Primordial Follicle Recruitment Research Articles

The Role of Anti-Müllerian Hormone in Ovarian Function.

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

The Impacts of Periconceptional Alcohol on Neonatal Ovaries and Subsequent Adult Fertility in the Rat.

Maternal exposures during pregnancy can impact the establishment of the ovarian reserve in offspring, the lifetime supply of germ cells that determine a woman's reproductive lifespan. However, despite alcohol consumption being common in women of reproductive age, the impact of prenatal alcohol on ovarian development is rarely investigated. This study used an established rat model of periconceptional ethanol exposure (PCEtOH; 12.5% v/v ethanol) for 4 days prior to 4 days post-conception. Ovaries were collected from neonates (day 3 and day 10), and genes with protein products involved in regulating the ovarian reserve analyzed by qPCR. Adult offspring had estrous cycles monitored and breeding performance assessed. PCEtOH resulted in subtle changes in expression of genes regulating apoptosis at postnatal day (PN) 3, whilst those involved in regulating growth and recruitment of primordial follicles were dysregulated at PN10 in neonatal ovaries. Despite these gene expression changes, there were no significant impacts on breeding performance in adulthood, nor on F2-generation growth or survival. This contributes additional evidence to suggest that a moderate level of alcohol consumption exclusively around conception, when a woman is often unaware of her pregnancy, does not substantially impact the fertility of her female offspring.

In Vitro Maturation, In Vitro Oogenesis, and Ovarian Longevity.

This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic cells. As an aside benefit we also have derived a better understanding of ovarian longevity from ovary transplant. We have found that primordial follicle recruitment is triggered by tissue pressure gradients. Increased pressure holds the follicle in meiotic arrest and prevents recruitment. Therefore recruitment occurs first in the least dense inner tissue of the cortico-medullary junction. Many oocytes can be obtained from human ovarian tissue and mature to metaphase 2 in vitro with no need for ovarian stimulation. Ovarian stimulation may only be necessary for removing the oocyte from the ovary, but this can also be accomplished by simple dissection at the time of ovary tissue cryopreservation. By using surgical dissection of the removed ovary, rather than a needle stick, we can obtain many oocytes from very small follicles not visible with ultrasound. A clearer understanding of ovarian function has come from in vitro oogenesis experiments, and that explains why IVM has now become so simple and robust. Tissue pressure (and just a few "core genes" in the mouse) direct primordial follicle recruitment and development to mature oocyte, and therefore also control ovarian longevity. There are three distinct phases to oocyte development both in vitro and in vivo: in vitro differentiation "IVD" which is not gonadotropin sensitive (the longest phase), in vitro gonadotropin sensitivity "IVG" which is the phase of gonadotropin stimulation to prepare for meiotic competence, and IVM to metaphase II. On any given day 35% of GVs in ovarian tissue have already undergone "IVD" and "IVG" in vivo, and therefore are ready for IVM.

The roles of anti-Müllerian hormone in breast cancer.

Anti-Müllerian hormone (AMH) is produced and secreted by granulosa cells of growing follicles, and its main role is to inhibit the recruitment of primordial follicles, reduce the sensitivity of follicles to follicle-stimulating hormone (FSH), and regulate FSH-dependent preantral follicle growth. It has become an effective indicator of ovarian reserve in clinical practice. Research on AMH and its receptors in recent years has led to a better understanding of its role in breast cancer. AMH specifically binds to anti-Müllerian hormone receptor II (AMHRII) to activate downstream pathways and regulate gene transcription. Since AMHRII is expressed in breast cancer cells and triggers apoptosis, AMH/AMHRII may play an important role in the occurrence, treatment, and prognosis of breast cancer, which needs further research. The AMH level is a potent predictor of ovarian function after chemotherapy in premenopausal breast cancer patients older than 35 years, either for ovarian function injury or ovarian function recovery. Moreover, AMHRII has the potential to be a new marker for the molecular typing of breast cancer and a new target for breast cancer treatment, which may be a link in the downstream pathway after TP53 mutation.

