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.

Zinc deficiency deteriorates ovarian follicle development and function by inhibiting mitochondrial function.

Zinc (Zn) is a crucial trace element essential for human growth and development, particularly for reproductive health. Previous research has shown a decrease in serum zinc concentration with age and individuals with conditions such as polycystic ovary syndrome (PCOS) and diabetes mellitus. However, the specific effects of zinc deficiency on the female reproductive system, especially ovarian function, are not fully understood. In our study, we observed a significant reduction in the total number of follicles and mature follicles in the zinc deficiency group. This reduction correlated with decreased level of anti-Mullerian hormone (AMH) and abnormal gene expression affecting hormone secretion regulation. Furthermore, we found that zinc deficiency disrupted mitochondrial dynamics, leading to oxidative stress in the ovaries, which further inhibited autophagy and increased ovarian apoptosis. These changes ultimately resulted in the failure of germinal vesicle breakdown (GVBD) and reduced oocyte quality. Meanwhile, administration of zinc glycine effectively alleviated the oocyte meiotic arrest caused by dietary zinc deficiency. In conclusion, our findings demonstrated that dietary zinc deficiency can affect hormone secretion and follicle maturation by impairing mitochondrial function and autophagy.

Increased Vasoactive Intestinal Peptide (VIP) in polycystic ovary syndrome patients undergoing IVF.

Introduction: Polycystic ovary syndrome (PCOS) is a common multifactorial and polygenic disorder of the endocrine system, affecting up to 20% of women in reproductive age with a still unknown etiology. Follicular fluid (FF) represents an environment for the normal development of follicles rich in metabolites, hormones and neurotransmitters, but in some instances of PCOS the composition can be different. Vasoactive intestinal peptide (VIP) is an endogenous autonomic neuropeptide involved in follicular atresia, granulosa cell physiology and steroidogenesis. Methods: ELISA assays were performed to measure VIP and estradiol levels in human follicular fluids, while AMH, FSH, LH, estradiol and progesterone in the plasma were quantified by chemiluminescence. UHPLC/QTOF was used to perform the untargeted metabolomic analysis. Results: Our ELISA and metabolomic results show: i) an increased concentration of VIP in follicular fluid of PCOS patients (n=9) of about 30% with respect to control group (n=10) (132 ± 28 pg/ml versus 103 ± 26 pg/ml, p=0,03) in women undergoing in vitro fertilization (IVF), ii) a linear positive correlation (p=0.05, r=0.45) between VIP concentration and serum Anti-Müllerian Hormone (AMH) concentration and iii) a linear negative correlation between VIP and noradrenaline metabolism. No correlation between VIP and estradiol (E2) concentration in follicular fluid was found. A negative correlation was found between VIP and noradrenaline metabolite 3,4-dihydroxyphenylglycolaldehyde (DOPGAL) in follicular fluids. Conclusion: VIP concentration in follicular fluids was increased in PCOS patients and a correlation was found with noradrenaline metabolism indicating a possible dysregulation of the sympathetic reflex in the ovarian follicles. The functional role of VIP as noradrenergic modulator in ovarian physiology and PCOS pathophysiology was discussed.

Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels.

PURPOSE: To determine correlations between chemicals in follicular fluid (FF) and follicular reproductive hormone levels. METHODS: The analysis was part of a larger cohort study to determine associations between exposure to EDCs and in vitro fertilization (IVF) outcomes. FF was aspirated from a single leading follicle per participant. Demographics and data on exposure to EDCs were self-reported by the participants using a questionnaire. The concentrations of estradiol (E2), progesterone (PG), anti-Mullerian hormone (AMH), and inhibin B, as well as that of 12 phthalate metabolites and 12 phenolic chemicals were measured in each FF sample. Multivariate linear regression model was used to identify the drivers of hormone levels based on participant's age, BMI, smoking status, and chemical exposure for the monitored chemicals detected in more than 50% of the samples. Benjamini-Hochberg false discovery rate (FDR) correction was applied on the resulting p values (q value). RESULTS: FF samples were obtained from 72 women (mean age 30.9 years). Most of the phthalates and phenolic substances monitored (21/24, 88%) were identified in FF. Ten compounds (7 phthalate metabolites, 3 phenols) were found in more than 50% of samples. In addition, there were positive associations between E2 levels and mono-n-butyl phthalate (MnBP) (beta = 0.01) and mono-isobutyl phthalate (MiBP) (beta = 0.03) levels (q value < 0.05). CONCLUSION: Higher concentrations of several phthalate metabolites, present among others in personal care products, were associated with increased E2 levels in FF. The results emphasize the need to further investigate the mechanisms of action of such EDCs on hormonal cyclicity and fertility in women.

