Evaluating Selective Quality Control in Mammalian Oogenesis: Evidence and Opportunities.

Background: Oocytes are a finite and non-renewable resource that are maintained in primordial follicle structures. The ovarian reserve is the totality of primordial follicles, present from birth, within the ovary and its establishment, size, and maintenance dictates the duration of the female reproductive lifespan. Understanding the cellular and molecular dynamics relevant to the establishment and maintenance of the reserve provides the first steps necessary for modulating both individual human and animal reproductive health as well as population dynamics. Summary: This review details the key stages of establishment and maintenance of the ovarian reserve, encompassing germ cell nest formation, germ cell nest breakdown, and primordial follicle formation and activation. Furthermore, we spotlight several formative single-cell sequencing studies that have significantly advanced our knowledge of novel molecular regulators of the ovarian reserve, which may improve our ability to modulate female reproductive lifespans. Key Messages: The application of single-cell sequencing to studies of ovarian development in mammals, especially when leveraging genetic and environmental models, offers significant insights into fertility and its regulation. Moreover, comparative studies looking at key stages in the development of the ovarian reserve across species has the potential to impact not just human fertility, but also conservation biology, invasive species management, and agriculture.

Fertility preservation in girls with Turner syndrome: limitations, current success and future prospects

Changes in plasma müllerian-inhibiting substance and brain-derived neurotrophic factor after chemotherapy in premenopausal women

Disclosure: Y. Zhou: None. K.M. Halloran: None. M. Bellingham: None. N.P. Evans: None. R.G. Lea: None. K.D. Sinclair: None. V. Padmanabhan: None. The ovarian capacity to provide fertilizable oocytes, regulate folliculogenesis as well as activate and progress primordial follicles into preovulatory follicles are key contributing factors to female fertility and reproductive aging. In sheep, as in humans, while genetics play a role in establishing the ovarian reserve during fetal development, developmental insults such as inappropriate exposure to native steroids or environmental chemicals (ECs) can adversely impact the establishment of the ovarian reserve and, additionally can affect folliculogenesis. Considering humans are exposed to multiple ECs simultaneously which could have additive, synergistic, or antagonistic effects, real-life EC exposure models are needed to assess risks posed by EC exposure. Offspring from sheep grazed on biosolids-treated pasture offers one such real-life exposure model. Studies with day 140 fetuses found maternal exposure to a real-life EC mixture via biosolids increased the proportion of unhealthy transitory follicles (Lea et al., Sci Rep. 2016;6:22279). The present study tested if biosolids exposure during fetal life would impact folliculogenesis, follicle activation and follicle atresia during their adult life. Ovaries were derived from adult EasyCare ewes (age 31.4 ± 0.5 months) whose mothers were grazed on biosolids-treated (BTP, n=10) or inorganic fertilizer-treated (Control, n=10) pastures from preconception through to birth. Ovarian morphometry was undertaken to assess the distribution and health status of follicles. Follicular atresia was evaluated via the expression of Caspase-3, an apoptosis marker. Data analysis utilized Student's t-test for normally distributed variables and Mann-Whitney U tests for non-parametric comparisons. There were no differences in the absolute counts or the percentages of follicle classes at all stages of folliculogenesis, the density of primordial follicles in cortical tissue, or the follicular activation rate. An increase in the percentage of unhealthy activated follicles (post-primordial) was noted in the BTP group (p = 0.014), predominantly in the transitory stage (p = 0.021). Consistent with this, a higher density of Caspase-3 positive primary follicles was observed in the BTP group (p = 0.033). These findings indicate the adverse impacts of fetal exposure to an EC mixture via maternal biosolid exposure on the survival of early-stage activated follicles in adult sheep. The lack of difference in ovarian reserve between the control and BTP groups suggests compensatory mechanisms may be in place to prevent premature depletion of the ovarian reserve. Whether EC mixture exposure via biosolids also has an adverse effect on the quality of the surviving activated follicles remains to be determined. Funding source: NIH R01 ES030374. Presentation: 6/3/2024

