Decline In Female Fertility Research Articles

Mechanism study of YangJing ZhongYu decoction on regulating mitochondrial dynamics of ovarian granular cells and improving diminished ovarian reserve

ObjectiveDiminished ovarian reserve (DOR) encompasses both reproductive and endocrine disorders, resulting in a decline in female fertility. This paper explored the mechanism of Yangjing Zhongyu Decoction (YJZYD) regulating mitochondrial dynamics of ovarian granulosa cells (GCs) to improve DOR.MethodsDOR patients were treated with YJZYD, with ovarian volume (OV), antral follicle count (AFC), and endometrial thickness (EMT) detected. C57BL/6 female mice were treated by cyclophosphamide (Cy) intraperitoneal injection and YJZYD solution daily gavage, with serum anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels determined. Ovarian GCs (KGN) were interfered with 4-Hydroperoxy-Cyclophosphamide (4-HC) and treated with the MAPK/ERK pathway inhibitor or activator.ResultsDOR patients showed increased levels of serum AMH, E2, OV, AFC and EMT, while reduced FSH and LH levels after YJZYD treatment. After Cy induction, DOR mice exhibited irregular estrous cycles, diminished serum AMH and E2 levels, elevated FSH and LH levels, reduced follicle number and atresia follicle number, disorderly arranged GCs, and severe interstitial fibrosis. After 4-HC treatment, KGN proliferation and Bcl-2, MFN1, and MFN2 were suppressed, while apoptotic rate, Bax, Cleaved-caspase-3, and p-Drp1 (Ser616) levels, and mitochondrial fission and quantity increased. YJZYD promoted 4-HC-treated KGN proliferation, boosted mitochondrial fusion, and inhibited apoptosis and mitochondrial fission via the MAPK/ERK pathway.ConclusionYJZYD promoted ovarian GC proliferation and mitochondrial fusion, suppressed cell apoptosis and mitochondrial fission, and effectively improved DOR in mice by activating the MAPK/ERK pathway, providing a theoretical basis for the clinical application value of YJZYD in DOR treatment.

Potential mechanisms and therapeutic strategies for LPS-associated female fertility decline.

As a major component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS) can be recognized by toll-like receptors (TLRs) and induce inflammation through MyD88 or the TIR domain-containing adapter-inducing interferon-β (TRIF) pathway. Previous studies have found that LPS-associated inflammatory/immune challenges were associated with ovarian dysfunction and reduced female fertility. However, the etiology and pathogenesis of female fertility decline associated with LPS are currently complex and multifaceted. In this review, PubMed was used to search for references on LPS and fertility decline so as to elucidate the potential mechanisms of LPS-associated female fertility decline and summarize therapeutic strategies that may improve LPS-associated fertility decline.

The maintenance of oocytes in the mammalian ovary involves extreme protein longevity

Women are born with all of their oocytes. The oocyte proteome must be maintained with minimal damage throughout the woman’s reproductive life, and hence for decades. Here we report that oocyte and ovarian proteostasis involves extreme protein longevity. Mouse ovaries had more extremely long-lived proteins than other tissues, including brain. These long-lived proteins had diverse functions, including in mitochondria, the cytoskeleton, chromatin and proteostasis. The stable proteins resided not only in oocytes but also in long-lived ovarian somatic cells. Our data suggest that mammals increase protein longevity and enhance proteostasis by chaperones and cellular antioxidants to maintain the female germline for long periods. Indeed, protein aggregation in oocytes did not increase with age and proteasome activity did not decay. However, increasing protein longevity cannot fully block female germline senescence. Large-scale proteome profiling of ~8,890 proteins revealed a decline in many long-lived proteins of the proteostasis network in the aging ovary, accompanied by massive proteome remodeling, which eventually leads to female fertility decline.