Analysis of the potential regulatory mechanisms of female and latent genital tuberculosis affecting ovarian reserve function using untargeted metabolomics.

Female and latent genital tuberculosis (FGTB and LGTB) in young women may lead to infertility by damaging ovarian reserve function, but the regulatory mechanisms remain unclear. In this study, we investigated the effects of FGTB and LGTB on ovarian reserve function and potential regulatory mechanisms by untargeted metabolomics of follicular fluid, aiming to provide insights for the clinical management and treatment approaches for afflicted women. We recruited 19 patients with FGTB, 16 patients with LGTB, and 16 healthy women as a control group. Clinical data analysis revealed that both the FGTB and LGTB groups had significantly lower ovarian reserve marker levels compared to the control group, including lower anti-Müllerian hormone levels (FGTB: 0.82 [0.6, 1.1] µg/L; LGTB: 1.57 [1.3, 1.8] µg/L vs. control: 3.29 [2.9, 3.5] µg/L), reduced antral follicular counts (FGTB: 6 [5.5, 9.5]; LGTB: 10.5 [7, 12.3] vs. control: 17 [14.5, 18]), and fewer retrieved oocytes (FGTB: 3 [2, 5]; LGTB: 8 [4, 8.3] vs. control: 14.5 [11.5, 15.3]). Conversely, these groups exhibited higher ovarian response marker levels, such as longer gonadotropin treatment days (FGTB: 12 [10.5, 12.5]; LGTB: 11 [10.8, 11.3] vs. control: 10 [8.8, 10]) and increased gonadotropin dosage requirements (FGTB: 3300 [3075, 3637.5] U; LGTB: 3037.5 [2700, 3225] U vs. control: 2531.25 [2337.5, 2943.8] U). All comparisons were statistically significant at P < 0.05. The results suggested that FGTB and LGTB have adverse effects on ovarian reserve and response. Untargeted metabolomic analysis identified 92 and 80 differential metabolites in the control vs. FGTB and control vs. LGTB groups, respectively. Pathway enrichment analysis revealed significant alterations in metabolic pathways in the FGTB and LGTB groups compared to the control group (P < 0.05), with specific changes noted in galactose metabolism, biotin metabolism, steroid hormone biosynthesis, and nicotinate and nicotinamide metabolism in the FGTB group, and caffeine metabolism, primary bile acid biosynthesis, steroid hormone biosynthesis, and glycerophospholipid metabolism in the LGTB group. The analysis of metabolic levels has revealed the potential mechanisms by which FGTB and LGTB affect ovarian reserve function, namely through alterations in metabolic pathways. The study emphasizes the importance of comprehending the metabolic alterations associated with FGTB and LGTB, which is of considerable relevance for the clinical management and therapeutic approaches in afflicted women.

Developmental programming of production and reproduction in dairy cows: IV. Association of maternal milk fat and protein percentage and milk fat to protein ratio with offspring's birth weight, survival, productive and reproductive performance and AMH concentration from birth to the first lactation period.

Although the association of maternal milk production with developmental programming of offspring has been investigated, there is limited information available on the relationship of maternal milk components with productive and reproductive performance of the offspring. Therefore, the present study was conducted to analyze the association of maternal milk fat and protein percentage and milk fat to protein ratio with birth weight, survival, productive and reproductive performance and AMH concentration in the offspring. In study I, data of birth weight, milk yield and reproductive variables of offspring born to lactating dams (n = 14,582) and data associated with average maternal milk fat percentage (MFP), protein percentage (MPP) and fat to protein ratio (MFPR) during 305-day lactation were retrieved. Afterwards, offspring were classified in various categories of MFP, MPP and MFPR. In study II, blood samples (n = 339) were collected from offspring in various categories of MFP, MPP and MFPR for measurement of serum AMH. Maternal milk fat percentage was positively associated with birth weight and average percentage of milk fat (APMF) and protein (APMP) and milk fat to protein ratio (FPR) during the first lactation, but negatively associated with culling rate during nulliparity in the offspring (P < 0.05). Maternal milk protein percentage was positively associated with birth weight, APMF, APMP, FPR and culling rate, but negatively associated with milk yield and fertility in the offspring (P < 0.05). Maternal FPR was positively associated with APMF and FPR, but negatively associated with culling rate, APMP and fertility in the offspring (P < 0.05). However, concentration of AMH in the offspring was not associated with MFP, MPP and MFPR (P > 0.05). In conclusion, the present study revealed that maternal milk fat and protein percentage and their ratio were associated with birth weight, survival, production and reproduction of the offspring. Yet it was a preliminary research and further studies are required to elucidate the mechanisms underlying these associations.