P-397 The effect of letrozole overlapped with gonadotropin on IVF outcomes in women with DOR or aged over 40 years old

Abstract Study question Evaluating the efficacy of modified letrozole protocol (mLP) for diminished ovarian reserve (DOR) or advanced age women. Summary answer The modified letrozole protocol provides another option for women with DOR or advanced age, especially those who experienced ovarian stimulation previously. What is known already For years, numerous methods have been conducted to obtain better outcomes in women with advanced age or DOR. Increasing doses of gonadotropin (Gn), exogenous luteinizing hormone (LH) or growth factor (GH) supplementation, and various kinds of controlled ovarian stimulation (COS) protocols were reported. However, there is still no consensus due to the contradictory reports of comparable reproductive outcomes. During ovarian stimulation, estrogen and androgen are important in the recruitment of primordial follicles and promotion of follicular growth at the preantral and antral stages across different species. Study design, size, duration This is a paired-match study including 243 women with DOR and 249 women aged over 40 years old who received in vitro fertilization (IVF) treatment. Participants/materials, setting, methods 123 women received stimulation with mLP. GnRH agonist (GnRH-a) long, GnRH antagonist (GnRH-anta), and mild stimulation protocol were used as controls with 123 women in each group. Clinical pregnancy rate (CPR) and cumulative clinical pregnancy rate (CCPR) were main outcomes. Main results and the role of chance CPR in the mLP group (38.46%) was significantly higher than mild stimulation (17.11%), but not significantly different from GnRH-a long (26.13%) and GnRH-anta (29.17%) group. CCPR showed an increasing trend in the mLP group (33.33%) although without significance when compared with controls. The CCRP of GnRH-a long, GnRH-anta, mild stimulation group were 21.68%, 29.03%, and 13.01%, respectively. In women with repeated cycles, mLP achieved higher available embryo rate and top-quality embryo rate. Further study showed positive correlation between testosterone and the number of oocytes retrieved in mLP group (r = 0.395, P< 0.01). Limitations, reasons for caution It is a retrospective study which resulted in minor bias among mLP and the other control protocols. Although we have already paired for age and AMH, the presence of biases cannot be totally excluded. Further prospective studies need to be done to validate the effect of mLP. Wider implications of the findings The modified letrozole protocol may be effective for women with DOR or advanced age, especially those who have experienced previous cycle failure. An increasing serum testosterone level may play a role and reflect follicular growth during ovarian stimulation. Trial registration number not applicable

Associations between urinary phthalate concentrations and antral follicle count among women undergoing in vitro fertilization.

Phthalates are ubiquitously used in a variety of products and have an adverse effect on folliculogenesis. However, previous epidemiological studies on the associations between phthalate exposure and antral follicle count (AFC) produced conflicting results. The present study aimed to evaluate the associations between urinary phthalate metabolite concentrations and AFC among women undergoing in vitro fertilization (IVF). We collected 525 urine samples and measured 8 phthalate metabolites from IVF patients. Poisson regression models were conducted to evaluate the associations between phthalate metabolite concentrations and AFC. In addition, participants were stratified into a younger group (< 35 years) and an older group (≥ 35 years) to explore the potential effect modification by age. We also performed sensitivity analyses by restricting our analyses to only infertile women diagnosed with tubal factor infertility to test the robustness of the results. Significant positive associations were observed among urinary MBP, MEOHP and ∑PAEs concentrations and AFC after adjusting for age, BMI, year of study and infertility diagnosis. Compared with women in the first tertile, women in the third tertile of MBP and MEOHP had 7.02% (95% CI: 1.18%, 12.9%) and 8.84% (95% CI: 2.83%, 14.9%) higher AFC, respectively, and women in the second and third tertiles of ∑PAEs had 6.19% (95% CI: 0.37%, 12.0%) and 9.09% (95% CI: 3.22%, 15.0%) higher AFC, respectively. In addition, MBP, MEOHP and ∑PAEs also had significant positive associations with AFC in trend tests for dose-response. In the age-stratified analysis, we found a stronger relationship between phthalate metabolite concentrations and AFC among older women and an inverse association among younger women. We observed similar results in the sensitivity analyses. We found positive associations between phthalate exposure and AFC, which support the idea that phthalate exposure may accelerate primordial follicle recruitment and lead to higher AFC in women undergoing IVF. More studies are needed to better understand their relationships.