Developmental exposure to environmental chemicals perturbs establishment and maintenance of the ovarian reserve across the reproductive lifetime, leading to premature follicle depletion and ovarian aging. Considering humans are exposed to a complex mixture of environmental chemicals, real-life models assessing their cumulative impact on the ovarian reserve are needed. Biosolids are a source of a real-life mixture of environmental chemicals. While earlier studies demonstrated that grazing pregnant sheep on biosolids-treated pastures did not influence establishment of the ovarian reserve in fetal life, its impact on subsequent depletion of ovarian reserve during reproductive life of offspring is unknown. We hypothesized that developmental exposure to biosolids accelerates depletion of ovarian reserve. Ovaries were collected from F1 juveniles (9.5weeks) and adults (2.5years) born to F0 ewes grazed on control inorganic fertilizer pastures or biosolids-treated pastures from before conception and throughout gestation. The impact on follicular density, activation rate, and anti-Müllerian hormone (mediator of activation) expression by immunohistochemistry was determined. Activation rate was increased in F1 biosolids-treated pastures juveniles with a corresponding reduction in primordial follicle density. In contrast, activation rate and ovarian reserve were similar between control and F1 biosolids-treated pastures adults. The density of anti-Müllerian hormone-positive antral follicles was lower in biosolids-treated pastures juveniles, whereas anti-Müllerian hormone expression tended to be higher in antral follicles of biosolids-treated pastures adults, consistent with the changes in the ovarian reserve. These findings of detrimental effects of developmental exposure to biosolids during juvenile life that normalizes in adults is supportive of a shift in activation rate likely related to peripubertal hormonal changes.

Aim. The research was conducted for the assessment of the impact of chronic salpingoophoritis on the ovarian reserve of women in various phases of reproductive age.Materials and methods. A prospective, controlled and open cohort study was performed in 2013-2018 (n=202). The main group consisted of women with chronic salpingoophoritis (ChrSO) who applied for preconception consultation (n=138). In accordance with the reproductive age phase, the main group was divided into subgroups: the early reproductive age period (ERP, n=44), the peak reproductive age period (PRP, n=56), the late reproductive period (LRP, n=38). The control group consisted of conditionally healthy women of reproductive age (n=64). The ovarian reserve (OR) was estimated on the basis of the serum level of antimullerian hormone (AMH), inhibin B, estradiol, follicle stimulating hormone (FSH), an ultrasoundbased assessment of the number of antral follicles (AF), and the ovarian volume. Results. The age of women ranged from 18 to 40 years. Based on the discriminant analysis, it was found that the main indicators determining the specificity of the OR in ChrSO, depending on the phase of reproductive age, are the number of antral follicles, estradiol level and AMH (Wilks’ lambda = 0.35503, p<0.0001). The specificity of the OR of women with ChrSO (difference from the control group), regardless of the phase of reproductive age, initially and when evaluated after 6 months, is determined by the number of AF and the level of estradiol and AMH; the number of AF and AMH is determined with a similar estimate after 12 months. The specificity of the OR in ChrSO, which is dependent on the reproductive age phase, has been proved through the analysis with the neural networks training(the proportion of correct answers is more than 80%). The linear relationships were established between the values of each OR parameter in women with ChrSO. Initially, when estimating after 6 and 12 months, linear regression equations were calculated, allowing the values of individual OR parameters to be calculated over 6 and 12 months.Conclusion. Chronic salpingoophoritis (ChrSO) is associated with a decrease in ovarian reserve in women of reproductive age. The effect of ChrSO on some parameters of the ovarian reserve depends on the age phase of the reproductive period, which increases with time (after 6, 12 months). The presence of ChrSO in women planning future pregnancies requires preventive and therapeutic measures aimed at preserving the ovarian reserve and the preferred implementation of fertility in early reproductive age before the ovarian reserve starts to decline.