Associations between follicular fluid trace elements and ovarian response during in vitro fertilization.

INTRODUCTION: Exposure to trace elements has been associated with ovarian response in experimental studies. We conducted a hypothesis-generating study of associations between ovarian follicular fluid (FF) trace elements and measures of ovarian response among women using in vitro fertilization (IVF). METHODS: We collected ovarian FF specimens from 56 women. We determined concentrations (µg/L) of 11 trace elements using inductively coupled plasma-tandem mass spectrometry. We estimated associations between women's FF trace elements per interquartile range difference, and measures of ovarian response using linear (peak estradiol (E2), baseline anti-mullerian hormone (AMH), and follicle stimulating hormone (FSH)) and negative binomial (baseline antral follicle count (AFC) and oocyte count) regression, adjusting for confounding factors. We used principal component analysis (PCA) to estimate the associations of the FF trace elements mixture. We also explored FF oxidative stress enzymes as causal mediators of the associations. RESULTS: Higher FF cobalt was associated with greater peak E2 (mean difference = 351.48 pg/mL; 95%CI: 21.76, 724.71) and AFC (rate ratio = 1.14; 95%CI: 1.01, 1.28), and higher FF copper was associated with greater peak E2 (mean difference = 335.66 pg/mL; 95%CI: 81.77, 753.08) and oocyte count (rate ratio = 1.19; 95%CI: 1.02, 1.43). Higher FF mercury was also associated with greater peak E2 (mean difference = 410.70 pg/mL; 95%CI: 61.90, 883.39). Higher FF lead was associated with lesser AFC (rate ratio = 0.85; 95%CI: 0.73, 0.98). Using PCA, the mixture of Sr, Hg, and As was associated with higher peak estradiol, AFC, and oocyte count. FF glutathione peroxidase, paraoxonase, and arylesterase activities were inconsistent mediators of the associations, but the effect estimates were imprecise. CONCLUSION: Our results suggest that essential and non-essential trace elements in FF were associated with ovarian response during IVF.

Fertility protection: a novel approach using pretreatment with mesenchymal stem cell exosomes to prevent chemotherapy-induced ovarian damage in a mouse model.

