days of treatment (P< 0.005, two-way ANOVA). N/A. Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not DHT. Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'. This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose. N/A.

Effects of Insulin on Porcine Neonatal Sertoli Cell Responsiveness to FSH In Vitro.

There is ongoing debate as to whether the decline of sperm production in recent times may be related to a parallel increase in the rate of obesity and diabetes. Lower anti-Müllerian hormone (AMH) and inhibin B secretion have been observed in young hyperinsulinemic patients compared to healthy controls, suggesting a Sertoli cell (SC) dysfunction. The pathophysiological mechanisms underlying SC dysfunction in these patients are poorly understood. To the best of our knowledge, no evidence is available on the effects of insulin on SC function. Therefore, this study was undertaken to assess the effects of insulin on basal and follicle-stimulating hormone (FSH)-stimulated SC function in vitro. To accomplish this, we evaluated the expression of AMH, inhibin B and FSHR genes, the secretion of AMH and inhibin B and the phosphorylation of AKT473 and SC proliferation on neonatal porcine SC after incubation with FSH and/or insulin. We found that similar to FSH, the expression and secretion of AMH is suppressed by insulin. Co-incubation with FSH and insulin decreased AMH secretion significantly more than with FSH alone. Insulin had no effect on the expression and secretion of the inhibin B gene, but co-incubation with FSH and insulin had a lower effect on inhibin B secretion than that found with FSH alone. FSH and/or insulin increased AKT473 phosphorylation and SC proliferation. In conclusion, the results of this study showed that insulin modulates SC function. We hypothesize that hyperinsulinemia may therefore influence testicular function even before puberty begins. Therefore, particular care should be taken to avoid the onset of hyperinsulinemia in children to prevent a future deleterious effect on fertility.

MON-202 Changes in the Expression of Epigenetic Enzymes Induced by Prenatal Testosterone Excess May Underlie the Antral Follicular Defects in the Sheep Model of PCOS

Prenatal testosterone (T) excess leads to oligo/anovulation and multifollicular ovary in sheep, characteristics of women with PCOS. Persistence of antral follicles - the result of reduced atresia, follicular growth arrest and premature luteinization, contribute at least in part to the multifollicular ovarian morphology. Decrease in aromatase (CYP19) in granulosa cells and 17alpha-hydroxylase (CYP17) in theca cells and increase in anti-Mullerian hormone (AMH) in granulosa cells may contribute to the antral follicular persistence. Observations that histone H3K9 hypermethylation decreases cumulus cell CYP19 (Journal of Assisted Reproduction and Genetics 33: 1105-1113, 2016), AMH gene hypermethylation reduces granulosa cell AMH (Oncotarget 6: 3627-3643, 2015), and inhibition of histone deacetylation reduces theca cell CYP17 expression (PloS One 7: e49553, 2012) suggest that epigenetic changes are involved. We hypothesized that prenatal T excess induced changes in expression of CYP17, CYP19 and AMH are facilitated by changes in expression of key epigenetic enzymes. Granulosa / theca cells were isolated via laser capture microdissection from antral follicles of 21 months-of-age control (n=5) and prenatal T-treated (100mg im. twice weekly from gestational day 30 to 90; term: 147 days; n=6) sheep. Expression of methylation / demethylation and / or acetylation / deacetylation enzymes were determined by real time PCR and data analyzed by Student’s t-test and Cohen’s effect size analysis. Prenatal T excess decreased (p < 0.05) histone demethylase KDM1A in granulosa and theca cells, histone acetylase (HDAC) 3 in theca cells, and increased histone methyltransferase SMYD3 in the theca cells. Prenatal T excess also induced large magnitude (1) increases in DNA methyltransferase (DNMT) 1, histone methyltransferases EZH2 and SUV39H1, and histone deacetylase HDAC1 in granulosa cells; (2) decrease in DNA methyltransferase DNMT3B, KDM1A, and HDAC3 in granulosa cells; (3) increase in SMYD3 and HDAC1 in theca cells, and (4) decrease in KDM1A and HDAC3 in theca cells. While decrease in KDM1A and increase in SUV39H1 leading to H3K9 hypermethylation may account for reduced granulosa cell expression of CYP19, downregulation of HDAC3 may underlie the decrease in thecal cell expression of CYP17. Decreased DNMT3B expression may contribute to hypomethylation of AMH gene and consequent increase in AMH expression in granulosa cells. These findings suggest that changes in expression of key genes involved in the development of multifollicular phenotype are likely mediated via granulosa and theca cell specific changes in epigenetic enzyme expression. Because the ovarian attributes of prenatal T-treated sheep parallel that seen in PCOS women, these observations may be of translational relevance. Supported by NIH PO1HD44232.