Hyperbaric Oxygen Treatment Ameliorates the Decline in Oocyte Quality and Improves the Fertility of Aged Female Mice.

The age-related decay in oocyte quality contributes to the gradual decline in fertility and the final occurrence of natural sterility. In this study, we aimed to investigate the effects of the hyperbaric oxygen treatment (HBOT) on oocyte quality in aging mouse oocyte. Eight- and forty-week-old female C57BL/6J mice were treated with HBO for 10days, and the quality of oocytes was analyzed. The results revealed that HBOT improved the age-related serum AMH levels. While compared with untreated aged mice, HBOT showed reduced follicular apoptosis and improved oocyte maturation, fertilization, and blastocyst formation in aged mice. HBO triggered changes in the microRNA expression in the ovaries of aged mice. In this study, 27 DEGs were identified in the HBOT mouse ovarian tissues, of which 9 were upregulated and 18 were downregulated. Notably, KEGG analysis revealed that these genes involved in different biological processes differed significantly in the ovary. Among these, the PI3K-Akt signaling was the most prominent pathway that controlled the recruitment and growth of primordial follicles. The calcium signaling pathway was found to be involved during the peri-implantation period. These results suggest that HBOT can be applied to improve the quality of oocytes, and it could be a potential clinical application to improve the fertility of aged female.

Clinical Utilities of Anti-Müllerian Hormone

The anti-Müllerian hormone (AMH) plays an essential role in sex determination in early embryonic development. Through a series of sequential steps that follows inheriting an XY chromosome, Sertoli cell differentiation upregulates the expression of AMH-suppressing Müllerian duct development and maintains the AMH at a high level until puberty. In females, the AMH is produced by granulosa cells of follicles beginning in the second half of fetal life and continues through adulthood, with a steady decline through the reproductive years and severe decline at menopause, until levels eventually become undetectable. The AMH is essential for the regulation of follicular maturation via the recruitment of primordial follicles throughout folliculogenesis. AMH serum concentration in women strongly correlates with ovarian reserve quantity and reflects ovulation potential. Because the AMH is expressed almost exclusively by growing follicles before FSH-dependent selection, it commonly serves as a marker for ovarian function in various clinical situations, including in the diagnosis and pathogenesis of polycystic ovarian syndrome, artificial reproductive technology, and predictions of menopause or premature ovarian failure.

BMP6 regulates AMH expression via SMAD1/5/8 in goat ovarian granulosa cells

Anti-Müllerian hormone (AMH) is produced by ovarian granulosa cells (GCs)and plays a major role in inhibiting the recruitment of primordial follicles and reducing the sensitivity of growing follicles to follicle-stimulating hormone (FSH). Bone morphogenetic protein 6 (BMP6) has similar spatiotemporal expression to AMH during follicular development, suggesting that BMP6 may regulate AMH expression. However, the specific mechanism by which BMP6 regulates AMH expression remains unclear. The objectives of this study were to examine the molecular pathway by which BMP6 regulates AMH expression. The results showed that BMP6 promoted the secretion and expression of AMH in goat ovarian GCs. Mechanistically, BMP6 upregulated the expression of sex-determining region Y-box 9 (SOX9) and GATA-binding factor 4 (GATA4), which was associated with the transcriptional initiation of AMH. AMH expression was significantly decreased by GATA4 knockdown. Moreover, BMP6 treatment promoted the phosphorylation of SMAD1/5/8, whereas inhibiting the SMAD1/5/8 signaling pathway significantly abolished BMP6-induced upregulation of AMH and GATA4 expression. Interestingly, the activation of SMAD1/5/8 alone did not affect the expression of AMH or GATA4. The results suggested that BMP6 upregulated GATA4 through the SMAD1/5/8 signaling pathway, which in turn promoted AMH expression.