BACKGROUND: Primary ovarian insufficiency refers to the loss of ovarian function before the age of 40 years and leads to amenorrhea and infertility. Primary ovarian insufficiency has diverse causes, but a common cause is exposure to gonadotoxic chemotherapy used in cancer treatment. Because of the risk for developing primary ovarian insufficiency, patients who want to preserve their fertility may consider various procedures for fertility preservation. However, current fertility preservation options are highly invasive, carry substantial risks, and have uncertain success rates. Recent studies from our group and others reported that mesenchymal stem cells and mesenchymal stem cell-derived exosomes can restore ovarian function in preclinical models of primary ovarian insufficiency by restoring damaged cells and inhibiting apoptosis. Although the restorative effect of mesenchymal stem cell-derived exosomes has been well reported in previous studies, the potential of mesenchymal stem cell-derived exosomes in preventing ovarian damage has not been fully elucidated. OBJECTIVE: This study hypothesized that the antiapoptotic potential of mesenchymal stem cell-derived exosomes may protect ovarian tissue from chemotherapy-induced damage. STUDY DESIGN: In this study, we delivered mesenchymal stem cell-derived exosomes directly into the ovaries of mice before administration of chemotherapy. A total of 60 mice were divided into 3 groups (20 per group), which were labeled the control, chemotherapy, and fertility protection groups. Only the fertility protection group mice received exosomes, whereas the control and chemotherapy group mice received saline. After exosome injection, the chemotherapy and fertility protection groups of mice were subjected to chemotherapy to induce ovarian damage. After chemotherapy, we evaluated the protective effects of exosome treatment on ovarian function, such as estrous cyclicity, serum hormone levels, and the fertility rate, by comparing these outcomes between the chemotherapy and fertility protection groups. These outcomes were also compared with those of the control group for comparison with outcomes under healthy conditions. RESULTS: After intraovarian injection of exosomes before chemotherapy, the mice were able to maintain their estrous cycle (4- to 5-day cyclicity), serum anti-müllerian hormone level (66.06±26.40 ng/mL, not significantly different from that of the healthy controls), folliculogenesis (32.2±11.3 in the chemotherapy group vs 46.4±14.1 in the fertility protection group; P<.05), expression of the steroidogenic acute regulatory protein gene (a the steroidogenesis marker) (0.44±0.11-fold expression in the chemotherapy group and 0.88±0.31-fold expression in the fertility protection group; P<.05), and fertility (2 of 8 in the chemotherapy group and 5 of 8 in the fertility protection group), thereby showing prevention of chemotherapy-induced damage. We found that exosome treatment before chemotherapy can preserve ovarian function and protect fertility through the overexpression of ATP synthase-binding cassette transporters, such as ABCB1b (10.17±17.75-fold expression in the chemotherapy group and 44.14±33.25-fold expression in the fertility protection group; P<.05) and ABCC10 (3.25±0.59-fold expression in the chemotherapy group and 5.36±1.86-fold expression in the fertility protection group; P<.05). CONCLUSION: In this study, we present a novel fertility protection method using mesenchymal stem cell-derived exosomes. We concluded that mesenchymal stem cell-derived exosomes are a promising and simple treatment option for fertility protection in reproductive-aged patients who are receiving gonadotoxic chemotherapy.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and ß (ERα and Erß), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Brca2(p.T1942fs/+) dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells.

Pathogenic variants of BRCA1/2 constitute hereditary breast and ovarian cancer (HBOC) syndrome, and BRCA1/2 mutant is a risk for various cancers. Whereas the clinical guideline for HBOC patients has been organized for the therapy and prevention of cancer, there is no recommendation on the female reproductive discipline. Indeed, the role of BRCA1/2 pathogenic variants in ovarian reserve has not been established due to the deficiency of appropriate animal models. Here, we used a rat model of Brca2(p.T1942fs/+) mutant of Sprague-Dawley strain with CRISPR-Cas9 editing to evaluate ovarian reserve in females. Fertility and ovarian follicles were evaluated and anti-Müllerian hormone (AMH) was measured at 8-32 weeks of age with a comparison between the wild-type and the mutant rats (MUT). MUT revealed a significantly smaller number of deliveries with fewer total pups. Furthermore, MUT showed a significant decrease in primordial follicles at 20 weeks and a low AMH level at 28 weeks. RNA-sequencing of the ovary at 10 weeks detected acceleration of the DNA damage repair pathway, which was accompanied by oxidative stress-induced DNA double-strand breaks, a decrease in PTEN, and an increase in mTOR in follicular granulosa cells. In conclusion, Brca2(p.T1942fs/+) dissipates primordial follicles via early activation of granulosa cells through oxidative stress, leading to earlier termination of fertility.

Correlation between ovarian follicular development and Hippo pathway in polycystic ovary syndrome.

BACKGROUND: For women of childbearing age, the biggest problem caused by polycystic ovary syndrome (PCOS) is infertility, which is mainly caused by anovulation, abnormal follicular development, proliferation of small antral follicles, and cystic follicles. The mechanism underlying its occurrence is not clear. The abnormal proliferation and development of follicles in PCOS patients is a complex process, which is affected by many factors. The objective of this study was to investigate the relationship between the Hippo pathway and follicular development in PCOS, and to further explore this relationship by using the YAP inhibitor verteporfin (VP). METHOD: 30 3-week-old BALB/C female rats were randomly divided into control group (n = 10), DHEA group (n = 10) and DHEA + VP group (n = 10). The morphology of ovary and the degree of follicular development were observed by HE staining, and the expression and location of AMH in ovarian follicles were observed by immunofluorescence. The ovarian reserve function index AMH, cell proliferation index PCNA and the ratio of Hippo pathway related proteins MST, LATS, YAP, P-YAP and P-YAP/YAP were detected by Western blot. RESULTS: After dividing 30 3-week-old female mice into control, dehydroepiandrosterone (DHEA; model of PCOS), and DHEA + VP groups, we found that the number of small follicles increased in the DHEA group compared to the control group. Additionally, in the DHEA group compared to the control group, anti-müllerian hormone (AMH; ovarian reserve index) increased, proliferating cell nuclear antigen (PCNA; cell proliferation index) decreased, and upstream (MST and LATS) and downstream (YAP and p-YAP) proteins in the Hippo pathway increased, though the p-YAP/YAP ratio decreased. VP ameliorated the increases in AMH, MST, LATS, YAP and p-YAP, but did not ameliorate the decrease in the p-YAP/YAP ratio. CONCLUSIONS: This study indicates that the increased small follicles in the ovaries and changes in ovarian reserve and cell proliferation may be closely related to Hippo pathway activation. This suggests that the Hippo pathway may be an important pathway affecting the proliferation and development of follicles and the occurrence of PCOS.