Transcriptome analysis of porcine oocytes during postovulatory aging

Decreased oocyte quality is a significant contributor to the decline in female fertility that accompanies aging in mammals. Oocytes rely on mRNA stores to support their survival and integrity during the protracted period of transcriptional dormancy as they await ovulation. However, the changes in mRNA levels and interactions that occur during porcine oocyte maturation and aging remain unclear. In this study, the mRNA expression profiles of porcine oocytes during the GV, MII, and aging (24 h after the MII stage) stages were explored by transcriptome sequencing to identify the key genes and pathways that affect oocyte maturation and postovulatory aging. The results showed that 10,929 genes were coexpressed in porcine oocytes during the GV stage, MII stage, and aging stage. In addition, 3037 genes were expressed only in the GV stage, 535 genes were expressed only in the MII stage, and 120 genes were expressed only in the aging stage. The correlation index between the GV and MII stages (0.535) was markedly lower than that between the MII and aging stages (0.942). A total of 3237 genes, which included 1408 upregulated and 1829 downregulated genes, were differentially expressed during porcine oocyte postovulatory aging (aging stage vs. MII stage). Key functional genes, including ATP2A1, ATP2A3, ATP2B2, NDUFS1, NDUFA2, NDUFAF3, SREBF1, CYP11A1, CYP3A29, GPx4, CCP110, STMN1, SPC25, Sirt2, SYCP3, Fascin1/2, PFN1, Cofilin, Tmod3, FLNA, LRKK2, CHEK1/2, DDB1/2, DDIT4L, and TONSL, and key molecular pathways, such as the calcium signaling pathway, MAPK signaling pathway, TGF-β signaling pathway, PI3K/Akt signaling pathway, FoxO signaling pathway, gap junctions, and thermogenesis, were found in abundance during porcine postovulatory aging. These genes are mainly involved in the regulation of many biological processes, such as oxidative stress, calcium homeostasis, mitochondrial function, and lipid peroxidation, during porcine oocyte postovulatory aging. These results contribute to a more in-depth understanding of the biological changes, key regulatory genes and related biological pathways that are involved in oocyte aging and provide a theoretical basis for improving the efficiency of porcine embryo production in vitro and in vivo.

Cryptochrome 1 regulates ovarian granulosa cell senescence through NCOA4-mediated ferritinophagy

Age-associated decreases in follicle number and oocyte quality result in a decline in female fertility, which is associated with increased infertility. Granulosa cells play a major role in oocyte development and maturation both in vivo and in vitro. However, it is unclear whether a reduction in cryptochrome 1 (Cry1) expression contributes to granulosa cell senescence, and further exploration is needed to understand the underlying mechanisms. In this study, we investigated the role of Cry1, a core component of the molecular circadian clock, in the regulation of senescence in ovarian granulosa cells. Western blotting and qRT-PCR showed that Cry1 expression was downregulated in aged human ovarian granulosa cells and was correlated with age and anti-Müllerian hormone (AMH) levels. RNA-seq analysis suggested that ferritinophagy was increased after Cry1 knockdown in KGN cells. MDA, iron, and reactive oxygen species (ROS) assays were used to detect cellular ferritinophagy levels. Ferroptosis inhibitors, iron chelators, autophagy inhibitors, and nuclear receptor coactivator 4 (NCOA4) knockdown alleviated KGN cell senescence induced by Cry1 knockdown. Immunofluorescence, immunoprecipitation, and ubiquitination assays indicated that Cry1 affected NCOA4 ubiquitination and degradation through HERC2, thereby affecting NCOA4-mediated ferritinophagy and causing granulosa cell senescence. KL201, a Cry1 stabilizer, enhanced ovarian function in naturally aged mice by reducing ferritinophagy. Our study reveals the potential mechanisms of action of Cry1 during ovarian aging and provides new insights for the clinical treatment of age-related fertility decline.

Roles of immune microenvironment in the female reproductive maintenance and regulation: novel insights into the crosstalk of immune cells.

Female fertility decline is an accumulative consequence caused by complex factors, among them, the disruption of the immune profile in female reproduction stands out as a crucial contributor. Presently, the effects of immune microenvironment (IME) on the female reproductive process have attracted increasing attentions for their dynamic but precisive roles. Immunocytes including macrophages, dendritic cells, T cells, B cells and neutrophils, with diverse subpopulations as well as high plasticity functioned dynamically in the process of female reproduction through indirect intercellular communication via specific cytokine release transduced by molecular signal networks or direct cell-cell contact to maintain the stability of the reproductive process have been unveiled. The immune profile of female reproduction in each stage has also been meticulously unveiled. Especially, the application of single-cell sequencing (scRNA-seq) technology in this process reveals the distribution map of immune cells, which gives a novel insight for the homeostasis of IME and provides a research direction for better exploring the role of immune cells in female reproduction. Here, we provide an all-encompassing overview of the latest advancements in immune modulation within the context of the female reproductive process. Our approach involves structuring our summary in accordance with the physiological sequence encompassing gonadogenesis, folliculogenesis within the ovaries, ovulation through the fallopian tubes, and the subsequent stages of embryo implantation and development within the uterus. Our overarching objective is to construct a comprehensive portrayal of the immune microenvironment (IME), thereby accentuating the pivotal role played by immune cells in governing the intricate female reproductive journey. Additionally, we emphasize the pressing need for heightened attention directed towards strategies that focus on immune interventions within the female reproductive process, with the ultimate aim of enhancing female fertility.

Epigallocatechin-3-gallate improves the quality of maternally aged oocytes.