O-244 Androgen levels and their predictive value in women with different ovarian responses

Abstract Study question Do androgen levels impact oocyte yield during ovarian stimulation and how predictive are they for distinct ovarian response? Summary answer Testosterone positively correlates with the number of oocytes retrieved in all patients. DHEA-S appears more specific by only correlating significantly in poor responding women. What is known already Androgens are involved in the initiation of primordial follicle recruitment and early development. Testosterone and DHEA-S increase the number of growing follicles by reducing follicular atresia. A reduction in androgen levels can be observed with diminished ovarian reserve and advanced age. To standardize the definition of POR Bologna criteria have been elaborated including age, laboratory or sonographic ovarian reserve and low ovarian response in previous stimulations. The mentioned markers have been reviewed concerning its predictive power for POR by the POSEIDON analysis and divided into four subgroups. To date androgen levels are not raised to stratify POR in these classifications. Study design, size, duration All women were recruited in the division for reproductive medicine at the University Hospital Heidelberg. The patients’ files were retrospectively analyzed for androgen levels before ovarian stimulation and outcome. A classification between poor and normal response was performed according to Bologna criteria. Participants/materials, setting, methods All patients provided written informed consent. Blood samples were analyzed from serum drawn on day 2 to 5 of the menstrual cycle before ovarian stimulation. Statistical analysis was performed with SPSS; statistical significance was set to p &amp;lt; = 0.05. Main results and the role of chance Valid data for analysis was available in 335 cycles, 238 were scaled in normal (NOR), 97 in poor ovarian response (POR). Statistics in both subgroups were performed concerning age, AMH, count of retrieved oocytes, DHEA-S and testosterone with significant lower AMH as well as androgen levels, oocyte yields and a higher median age in the POR group. An expected significant positive correlation between AMH and retrieved oocytes was confirmed in both subgroups (p=.000 and .004). Increasing age negatively correlates with the number of retrieved oocytes after ovarian stimulation in NOR (p=.003), but not in POR (p=.441) patients suggesting age as less powerful predictive marker for oocyte yield in those patients. In addition, a positive correlation for testosterone and retrieved oocytes merely reached significance in all analyzed cycles independently of different ovarian responses. On the contrary DHEA-S levels and oocyte counts cohered with a marginal significance (p=.05) only in POR patient cycles. Serum levels for DHEA-S positively correlated with the variable “oocyte count ≥ 3” in the POR group (OR 1.006, 95% CI, 0.999- 1.014 Limitations, reasons for caution The results are based on a limited sample size. Therefore, results need to be proven in further studies including further aspects affecting ovarian response. Wider implications of the findings The significant correlation between pre-stimulatory DHEA-S levels in POR for oocyte yield on the contrary to NOR, supports the experimental supplementation with DHEA in this subgroup, aiming to improve outcomes during ovarian stimulation. Testosterone is less specific for estimating ovarian response in both subgroups. Trial registration number n.A.

Anti-Müllerian Hormone Inhibits FSH-Induced Cumulus Oocyte Complex In Vitro Maturation and Cumulus Expansion in Mice.