MON-208 Association Study of AMH Promoter Polymorphisms and Serum AMH Levels in PCOS Patients

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women, is diagnosed based on three criteria, including a polycystic ovarian morphology (PCOM). Although the etiology of PCOS is not fully understood, a genetic component has been demonstrated. Women with PCOS have elevated serum Anti-Müllerian Hormone (AMH) levels, a hormone known to reflect the ovarian reserve. Furthermore, the AMH production per follicle is suggested to be higher in PCOS patients and this combined implies an aberrant regulation of ovarian AMH expression. Currently, some transcription factors, such as FOXL2 and SF1, have been demonstrated to play a role in the ovarian regulation of AMH promoter activity. However, still little is known about AMH gene regulation, particularly in women with PCOS. Hence, the aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) in the AMH promoter on serum AMH levels in a cohort of PCOS patients. A candidate gene association study was conducted. All two-allelic SNPs located in or nearby the AMH promoter (Chr19:2,245,353 – 2,250,827bp) with a minor allele frequency > 1% and an imputation quality r2 threshold above 0.75 were included. We included Caucasian PCOS women, diagnosed based on the Rotterdam criteria. AMH levels were measured with the picoAMH assay (Ansh Labs®, Houston, Texas, USA). The association between SNPs and serum AMH levels was assessed by linear regression with Wald test of significance, allele carrier model analysis with one-way analysis of covariance (ANCOVA). All statistical models were adjusted for age, BMI and total follicle count (TFC). AMH levels were natural log transformed to obtain a normal distribution. A p-value below 8.51e-03 was considered as statistically significant, based on Matrix Spectral Decomposition with an effective number of independent variables (VeffLi) of 6. All analyses were performed using R studio. We assessed 11 SNPs in 700 Caucasian PCOS women. Human AMH promoter polymorphism rs10406324 (-220 A/G) was associated with serum levels of AMH in the linear regression analysis (β = 0.52, p=6.08e-07). This association was also present in the dominant carrier model (AA: 9.85 ng/ml ± 0.27 (n=645) vs AG/GG: 7.60ng/ml ± 0.84 (n=54/n=1), p=6.48e-05, F = 16.2). The association of TFC and the polymorphism rs10406324, corrected for Age, BMI and AMH, showed an inverse trend compared to serum AMH levels (β=-8.00, p=3.75e-03), however this result was not significant in the carrier model (AA: 42.2 ± 0.88 (n=645) vs AG/GG: 43.6 ± 2.41 (n=54/n=1), p=0.60, F = 0.281). Moreover, this SNP was not in LD with any other SNP in the selected region. The human AMH promoter polymorphism rs10406324 is associated with serum AMH levels in Caucasian PCOS women. Replication and functional analyses are necessary to further elucidate the mechanisms by which this SNP affects the AMH promoter activity.

Effect of electroacupuncture on anti-Mullerian hormone expression in rats with polycystic ovarian syndrome

To observe whether the effect of electroacupuncture (EA) on improving sex hormone disorders and follicle development is by decreasing the expression of anti-Mullerian hormone (AMH) in rats with experimental polycystic ovarian syndrome (PCOS). Forty rats were randomly divided into four groups, a normal group (NG), a model group (MG), an EA at acupoints group (EAAG), and an EA at non-acupoints group (EANAG), with 10 rats in each group. The rats in the EAAG and EANAG were intervened by EA treatment for consecutive 14 d. Zhongji (CV 3) and Guanyuan (CV 4) were selected as the acupoints in the EAAG, and the tip of the tail and 1 cm up from the tail tip were selected as the non-acupoints in the EANAG. After treatment, the histomorphological changes of the ovary, the levels of aromatase P450 (P450arom), testosterone and estradiol in the ovarian tissues, and the expressions of follicle stimulating hormone (FSH) and AMH were observed. After treatment, compared with the MG and EANAG, the expression of AMH decreased (P<0.05), the levels of P450arom and estradiol increased significantly, and the level of testosterone decreased significantly (all P<0.01) in the EAAG. Additionally, several normal follicles were present and the number of cystically dilated follicles decreased in the EAAG. Compared with the MG and EANAG, the EAAG obviously had more follicular granulosa cells. EA can down-regulate the abnormally increased expression of AMH to improve sex hormone disorders and follicle development in PCOS rats.