Disclosure: K.M. Halloran: None. J.N. Ciarelli: None. Y. Zhou: None. M. Bellingham: None. R.G. Lea: None. N.P. Evans: None. K.D. Sinclair: None. P. Smith: None. V. Padmanabhan: None. Establishment of the fetal ovarian follicular reserve is a determinant of lifelong fertility. Environmental chemicals (ECs) are implicated in perturbation of early follicle development which may negatively impact adult reproductive function. Considering humans are exposed to a complex cocktail of ECs that may have additive, synergistic and/or antagonistic effects, real-life exposure models are needed to assess the impact of mixtures of ECs on ovarian reserve. Biosolids contain a complex mixture of ECs and earlier studies have shown that grazing pregnant sheep on biosolids-treated pastures (BTP) did not influence the ovarian follicular pool of fetal day 110 lambs. In contrast, an increased proportion of healthy primordial and reduced proportion of healthy transitory follicles, without an impact on their cortical density, was observed in late gestation fetuses (day 140). Collectively this suggests that in BTP sheep, while establishment of an ovarian reserve is not affected its depletion across the reproductive life span may be affected. In this study we tested the hypothesis that prenatal biosolid exposure would affect follicular depletion and negatively impact ovarian reserve in postnatal lambs. Ewes were grazed on pastures treated with either inorganic fertilizer (control; C) or biosolids (BTP) beginning a month before mating and throughout gestation until birth. After birth, ewes and lambs were moved to C pastures. Female lambs (10 C and 11 BTP) were euthanized at 8 weeks of age. Ovarian follicles were classified by stage (primordial, transitory, primary, preantral, and antral) and health status. The impact of exposure to mixture of ECs on follicular cortical density (n/mm2), distribution, health status, and activation rate were assessed. Anti-müllerian hormone (AMH) expression, which correlates with the number of growing follicles, was quantified by immunohistochemical staining. BTP lambs had reduced density (P=0.07) and proportion (P=0.001) of primordial follicles. Activation rate was increased in the BTP group (P=0.01). The density of unhealthy follicles was reduced in the primordial (P=0.04) and transitory (P=0.09) stages, but increased in preantral (P=0.02) and antral (P=0.09) stages. The percentage of unhealthy follicles was reduced in transitory (P=0.09), but increased in preantral (P=0.01) and antral (P=0.07) stages. Consistent with this, increased AMH staining intensity was evident in transitory plus primary follicles (P<0.05). Collectively these results provide evidence that lambs born to BTP ewes have a reduced ovarian follicular pool, increased follicular activation, and increased unhealthy activated follicles at 8 weeks of age. These findings demonstrate the detrimental effects of ECs in biosolids on ovarian health leading to premature depletion of follicular pool and raise concerns regarding the quality of activated follicles. Funding: NIH R01 ES030374 Presentation: 6/1/2024