Simple SummaryAnti-Müllerian hormone (AMH) is a homodimeric glycoprotein composed of two identical subunits, which inhibits the recruitment of primordial follicles and the development of antral follicles in females. Anti-Müllerian hormone can be used as a diagnostic and prognostic marker for ovarian reserve, superovulation, embryo quality, and conception rate. However, few studies have focused on the effect of AMH on oocyte maturation. In the present study, we found Anti-Müllerian hormone has no effect on the nuclear maturation and cumulus expansion of cumulus oocyte complexes (COCs), whereas it has an inhibitory effect on follicle-stimulating hormone (FSH)-stimulated COCs nuclear maturation and cumulus expansion. These findings expand our knowledge of the functional role of AMH in modulating folliculogenesis.Anti-Müllerian hormone (AMH) is secreted by the ovaries of female animals and exerts its biological effects through the type II receptor (AMHR2). AMH regulates follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH. Despite the considerable research on the actions of AMH in granulosa cells, the effect of AMH on the in vitro maturation of oocytes remains largely unknown. In the current study, we showed that AMH is only expressed in cumulus cells, while AMHR2 is produced in both cumulus cells and oocytes. AMH had no significant effect on COCs nuclear maturation, whereas it inhibited the stimulatory effects of FSH on COCs maturation and cumulus expansion. Moreover, AMH treatment effectively inhibited the positive effect of FSH on the mRNA expressions of Hyaluronan synthase 2 (Has2), Pentraxin 3 (Ptx3), and TNF-alpha-induced protein 6 (Tnfaip 6) genes in COCs. In addition, AMH significantly decreased the FSH-stimulated progesterone production, but did not change estradiol levels. Taken together, our results suggest that AMH may inhibit the effects of FSH-induced COCs in vitro maturation and cumulus expansion. These findings increase our knowledge of the functional role of AMH in regulating folliculogenesis.

Chronic exposure to propylparaben at the humanly relevant dose triggers ovarian aging in adult mice

Parabens, a type of endocrine-disrupting chemicals, are widely used as antibacterial preservatives in food and cosmetics in daily life. Paraben exposure has gained particular attention in the past decades, owing to its harmful effects on reproductive function. Whether low-dose paraben exposure may cause ovarian damage has been ignored recently. Here, we investigated the effects of chronic low-dose propylparaben (PrPB) exposure on ovarian function. Female C57BL/6J mice were exposed to PrPB at a humanly relevant dose for 8 months. Our results showed that chronic exposure to PrPB at a humanly relevant dose significantly altered the estrus cycle, hormone levels, and ovarian reserve, accelerating ovarian aging in adult mice. These effects are accompanied by oxidative stress enrichment, leading to steroidogenesis dysfunction and acceleration of primordial follicle recruitment. Notably, melatonin supplementation has been shown to protect against PrPB-induced steroidogenesis dysfunction in granulosa cells. Here, we report that daily chronic PrPB exposure may contribute to ovarian aging by altering oxidative stress-mediated JNK and PI3K-AKT signaling regulation, and that melatonin may serve as a pharmaceutical candidate for PrPB-associated ovarian dysfunction.

Exposure to environmentally relevant concentrations of ambient fine particulate matter (PM2.5) depletes the ovarian follicle reserve and causes sex-dependent cardiovascular changes in apolipoprotein E null mice

BackgroundFine particulate matter (PM2.5) exposure accelerates atherosclerosis and contains known ovotoxic chemicals. However, effects of exposure to PM2.5 on the finite ovarian follicle pool have hardly been investigated, nor have interactions between ovarian and cardiovascular effects. We hypothesized that subchronic inhalation exposure to human-relevant concentrations of PM2.5 results in destruction of ovarian follicles via apoptosis induction, as well as accelerated recruitment of primordial follicles into the growing pool. Further, we hypothesized that destruction of ovarian follicles enhances the adverse cardiovascular effects of PM2.5 in females.ResultsHyperlipidemic apolipoprotein E (Apoe) null ovary-intact or ovariectomized female mice and testis-intact male mice were exposed to concentrated ambient PM2.5 or filtered air for 12 weeks, 5 days/week for 4 h/day using a versatile aerosol concentration enrichment system. Primordial, primary, and secondary ovarian follicle numbers were decreased by 45%, 40%, and 17%, respectively, in PM2.5-exposed ovary-intact mice compared to controls (P < 0.05). The percentage of primary follicles with granulosa cells positive for the mitosis marker Ki67 was increased in the ovaries from PM2.5-exposed females versus controls (P < 0.05), consistent with increased recruitment of primordial follicles into the growing pool. Exposure to PM2.5 increased the percentages of primary and secondary follicles with DNA damage, assessed by γH2AX immunostaining (P < 0.05). Exposure to PM2.5 increased the percentages of apoptotic antral follicles, determined by TUNEL and activated caspase 3 immunostaining (P < 0.05). Removal of the ovaries and PM2.5-exposure exacerbated the atherosclerotic effects of hyperlipidemia in females (P < 0.05). While there were statistically significant changes in blood pressure and heart rate variability in PM2.5-compared to Air-exposed gonad-intact males and females and ovariectomized females, the changes were not consistent between exposure years and assessment methods.ConclusionsThese results demonstrate that subchronic PM2.5 exposure depletes the ovarian reserve by increasing recruitment of primordial follicles into the growing pool and increasing apoptosis of growing follicles. Further, PM2.5 exposure and removal of the ovaries each increase atherosclerosis progression in Apoe-/- females. Premature loss of ovarian function is associated with increased risk of osteoporosis, cardiovascular disease and Alzheimer’s disease in women. Our results thus support possible links between PM2.5 exposure and other adverse health outcomes in women.