Anti-Müllerian Hormone Expression in Endometrial Cancer Tissue.

Anti-Müllerian hormone (AMH) is a commonly known factor secreted by Sertoli cells, responsible for regression of the Müllerian ducts in male fetuses. AMH has also other functions in humans. In vivo and in vitro studies have shown that AMH inhibits cell cycle and induces apoptosis in cancers with AMH receptors. The aim of the study was to assess whether the tissue of pre-cancerous states of endometrium (PCS) and various histopathologic types of endometrial cancer (EC) exhibit the presence of AMH. We aimed to investigate whether the potential presence of the protein concerns menopausal women or those regularly menstruating, and whether is related to cancers with a good or a bad prognosis, as well as what other factors may influence AMH expression. The undertaken analysis was carried out on tissues retrieved from 232 women who underwent surgical treatment for PCS and EC. Tissues were prepared for immunohistochemical assessment with the use of a tissue microarrays method. AMH expression was confirmed in 23 patients with well differentiated endometrioid adenocarcinoma (G1), moderately differentiated endometrioid adenocarcinoma (G2), clear cell carcinoma (CCA) and nonatypical hyperplasia. AMH was not found in EC tissues in regularly menstruating women. An appropriately long mean period of breastfeeding in line with a prolonged period of hormonal activity had a positive effect on AMH expression. Our results may suggest that AMH is a factor which protects the organism against cancer, and should be further investigated as a potential prognosis marker and a therapeutic agent.

Sex biased expression of anti-Mullerian hormone (amh) gene in a live bearing fish, Gambusia holbrooki: Evolutionary implications and potential role in sex differentiation.

The amh, a member of transforming growth factor-β (TGF-β) family, is known to play a critical role in vertebrate male sex differentiation, with its paralogue/s evolving to determine sex in few heterogametic (XX/XY) teleosts. However, it remains relatively unexplored in the reproductively unique live bearing teleosts. Therefore, this study comparatively examined the structure and content of G. holbrooki amh as well as characterised its expression. A paralogous Y-specific amh (amhy) was not detected, suggesting an unlikely role in sex determination. Two transcripts (1.4 and 1.5 kb) were detected in adults: the larger (1.5 kb) retaining intron 5, coding for a truncated AMH-N and no TGF-β domain. The small (1.4 kb) transcript, had both domains intact and clustered with members of Poeciliidae. In contrast to other vertebrates, a higher conservation between the N- rather than the C- terminus of amh in Poeciliidae was observed, suggesting an adaptation that may be unique to live bearing teleosts. The amh expression was 6 times higher in brain of both sexes and testis compared with ovaries (p = .001). Intriguingly, female splenic tissues showed 10 times higher expression (p = .006) and such female bias splenic expression has not been reported in any teleosts. Ontogenic expression was 25 times higher in male embryos at gastrulation stage (p = .001), much earlier than those reported in egg-laying teleosts. Such heightened expression in male embryos suggests a repressive role associated with proliferation and migration of primordial germ cells (PGCs) that are known to occur earlier at blastulation in teleosts-potentially influencing gonadal fate.

Umbilical Cord Blood Mesenchymal Stem Cells as an Infertility Treatment for Chemotherapy Induced Premature Ovarian Insufficiency.