Exploring AMH levels, homeostasis parameters, and ovarian primordial follicle activation in pubertal infected sheep on a high-protein diet.

"Exploring AMH Levels, Homeostasis and Primordial Follicle Activation in Pubertal Infected Sheep on a High Protein Diet ". The first activation wave of ovarian primordial follicles is part of the onset of puberty and fertility. Abomasal helminth infection may cause an undesirable delay in puberty manifestation. Helminth-infected animals demand a higher amount of protein in their diet to repair the damage caused by the parasite in sheep's tissues, replenish the blood losses, and build the host's immune response. Helminths become resistant to drug therapy shortly after being exposed to a new treatment. Besides, there is the possibility of contamination by anthelmintic drugs in ovine products, possibly affecting human health and the environment. This study's objective was to evaluate if ovarian and clinical parameters can be improved by supplementing their diet with protein, offering a more sustainable management approach than relying on anthelmintic usage. We used a 2 × 2 factorial model where eighteen ewe lambs (Ovis aries) between 6 and 7 months old - born to the same ram - were fed one of two diet protein levels (12% or 19%). After 35 days on this diet, they were infected or left uninfected with 10,000 Haemonchus contortus L3 larvae. We evaluated Anti-Mullerian Hormone serum levels, blood cells and biochemical parameters at four different time points. Following 42 days of infection and 77 days on the diet, the lambs had their left ovaries removed, and we examined ovarian morphometrics through histological analysis. The groups Supplemented Protein-Infected(n = 5), Control Protein- Infected(n = 5), Supplemented Protein-Not Infected (n = 4) and Control Protein-Not Infected (n = 4) did not differ in their bodyweight gain. In the factorial ANOVA analysis examining the relationship between plasma protein, diet, and infection, the protein level of the diet showed significance (p = 0.02). Primordial follicle size varied with the interaction between diet and infection (p < 0.05), and oocyte size was affected by the level of protein in the diet (p = 0.047). Additionally, to understand how all homeostasis parameters relate to the primordial follicle and oocyte size, we applied an explanatory linear mixed model. In conclusion, serum AMH levels remained stable despite the infection and variations in diet protein levels, indicating its reliability as a marker for ovarian reserve in pubertal sheep. The number of blood cells, biochemical parameters, and primordial follicle activation were affected by both diet and infection.

Investigations of the function of AMH in granulosa cells in hens.

Anti-mullerian hormone (AMH) plays a crucial role in follicle regulation in mammals by preventing premature primordial follicle activation and restricting follicle development through reduction of FSH sensitivity and inhibition of FSH-induced increase of steroidogenic enzymes. AMH is produced by granulosa cells from growing follicles and expression declines at the time of selection in both mammalian and avian species. The role of AMH in chicken granulosa cells remains unclear, as research is complicated because mammalian AMH is not bioactive in chickens and there is a lack of commercially available chicken AMH. In the current experiments, we used RNA interference to study the role of AMH on markers of follicle development in the presence and absence of FSH. Cultured chicken granulosa cells from 3-5 mm follicles and 6-8 mm follicles, the growing pool from which follicle selection is thought to occur, were used. Transfection with an AMH-specific siRNA significantly reduced AMH mRNA expression in granulosa cells from 3-5 mm and 6-8 mm follicles. Genes of interest were only measured in granulosa cells of 3-5 mm follicles due to low expression of AMH mRNA at the 6-8 mm follicle stage. Knockdown of AMH mRNA did not affect markers of follicle development (follicle stimulating hormone receptor, FSHR; steroidogenic acute regulatory protein, STAR; cytochrome P450 family 11 subfamily A member 1, CYP11A1; bone morphogenetic protein receptor type 2, BMPR2) or FSH responsiveness in granulosa cells from 3-5 mm follicles, indicating that AMH does not regulate follicle development directly by affecting markers of steroidogenesis, FSHR or BMPR2 at this follicle stage in chickens.

Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development.

We previously showed that the anti-Müllerian hormone (AMH), infiltrating from the testis to the mesonephros reaches the cranial and middle regions of the Müllerian duct (MD) and induces their regression using an organ culture in mice. However, it is difficult to maintain structural integrity, such as the length and diameter and normal direction of elongation of the caudal region of the MD, in conventional organ culture systems. Therefore, the pathway of AMH to the caudal MD region remains uncharted. In this study, we established an organ culture method that can maintain the morphology of the caudal region of the MD. The gonad-mesonephros complex, metanephros, and urinary bladder of mouse fetuses at 12.5 dpc attached to the body trunk were cultured on agarose gels for 72 hr. The cultured caudal region of the mesonephros was elongated along the body trunk, and the course of the mesonephros was maintained in many individuals. In males, mesenchymal cells aggregated around the MD after culture. Moreover, the male MD diameter was significantly smaller than the female. Based on these results, it was concluded that the development of the MD was maintained in the present organ culture system. Using this culture system, AMH infiltration to the caudal region of the MD can be examined without the influence of AMH in the blood. This culture system is useful for clarifying the regression mechanism of the caudal region of the MD.

Cluster analysis reveals a homogeneous subgroup of PCOS women with metabolic disturbance associated with adverse reproductive outcomes.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a heterogeneous and complex reproductive endocrinological disease that could lead to infertility. There were many attempts to classify PCOS but it remains unclear whether there is a specific subgroup of PCOS that is associated with the best or worst reproductive outcomes of assisted reproductive techniques (ART). METHODS: Infertile PCOS patients who underwent their first cycle of in vitro fertilization (IVF) in West China Second University Hospital, Sichuan University from January 2019 to December 2021 were included. Basic clinical and laboratory information of each individual were extracted. Unsupervised cluster analysis was performed. Controlled ovarian stimulation parameters and reproductive outcomes were collected and compared between the different clusters of PCOS. RESULTS: Our analysis clustered women with PCOS into "reproductive", "metabolic", and "balanced" clusters based on nine traits. Reproductive group was characterized by high levels of testosterone (T), sex hormone-binding globulin (SHBG), follicular stimulation hormone (FSH), luteinizing hormone (LH), and anti-Müllerian hormone (AMH). Metabolic group was characterized by high levels of body mass index (BMI), fasting insulin, and fasting glucose. Balanced group was characterized by low levels of the aforementioned reproductive and metabolic parameters, except for SHBG. Compared with PCOS patients in reproductive and balanced clusters, those in metabolic cluster had lower rates of good quality day 3 embryo and blastocyst formation. Moreover, PCOS patients in the reproductive cluster had greater fresh embryo transfer (ET) cancelation rate and clinical pregnancy rate after fresh ET than metabolic cluster (odds ratio [OR] = 3.37, 95% confidence interval [CI]: 1.77-6.44, and OR = 6.19, 95% CI: 1.58-24.24, respectively). And compared with PCOS of metabolic cluster, PCOS of balanced cluster also had higher chance for fresh ET cancelation (OR = 2.83, 95% CI: 1.26-6.35). CONCLUSION: Our study suggested that PCOS patients in metabolic cluster may be associated with adverse reproductive outcomes and might need individualized treatment and careful monitoring before and during ART.

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.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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. TRIAL REGISTRATION NUMBER: N/A.

Sertoli Cell-Specific Activation of Transforming Growth Factor Beta Receptor 1 Leads to Testicular Granulosa Cell Tumor Formation.