Association Between AMH Levels and Fertility/Reproductive Outcomes Among Women Undergoing IVF: A Retrospective Study

Background:Anti-mullerian hormone (AMH) is a marker for predicting ovarian response to gonadotropin stimulation. It plays an important role in ovarian primordial follicle recruitment and dominant follicle selection. Therefore, the present study evaluated the AMH levels and their association with fertility/reproductive outcomes among women undergoing IVF.Methods:A retrospective study was conducted on 665 women in GarbhaGudi Institute of Reproductive Health and Research in India from October 2018 to 2019. Subjects were divided into ≥1.1 and ≤1.1 AMH level groups. Data on age, luteinizing hormone; LH (mIU/L), follicle-stimulating hormone values; FSH (mIU/ml), LH value, oocytes retrieved, and oocytes fertilization were collected. AMH category was considered as the primary explanatory variable. Independent sample t-test and chi-square tests were performed. The p<0.05 was considered statistically significant.Results:Couple’s age, FSH values (mIU/ml), number of large follicles, matured oocytes, fertilized oocytes, and cleaved embryos were statistically significant (p<0.001) among subjects with ≥1.1 AMH values. Percentage of women with successful embryo transfer was slightly higher among AMH category 1.1 (p=0.09). Fertilization rate (86.67±20.08 vs. 83.64±21.39, p=0.18) and clinical pregnancy rate (43.38% vs. 36.36%, p=0.19) were slightly higher among women with AMH level of ≥1.1 as compared to AMH of <1.1. Live birth rate was slightly higher among women with AMH level of 1.1 (25.85% vs. 22.22%, p=0.45). Also, the number of fertilized oocytes was associated with clinical pregnancy rate (aOR=1.20, 95%CI 1.09–1.33).Conclusion:Women with ≥1.10 serum AMH levels had more number of retrieved oocytes, good oocyte quality, increased embryo transfer, and fertilization rates.

In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity

Research questionIs it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? DesignA total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. ResultsEvery woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. ConclusionsThe authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight ‘core genes’ control primordial follicle recruitment and development.

Translational Physiology of Anti-Müllerian Hormone: Clinical Applications in Female Fertility Preservation and Cancer Treatment.

BackgroundWhilst the ability of AMH to induce the regression of the Müllerian ducts in the male fetus is well appreciated, AMH has additional biological actions in relation to steroid biosynthesis and ovarian follicle dynamics. An understanding of the physiology of AMH illuminates the potential therapeutic utility of AMH to protect the ovarian reserve during chemotherapy and in the treatment of female malignancies. The translation of the biological actions of AMH into clinical applications is an emerging focus of research, with promising preliminary results.Objective and RationaleStudies indicate AMH restrains primordial follicle development, thus administration of AMH during chemotherapy may protect the ovarian reserve by preventing the mass activation of primordial follicles. As AMH induces regression of tissues expressing the AMH receptor (AMHRII), administration of AMH may inhibit growth of malignancies expressing AMHR II. This review evaluates the biological actions of AMH in females and appraises human clinical applications.Search MethodsA comprehensive search of the Medline and EMBASE databases seeking articles related to the physiological functions and therapeutic applications of AMH was conducted in July 2021. The search was limited to studies published in English.OutcomesAMH regulates primordial follicle recruitment and moderates sex steroid production through the inhibition of transcription of enzymes in the steroid biosynthetic pathway, primarily aromatase and 17α-hydroxylase/17,20-lyase. Preliminary data indicates that administration of AMH to mice during chemotherapy conveys a degree of protection to the ovarian reserve. Administration of AMH at the time of ovarian tissue grafting has the potential to restrain uncontrolled primordial follicle growth during revascularization. Numerous studies demonstrate AMH induced regression of AMHR II expressing malignancies. As this action occurs via a different mechanism to traditional chemotherapeutic agents, AMH has the capacity to inhibit proliferation of chemo-resistant ovarian cancer cells and cancer stem cells.Wider ImplicationsTo date, AMH has not been administered to humans. Data identified in this review suggests administration of AMH would be safe and well tolerated. Administration of AMH during chemotherapy may provide a synchronistic benefit to women with an AMHR II expressing malignancy, protecting the ovarian reserve whilst the cancer is treated by dual mechanisms.