Background: Premature ovarian insufficiency (POI) is a challenging disease, with limited treatment options at the moment. Umbilical cord blood mesenchymal stem cells (UCMSCs) have demonstrated promising regenerative abilities in several diseases including POI. Materials and Method: A pre-clinical murine case versus vehicle control randomized study. Two experiments ran in parallel in each of the three groups. The first was to prove the ability of UCMSCs in restoring ovarian functions. The second was to prove improved fertility. A total of 36 mice were randomly assigned; 6 mice into each of 3 groups for two experiments. Group 1 (control), group 2 (sham chemotherapy), group 3 (stem cells). Results: In the first experiment, post-UCMSCs treatment (group 3) showed signs of restored ovarian function in the form of increased ovarian weight and estrogen-dependent organs (liver, uterus), increased follicular number, and a significant decrease in FSH serum levels (p < 0.05) compared to group 2, and anti-Mullerian hormone (AMH) serum levels increased (p < 0.05) in group 3 versus group 2. Immuno-histochemistry analysis demonstrated a higher expression of AMH, follicle stimulating hormone receptor (FSHR) and Inhibin A in the growing follicles of group 3 versus group 2. In the second experiment, post-UCMSCs treatment (group 3) pregnancy rates were higher than group 2, however, they were still lower than group 1. Conclusion: We demonstrated the ability of UCMSCs to restore fertility in female cancer survivors with POI and as another source of stem cells with therapeutic potentials.

Genes for anti-Müllerian hormone and androgen receptor are underexpressed in human cumulus cells surrounding morphologically highly graded oocytes.

Objectives:The aim of this study was to investigate the expression of genes crucial for the quality of the oocyte and whether expression levels of these genes in cumulus cells can be biological markers for the quality of the oocyte, zygote or embryo, or even for achievement of pregnancy after the assisted reproductive technology procedure. We examined the expression profile of the anti-Müllerian hormone (AMH) gene and its respective receptors: anti-Müllerian hormone receptor type 2 (AMHR2), follicle-stimulating hormone receptor (FSHR) and androgen receptor (AR) in cumulus cells (CCs) surrounding the oocyte, as well as AMH concentrations in follicular fluid of the associated follicle. The obtained gene expression levels were correlated with the morphological quality of the associated oocyte, zygote and embryo as well as with assisted reproductive technology outcome following the intracytoplasmic sperm injection procedure.Methods:This study involved 129 cumulus cells and 35 follicular fluid samples, taken from 58 patients undergoing the intracytoplasmic sperm injection procedure. Oocytes, zygotes and embryos were assessed for morphological quality. The relative gene expression of AMH, AMHR2, FSHR and AR was calculated using the delta–delta Ct method. Anti-Müllerian hormone concentrations in follicular fluids were measured by enzyme-linked immunosorbent assay.Results:The results yielded suggest a relationship between AMH, AR and oocyte morphology: AMH and AR gene expression levels in CCs surrounding morphologically optimal oocytes were significantly lower than in CCs surrounding oocytes with suboptimal morphology (p = 0.011 and p = 0.008, respectively). Statistically significant positive correlation was found between mRNA expression levels of AMH and FSHR (p < 0.001), AMH and AR (p = 0.001), AMHR2 and FSHR (p < 0.001), AMHR2 and AR (p < 0.001), as well as between FSHR and AR (p < 0.001).Conclusion:Assessed results point to AMH and AR relation with oocyte maturity, but not with its fertilization potential, or with embryo quality.

Bovine gonadotrophs express anti-Müllerian hormone (AMH): comparison of AMH mRNA and protein expression levels between old Holsteins and young and old Japanese Black females.

Anti-Müllerian hormone (AMH) is secreted from ovaries and stimulates gonadotrophin secretion from bovine gonadotroph cells. Other important hormones for endocrinological gonadotroph regulation (e.g. gonadotrophin-releasing hormone, inhibin and activin) have paracrine and autocrine roles. Therefore, in this study, AMH expression in bovine gonadotroph cells and the relationships between AMH expression in the bovine anterior pituitary (AP) and oestrous stage, age and breed were evaluated. AMH mRNA expression was detected in APs of postpubertal heifers (26 months old) by reverse transcription-polymerase chain reaction. Based on western blotting using an antibody to mature C-terminal AMH, AMH protein expression was detected in APs. Immunofluorescence microscopy utilising the same antibody indicated that AMH is expressed in gonadotrophs. The expression of AMH mRNA and protein in APs did not differ between oestrous phases (P>0.1). We compared expression levels between old Holsteins (79.2±10.3 months old) and young (25.9±0.6 months old) and old Japanese Black females (89.7±20.3 months old). The APs of old Holsteins exhibited lower AMH mRNA levels (P<0.05) but higher AMH protein levels than those of young Japanese Black females (P<0.05). In conclusion, bovine gonadotrophs express AMH and this AMH expression may be breed-dependent.