The transforming growth factor ß (TGFß) superfamily, consisting of protein ligands, receptors, and intracellular SMAD transducers, regulates fundamental biological processes and cancer development. Our previous study has shown that sustained activation of TGFß receptor 1 (TGFBR1) driven by anti-Mullerian hormone receptor type 2 (Amhr2)-Cre in the mouse testis induces the formation of testicular granulosa cell tumors (TGCTs). As Amhr2-Cre is expressed in both Sertoli cells and Leydig cells, it remains unclear whether the activation of TGFBR1 in Sertoli cells alone is sufficient to induce TGCT formation. Therefore, the objective of this study was to determine whether Sertoli cell-activation of TGFBR1 drives oncogenesis in the testis. Our hypothesis was that overactivation of TGFBR1 in Sertoli cells would promote their transdifferentiation into granulosa-like cells and the formation of TGCTs. To test this hypothesis, we generated mice harboring constitutive activation of TGFBR1 in Sertoli cells using anti-Mullerian hormone (Amh)-Cre. Disorganized seminiferous tubules and tumor nodules were found in TGFBR1CA; Amh-Cre mice. A histological analysis showed that Sertoli cell-specific activation of TGFBR1 led to the development of neoplasms resembling granulosa cell tumors, which derailed spermatogenesis. Moreover, TGCTs expressed granulosa cell markers including FOXL2, FOXO1, and INHA. Using a dual fluorescence reporter line, the membrane-targeted tdTomato (mT)/membrane-targeted EGFP (mG) mouse, we provided evidence that Sertoli cells transdifferentiated toward a granulosa cell fate during tumorigenesis. Thus, our findings indicate that Sertoli cell-specific activation of TGFBR1 leads to the formation of TGCTs, supporting a key contribution of Sertoli cell reprogramming to the development of this testicular malignancy in our model.

Melamine exposure during the weaning period negatively affects ovarian reserve.

In this study, it was aimed to investigate the effects of melamine exposure since the weaning period on ovarian tissue and ovarian reserve. Melamine is illegally added to milk and formula to provide high false protein positivity. Female rats (the weaning period = 21 days old, n = 18) were divided into 3 groups. 0.1 mL saline was applied to the control group by gavage for 21 days. Fifty mg/kg and seventy-five mg/kg melamine was dissolved in 0.1 mL of saline and applied by gavage for 21 days, respectively. At the end of the experiment, plasma anti-Mullerian hormone (AMH) was measured, follicle count and ovarian diameter measurement were performed in the right ovaries, and flow cytometric analysis for apoptosis was performed in the left ovaries. While a statistically significant decrease was not observed in the number of the follicle and ovarian diameter between the control and melamine-treated groups (p > 0.05), a significant decrease in the corpus luteum and a significant increase in the number of atretic follicles were observed (p < 0.05). Apoptosis (Annexin V) increased in both melamine groups and AMH plasma level decreased significantly in the 75 mg/kg group (p < 0.05). Melamine exposure from the weaning (early postnatal) period may cause a decrease in ovarian reserve in parallel with a dose increase.

SMAD signaling pathway is disrupted by BPA via the AMH receptor in bovine granulosa cells†.

Significant events that determine oocyte competence occur during follicular growth and oocyte maturation. The anti-Mullerian hormone, a positive predictor of fertility, has been shown to be affected by exposure to endocrine disrupting compounds, such as bisphenol A and S. However, the interaction between bisphenols and SMAD proteins, mediators of the anti-Mullerian hormone pathway, has not yet been elucidated. AMH receptor (AMHRII) and downstream SMAD expression was investigated in bovine granulosa cells treated with bisphenol A, bisphenol S, and then competitively with the anti-Mullerian hormone. Here, we show that 24-h bisphenol A exposure in granulosa cells significantly increased SMAD1, SMAD4, and SMAD5 mRNA expression. No significant changes were observed in AMHRII or SMADs protein expression after 24-h treatment. Following 12-h treatments with bisphenol A (alone or with the anti-Mullerian hormone), a significant increase in SMAD1 and SMAD4 mRNA expression was observed, while a significant decrease in SMAD1 and phosphorylated SMAD1 was detected at the protein level. To establish a functional link between bisphenols and the anti-Mullerian hormone signaling pathway, antisense oligonucleotides were utilized to suppress AMHRII expression with or without bisphenol exposure. Initially, transfection conditions were optimized and validated with a 70% knockdown achieved. Our findings show that bisphenol S exerts its effects independently of the anti-Mullerian hormone receptor, while bisphenol A may act directly through the anti-Mullerian hormone signaling pathway providing a potential mechanism by which bisphenols may exert their actions to disrupt follicular development and decrease oocyte competence.

Decreased growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions in the ovary via ulipristal acetate.

OBJECTIVE: Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors. METHODS: In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA. RESULTS: Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05). CONCLUSION: Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.