O-218 The effect of mTOR activation on human primordial follicle activation during in-situ culture

Abstract Study question What is the effect of mTOR pathway activation on human primordial follicles in-situ activation and subsequent development following tissue cryopreservation? Summary answer Temporary treatment of cryopreserved human ovarian tissue with mTOR activators cause the initiation of primordial follicle development and influence steroidogenesis. What is known already In-vitro activation of primordial follicles to produce mature oocytes provides an alternative technique for fertility preservation. The employment of different stimulators of PI3K pathway has been successfully used to activate resting follicles during culture or prior to grafting in patients with premature ovarian insufficiency. The addition of phosphatidic acid (PA) and propranolol (PP), as mTOR stimulators, in the culture medium has promoted primordial follicle activation morphologically in mouse and human ovaries. Molecular and functional evaluations of primordial follicle activation after treatment with the mentioned stimulators has not been conducted. Study design, size, duration Ovarian tissues which were donated from 6 transsexual patients (23-35 years old), were dissected and cryopreserved with slow-freezing technique. After thawing, they were cut into 1x1x1 mm fragments and incubated with different stimulators in three groups: 1) Control (without stimulators), 2) PA (200µM), and 3) PA+PP (200µM &amp; 50µM respectively). In groups two and three, ovarian fragments were cultured for 24 hours in presence of stimulators and then cultured for additional 6 days without stimulators. Participants/materials, setting, methods The cultured ovarian fragments were directly processed for Hematoxylin and Eosin staining and Western blot analysis. The proportion of morphologically normal and degenerated primordial and growing follicles and 17β-estradiol (E2) level in the culture medium were compared after 1 and 7 days of culture to assess follicular development and function. Western blot analysis for phosphorylated and non-phosphorylated status of FOXO3a and RSP6 proteins expression were compared after 24 hours of incubation. Main results and the role of chance The proportion of primordial and growing follicles were not significantly different in the experimental groups after 24 hours of incubation with either of the stimulators. Western blot analyses indicated a significant reduction of FOXO3a in the PA+PP group compared to the control group. The phosphorylation level of RPS6 protein did not significantly change in either of the groups. The proportion of transitional follicles were significantly higher in the PA group compared to other groups after 7 days of culture. The E2 secretion level was significantly higher at the last day of culture compared to day 1 for all groups. At the end of the culture period, E2 levels was significantly higher in both PA and PA+PP groups compared to the control group. Limitations, reasons for caution Due to ovarian fragmentation before culture, the HIPPO pathway downstream molecules should have also been evaluated by western blot, which was not contributed in this study. Wider implications of the findings The results demonstrate the beneficial effect of mTOR signaling to accelerate early primordial follicle recruitment in cryopreserved-thawed human ovarian fragments. Trial registration number not applicable

Pharmacological inhibition of the PI3K/PTEN/Akt and mTOR signalling pathways limits follicle activation induced by ovarian cryopreservation and in vitro culture