Study question Does exposure to chemotherapy agents used to treat cancer during pregnancy cause significant decrease in early establishment of the ovarian reserve? Summary answer Significant oocyte loss is noted following exposure of early mouse ovaries to doxorubicin. This apoptotic decline is not seen with cisplatin, docetaxel or paclitaxel exposure. What is known already Chemotherapy has been shown to adversely affect the ovarian reserve of pre-pubertal and reproductive age females. However, little is known regarding the effects in the next generation following treatment in pregnancy beyond observational population studies that examined rates of birth defects and developmental delays. Reproductive function in offspring develops over time and would be difficult to quantify in these short-term studies. An ex-vivo mouse ovary culture offers the ability to closely mimic maternal treatment level serum concentrations and directly evaluate the impact on oocyte number and cell death markers. Study design, size, duration In mice, the ovarian reserve is matured postnatally, mimicking the biologic second trimester activity of the human ovary. Wild-type C57bl/6 pups were collected on postnatal day 0. Ovaries were cultured in hanging well organ culture media with addition of DMSO or a chemotherapy agent. Immunofluorescence was used to quantify oocyte number and density. Power calculation showed a N of 11 per drug would be needed to demonstrate a decrease of ⅔ or more. Participants/materials, setting, methods: 83 ovaries were cultured, sectioned and analyzed in duplicate. Planned analysis at serum max and mid concentrations was performed at 48 hours and 5 days. Given clinically variation in concentrations of cisplatin, additional concentration samples were added. After noting the degradation following exposure to doxorubicin, additional samples at earlier time points of 12 and 24 hours of exposure were added for dynamic evaluation. Means were calculated and then compared using a 2-way ANOVA. Main results and the role of chance Doxorubicin exposure during establishment of the ovarian reserve resulted in a significant loss of oocyte number and density. At 12 hours a 22% decrease was noted; this increased to a loss of 91% of oocytes by 48 hours of exposure. The oocyte density fell from 693 oocytes/ mm² in the control to only 63 oocytes/ mm² in the serum max concentration (p = 0.003). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed early impact within the stroma by 12 hours with widespread apoptotic changes by 24 hours. Treatment with cisplatin resulted in a phenotypic change in the oocyte population with preservation of smaller, more peripheral cells. The average oocyte density remained similar to control even at the highest clinical concentrations, 536 oocytes/ mm² compared to 570 oocytes/ mm² (p = 0.772). Docetaxel and paclitaxel demonstrated an increase in oocyte number and density, though not enough to reach significance. The oocyte density 5 days following docetaxel exposure was 802 oocytes/ mm², a 41% increase (p = 0.12). The oocyte density 5 days following paclitaxel exposure was 780 oocytes/ mm², a 37% increase (p = 0.817). The drug exposure did impact stromal cells, as noted in TUNEL images. Limitations, reasons for caution This ex-vivo mouse model offers tight control of chemotherapy concentration, it does not account for filtration and modification by the placenta. Longer term cultures may also demonstrate that temporary arrest in small oocytes, such as those exposed to cisplatin, do not thrive and later progress to apoptosis. Wider implications of the findings: Doxorubicin is employed as the most common chemotherapy during pregnancy. Utilization may be to the serious detriment of the younger generation. If other alternatives are clinically effective they should be considered. No other models have explored the effect of fetal ovary exposure to chemotherapy on the establishment of ovarian reserve. Trial registration number Not applicable

The production of the oocyte in mammals begins in the fetal stage with the formation of primordial germ cells (PGCs). PGCs differentiate through the process of oocyte differentiation to become primary oocytes. After the emergence of primary oocytes, the oocytes become quiescent and enclosed by somatic follicle cells to form the ovarian reserve. The ovarian reserve forms through a highly conserved process in mammals and serves as the only resource to sustain and prolong female reproductive life. Primary oocytes are activated periodically from the ovarian reserve when puberty occurs. Once a primary oocyte is activated in the ovary, it has two fates available to it, continue through oocyte development and potentially be ovulated or face cell death. In my master's thesis I explore the cellular mechanisms of the mouse ovary spanning oocyte differentiation to oocyte development. In chapter one, I investigate oocyte differentiation, specifically cell fate determination in fetal gametogenesis. During mammalian fetal gametogenesis, both male and female germ cells are connected via intercellular bridges yet experience major differences in cell fate determination. In mouse fetal testes, male germ cells arrest in G0/G1 after embryonic day (E)14.5 and differentiate into gonocytes postnatally. In contrast mouse female germ cells enter meiosis after E14.5 and initiate oocyte differentiation. During oocyte differentiation, two fates are possible, ~80 percent of the germ cells donate organelles and undergo cell death; ~20 percent of the germ cells collect organelles from sister germ cells and become primary oocytes. Due to the small percentage of germ cells that become primary oocytes, this led us to investigate the differences in cell fates between female and male germ cells using single cell RNA sequencing. The findings highlight key features in female and male germ cell transcriptomics. This data helps us understand cellular mechanism differences in female and male germ cell populations and how it relates to differential cell fates. In chapter two, I investigate oocyte development, specifically whether quiescent primary oocytes experience cellular senescence and non-apoptosis cell death in the mouse ovary, a process that may lead to primary ovarian insufficiency (POI) and physiological ovarian aging. Cellular senescence is characterized by cell cycle arrest and production of the senescence associated secretory phenotype (SASP) associated with tissue aging. The activation of primary oocytes from the ovarian reserve is an irreversible process that forces the oocytes to develop via folliculogenesis or undergo cell death. In this project we used a mouse model with POI phenotypes caused by Pten depletion specifically in the oocyte. We examined quantitatively cellular senescence and cell death (apoptosis and ferroptosis) in 2 weeks and 4 weeks old mutant mouse ovaries, when primary oocyte loss take place. Our data demonstrated that overactivation of primary oocytes contributes to a reduction in the ovarian reserve in mutant ovaries; and an increased number of senescent primary oocytes were found in mutant ovaries as well. Although it appeared in a low number, it was found that all primary oocytes that undergo ferroptosis were senescent. This data drives what is known about cellular mechanisms in oocytes further by providing quantitative results of follicle loss, cellular senescence, and cell death markers in the primary oocyte.