159 Effect of bisphenol A and bisphenol S on AMH and AMHR mRNA expression during in vitro bovine oocyte maturation and early embryo development

Exposure to chemicals with known endocrine-disrupting effects, such as bisphenol A (BPA) and bisphenol S (BPS), leads to repercussions on oocyte development and, ultimately, on fertility. Bisphenol A is a plasticizer used worldwide that has been detected in blood, urine, tissue samples and follicular fluid. Due to its widely reported detrimental effects, BPA has been substituted with its analogue BPS. Previous experiments in our laboratory have shown that exposure of bovine oocytes to physiologically relevant doses of BPA resulted in spindle abnormalities, reduced meiosis progression, decreased blastocyst rate and gene expression changes. However, the effects of BPS have not yet been investigated. Anti-Müllerian hormone (AMH) has been reported to be a good marker of ovarian reserve and oocyte developmental capability and is commonly used in assisted reproduction for diagnostic measurements. There is evidence that women undergoing IVF with higher BPA levels have lower AMH levels and pregnancy success. The aim of this study was to assess the effect of BPA and BPS on AMH and its receptor as measures of oocyte developmental capability. Abattoir-derived bovine cumulus-oocyte complexes (COC) were matured in vitro in 4 groups: (1) control, (2) vehicle (0.1% ethanol), (3) BPA (0.05mg mL−1 in 0.1% ethanol), and (4) BPS (0.05mg mL−1 in 0.1% ethanol). Pools of 30 COC, 30 denuded oocytes, and cumulus cells corresponding to denuded oocytes were collected for each of the 4 experimental groups, and a minimum of 4 biological replicates were used for each analysis. Anti-Müllerian hormone and AMH receptor mRNA expression was measured in COC, denuded oocytes, and their corresponding cumulus cells using quantitative real-time PCR. Statistical analyses were performed using 1-way ANOVA. Results showed a decrease (P&amp;lt;0.05) in AMH mRNA expression in BPA-treated oocytes (without cumulus cells). In addition, there was an increase (P&amp;lt;0.05) in mRNA AMH receptor levels in COC when treated with BPS. Finally, analyses on cumulus cells alone showed an increase (P&amp;lt;0.05) in the AMH receptor mRNA levels in BPA-treated cells. These results suggest that BPA has an effect on AMH mRNA transcript levels in oocytes, while affecting the receptor expression in cumulus cells. Conversely, BPS affects AMH indirectly, increasing the mRNA levels of its receptor only. Further investigation of the effects of BPA and BPS on AMH expression in in vitro-produced blastocysts derived from treated oocytes will aid in understanding the potential consequences of exposure to BPA and its analogues on early embryonic development.

Elevation of antimüllerian hormone in women with polycystic ovary syndrome undergoing assisted reproduction: effect of insulin

To measure blood and follicular antimüllerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) undergoing assisted reproductive technologies (ART) and to examine the direct action of insulin on AMH expression in human granulosa cells. Prospective clinical and experimental study. University Hospital-based laboratory. Women with (n = 86) and without (n = 172) PCOS in ART. Blood, follicular fluid, and luteinized granulosa cells were collected from PCOS and non-PCOS women in ART. Hormone levels in blood and fluid, and gene expression in granulosa cells. Serum levels of AMH were elevated and inversely correlated with embryo cleavage rate in PCOS women in ART. Significant higher levels of AMH were also found in small and large follicles collected from PCOS women compared with non-PCOS women. Luteinized granulosa cells from PCOS women showed higher expression of AMH and its receptor AMHR2. Direct effect of insulin in increasing the expression of AMH in the isolated luteinized granulosa cells was observed, with the PCOS granulosa cells responding to a high dose of insulin. Cotreatment with AMH attenuated insulin-induced aromatase expression in the luteinized granulosa cells. These results suggest that insulin may contribute to AMH elevation in PCOS and that AMH counteracts insulin-promoted aromatase expression in granulosa cells.