BackgroundCryopreservation and transplantation of ovarian tissue (OTCTP) represent a promising fertility preservation technique for prepubertal patients or for patients requiring urgent oncological management. However, a major obstacle of this technique is follicle loss due to, among others, accelerated recruitment of primordial follicles during the transplantation process, leading to follicular reserve loss in the graft and thereby potentially reducing its lifespan. This study aimed to assess how cryopreservation itself impacts follicle activation.ResultsWestern blot analysis of the PI3K/PTEN/Akt and mTOR signalling pathways showed that they were activated in mature or juvenile slow-frozen murine ovaries compared to control fresh ovaries. The use of pharmacological inhibitors of follicle signalling pathways during the cryopreservation process decreased cryopreservation-induced follicle recruitment. The second aim of this study was to use in vitro organotypic culture of cryopreserved ovaries and to test pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR pathways. In vitro organotypic culture-induced activation of the PI3K/PTEN/Akt pathway is counteracted by cryopreservation with rapamycin and in vitro culture in the presence of LY294002. These results were confirmed by follicle density quantifications. Indeed, follicle development is affected by in vitro organotypic culture, and PI3K/PTEN/Akt and mTOR pharmacological inhibitors preserve primordial follicle reserve.ConclusionsOur findings support the hypothesis that inhibitors of mTOR and PI3K might be an attractive tool to delay primordial follicle activation induced by cryopreservation and culture, thus preserving the ovarian reserve while retaining follicles in a functionally integrated state.

Evaluation of the Effects of Platelet-Rich Plasma on Follicular and Endometrial Growth: A Literature Review.

Platelet-rich plasma (PRP) has been used in several areas of medicine due to its ability to promote tissue regeneration by growth factors and cytokines. This review addresses the use of PRP to rejuvenate ovarian follicles and increase the thickness of the endometrium to receive an embryo. PRP is obtained from the patient's own blood (autologous blood) - a fact that determines a lower chance of rejection reactions. Alpha granules of platelets provide and release supra physiological amounts of growth factors and cytokines, which provide a regenerative stimulus in tissues with low healing potential. In the ovary, PRP and its growth factors stimulate vascularization and recruitment of available primordial follicles that could no longer be otherwise stimulated. The rejuvenation of the ovary by PRP infusion aims to obtain new oocytes in ovaries with low numbers of follicles or low follicular reserve markers. In the preparation of the endometrium, PRP is used for its several growth factors that allow tissue proliferation and endometrial thickening, especially in cases of endometria that are difficult to prepare or that fail to reach an adequate minimum thickness (>7mm). To date, there are few studies of greater expression in the literature that support the use of PRP with the two purposes described above. Thus, although promising, the technique must still be validated by larger clinical trials.

Ascorbic acid ameliorates dysregulated folliculogenesis induced by mono-(2-ethylhexyl)phthalate in neonatal mouse ovaries via reducing ovarian oxidative stress.

Phthalates, including di-(2-ethylhexyl)phthalate (DEHP), are common industrial chemicals in the environment. Recent evidence indicates that DEHP and its active metabolite mono-(2-ethylhexyl)phthalate (MEHP) negatively modulate reproductive functions and induce reactive oxygen species. Ascorbic acid (AA) is a dietary requirement for primates, and it acts as a potent free radical scavenger to protect tissues against oxidative stress. In this study, to investigate the toxic effects of MEHP on the follicle development and the beneficial role of AA, neonatal mouse ovaries were treated with different concentrations of MEHP with or without AA for 6days. Then, the follicle constitution and oxidative status were compared in different groups. Results showed MEHP accelerated primordial follicle recruitment by increasing the percentage of primary and secondary follicles and decreasing the percentage of primordial follicles in the ovaries. Moreover, MEHP-induced ovarian oxidative stress by significantly increasing malondialdehyde (MDA) concentration and the expression of GSS and SOD1. When ovaries were co-administrated with MEHP and AA, follicle constitution was normalized, and the oxidative status was significantly decreased. These results suggested that AA ameliorated MEHP-induced ovarian oxidative stress and follicular dysregulation, which attested the clinical significance of AA for ovary protection in the case of MEHP exposure.