Currently, three crucial questions regarding the reliability of ovarian reserve measures in women with ovarian endometrioma during the reproductive age are being discussed. Firstly, the effects of endometriotic cystectomy on short and long-term ovarian reserve. Secondly, the accuracy of serum anti-Müllerian hormone (AMH) and antral follicle count (AFC) in estimating ovarian reserve in these cases. Thirdly, the impact of endometrioma itself on the ovarian reserve over time in such cases. The purpose of the present review is to critically assess available systematic reviews and meta-analyses that have explored these questions. Nine eligible reviews were found following a systematic search on PubMed.com and similarly assessed. These reviews varied considerably regarding the level of evidence, as per an identical comprehensive scoring system. Moderate to high-quality evidence demonstrates that endometriotic cystectomy, by the stripping technique, adversely affects ovarian reserve in the short and long term, up to 9-18 months post-surgery. Damage to ovarian reserve was considerable but more pronounced in bilateral cases than unilateral cases, equivalent to 39.5% and 57.0%, respectively. Repeat endometriotic cystectomy is detrimental to ovarian reserve. The impact of endometrioma diameter on ovarian reserve before or after surgery is still unclear. Moderate to high-quality evidence, relying on simultaneous assessment of both ovarian reserve measures, shows that AMH is sensitive while AFC is not in cases undergoing ovarian cystectomy. AMH should be the biomarker of choice for counseling and managing women with endometrioma in their reproductive age, especially before surgery. While there is some evidence to show that endometrioma per se may harm ovarian reserve, this evidence is not robust, and there is good-quality evidence to challenge this notion. It is necessary to conduct further targeted RCTs, systematic reviews, and meta-analyses based on solid methodological grounds to increase the level of evidence, refine quantitative estimates, investigate open questions, and decrease heterogeneity.

BACKGROUND Oocyte number is established early in life before a gradual loss of this ovarian reserve during reproductive life until oocyte availability becomes limiting at the menopause. Although there is a large genetic component to the ovarian reserve achieved before birth, other influences including the maternal endocrine and nutritional milieu, and environmental factors may represent important developmental determinants. Environmental and nutritional factors may also modify the downward trajectory of ovarian reserve in adult life. The combination of these early and later life influences has the potential to lead to diminished ovarian reserve, compromising fertility in later reproductive years and altering age at natural menopause. METHODS Literature searches of the ISI Web of Knowledge database were carried out using the main terms 'ovarian reserve' and 'menopause AND age' in conjunction with a range of other terms encompassing a variety of factors with potential effects on ovarian reserve. The various searches were inspected manually and the relevant papers selected for critical analysis and interpretation. RESULTS Evidence was identified supporting the view that elevated prenatal androgens have an adverse effect on the early establishment of ovarian reserve, although the implications for ovarian reserve in the polycystic ovary syndrome (which may also be programmed through prenatal androgen exposure) remain uncertain. Recent evidence is cited suggesting that effects of maternal nutrient restriction on ovarian reserve may also involve changes in prenatal androgen exposure. A general rationale is developed through examination of evidence which emphasizes the roles of the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER) systems in ovarian reserve modulation. Because of their similarity to the natural ligands, many environmental compounds have the ability to bind to these receptors (albeit at lower affinities) and thereby have the potential to influence either the initial setting of ovarian reserve during development or the trajectory of ovarian reserve during adult life. For example, exposure to compounds in cigarette smoke may accelerate loss of ovarian reserve in smokers leading to diminished ovarian reserve, earlier age at last child and earlier menopause. Socioenocomic factors are clearly associated with age at natural menopause, with correlations with economic status and education level. However, such effects in western societies are in general small, and the underlying mechanisms remain unclear. CONCLUSIONS Exposure to many environmental compounds, particularly to those that leach from plastics and other synthetic materials, is commonplace in modern societies to the extent that many are found at measurable concentrations in body fluids within most of the population. Relating fluid levels of individual compounds to parameters reflecting ovarian reserve in selected populations appears to be an effective way forward and, indeed, some early-stage findings do show some cause for concern. There is a pressing need for the development of practical advice enabling women to minimize their intake of AHR/ER ligands, perhaps through dietary/cosmetic choices or improved food packaging.

The effect of maternal separation stress-induced depression on ovarian reserve in Sprague Dawley Rats: The possible role of imipramine and agmatine through a mTOR signal pathway

The reproducibility of serum anti-Müllerian hormone in subfertile women: within and between patient variability

Biomarkers of ovarian response: current and future applications

Study question How do Indian healthcare professionals describe their clinical experience with and perspectives on AMH testing in Indian women seeking fertility treatments including fertility preservation? Summary answer The HCPs cautioned against AMH testing as a screening tool in presumed fertile Indian women due to its anticipated impact on women’s arranged-marriage prospects. What is known already AMH test is being increasingly used to assess women’s ovarian reserve (OR) while planning fertility treatments or to guide decisions about fertility preservation (FP). There is weak evidence suggesting that serum AMH level and fertility treatment outcomes vary in different population groups. Surveys with women in reproductive age (e.g. the US, Ireland, the Netherlands) indicate that a majority wants to know their OR to aid reproductive decision making. As yet, both globally and in an Indian context, there are only few qualitative studies exploring the views of HCPs on the OR assessment in clinical practice and its socio-cultural implications. Study design, size, duration This paper reports the findings of an exploratory qualitative research aimed at understanding whether and how elective fertility preservation could influence reproductive autonomy of Indian women. Between June 2018 and April 2019, IVF specialists and obstetricians practicing in ten cities across five Indian states were interviewed in English (language commonly spoken) using a semi-structured interview guide. The discussion about OR assessment with AMH-testing was initiated by the participants indicating its significance in their clinical practice. Participants/materials, setting, methods The study sample included 17 male and 15 female HCPs, the majority (18/32) was practicing in Mumbai. Twenty-six of them were in private practice while six worked as OBGYNs in publicly funded teaching hospitals. Twenty-six participants were interviewed in their clinics and the remaining six using Skype or telephone. After several rounds of immersive reading, the interview sections on OR and AMH-test were analyzed inductively using Braun and Clarke’s thematic analysis. Main results and the role of chance Several participants reported that many of their patients present with decreased OR (DOR) at a younger age and need higher dosages of hormones for ovulation induction compared to the dosages mentioned in international guidelines. They corroborated this experience with a few peer-reviewed articles indicating a six-years age difference in OR of Indian women undergoing IVF compared to Spanish women. A majority of participants advocated for the rational use of OR assessment in IVF patients but warned against its indiscriminate use or interpretation out of context due to concerns about overdiagnosis of ovarian factor infertility and overtreatment with IVF with donor eggs. Although the physicians who had performed elective FP perceived AMH test as a simple, affordable and empowering tool to guide FP decisions, most participants were critical of using AMH-test as a screening tool in young, presumed fertile women completing university education. They were concerned that a diagnosis of DOR as a result of such screening in this population in the Indian context will adversely impact women’s chances of marriage and might further increase pressure on women to get married and complete their childbearing early even if they are not ready for it. Limitations, reasons for caution This is the first qualitative study assessing views of Indian HCPs on AMH testing. These results are indicative rather than a representation of views of Indian HCPs. Almost half of the contacted HCPs did not respond to interview requests; we do not know whether they had different views. Wider implications of the findings The insights on clinical implications of AMH testing in India are relevant to other societies beyond the Euro-American and Australian context where AMH testing will increase in the future. The socio-cultural implications of ‘routine’ AMH testing in India urges us to be aware of similar implications in other cultural contexts. Trial registration number Not applicable