The decline in female fertility as age advances is intricately linked to the diminished developmental potential of oocytes. Despite this challenge, the strategies available to enhance the quality of aged oocytes remain limited. Epigallocatechin-3-gallate (EGCG), characterised by its anti-inflammatory, antioxidant and tissue protective properties, holds promise as a candidate for improving the quality of maternally aged oocytes. In this study, we explored the precise impact and underlying mechanisms of EGCG on aged oocytes. EGCG exhibited the capacity to enhance the quality of aged oocytes both in vitro and in vivo. Specifically, the application of EGCG in vitro resulted in noteworthy improvements, including an increased rate of first polar body extrusion, enhanced mitochondrial function, refined spindle morphology and a reduction in oxidative stress. These beneficial effects were further validated by the improved fertility observed among aged mice. In addition, our findings propose that EGCG might augment the expression of Arf6. This augmentation, in turn, contributes to the assembly of spindle-associated F-actin, which can contribute to mitigate the aneuploidy induced by the disruption of spindle F-actin within aged oocytes. This work thus contributes not only to understanding the role of EGCG in bolstering oocyte health, but also underscores its potential as a therapeutic intervention to address fertility challenges associated with advanced age.

OR19-04 Hypoglycosylated FSH Enhances Ovarian Follicle Development and Gamete Quality

Abstract Disclosure: A. Converse: None. T. Kumar: None. F.E. Duncan: None. Gamete quality depends on the proper growth and maturation of the ovarian follicle, which consists of the oocyte and its supporting somatic cells. FSH is essential for follicle growth as female Fshb and Fshr null mice are infertile due to arrested follicle development. Macroheterogeneity in N-glycosylation on the FSHß-subunit confers differential bioactivities with hypoglycosylated FSH21 exhibiting higher FSHR binding affinity and bioactivity compared to fully glycosylated FSH24. In females, there is a shift from the highly bioactive FSH21 to FSH24 with increasing age which occurs coordinately with the age-dependent decline in female fertility. In this study, we used an encapsulated in vitro follicle growth assay to test the hypothesis that FSH glycoforms have differential abilities to promote follicle development and gamete quality. Early-stage (preantral) ovarian follicles were isolated from CD-1 mice, cultured in media containing 10 ng/ml of either FSH21 or FSH24 for up to 12 days, and then ex vivo ovulation was induced. While both glycoforms maintained >75% follicle survival through day 12, follicles treated with FSH21 were significantly larger than FSH24-treated follicles between days 4 and 12 of culture. Over the 12 day culture, FSH21-treated follicles grew from 110.9 ± 9.2 µm to 251.3 ± 24.4 µm, while FSH24-treated follicles grew from 109.3 ± 7.1 µm to 229.7 ± 15.4 µm. This difference in growth correlated with a 13.4 and 4.5-fold increase in estradiol secretion by FSH21-treated follicles compared to FSH24 treatment on days 8 and 12, respectively. After ex vivo maturation, FSH21-treated follicles produced a higher proportion (76.9%) of meiotically competent gametes that reached the metaphase II (MII)-arrested state compared to FSH24-treated follicles (62.5%). Furthermore, normal meiotic spindle configuration was observed in 76% of MII eggs from the FSH21 cohort but only 50% of the FSH24 cohort. These results indicate that FSH21 is more bioactive than FSH24 in promoting follicle development and high-quality oocytes. Mechanistically, this is likely due in part to the differential effects of FSH21 and FSH24 on the formation of transzonal projections (TZPs), which are cytoplasmic filaments essential for granulosa-oocyte communication. By 24 hrs, FSH21-treated follicles had more TZPs than FSH24-treated follicles, as demonstrated by increased actin staining (1.6x) and Myo10 foci (5.1x) in the zona pellucida region. Thus, the early establishment of TZPs and increased somatic-oocyte communication in FSH21-treated follicles ultimately results in a more developmentally competent gamete. In addition, we anticipate that ongoing transcriptomic analysis of 24 hr FSH glycoform-treated follicles will highlight other processes involved in early follicle development that contribute to the observed differential effects on oocyte quality. Presentation Date: Saturday, June 17, 2023

Let's Talk About Sex: Tyrolean Online Study on Reproductive Health Issues.

The way pupils and university students talk about sex does not reflect their real understanding of reproductive health issues. Therefore, we developed a survey for pupils and students in Tyrol, Austria, to evaluate the current state of knowledge. Two questionnaires with 39 items for pupils (n=369) and 53 items for university students (n=537) were developed, and an online survey was carried out in Tyrol, Austria, between April and July 2022. A sum score for each correct answer to ten identical items in both questionnaires was used to compare groups (range: 0-11 points), with analysis performed using independent sample t-test, analyses of variance (ANOVAs) and chi-square test. With regards to reproductive health issues and fertility awareness, female vs. male participants (p<0.001), university students vs. pupils (p<0.001) and medical students vs. other students (p<0.001) had greater knowledge. Participants who had already had sexual intercourse showed a broader knowledge of contraception and fertility (p<0.001).The age at which the decline of female fertility becomes relevant was misstated by the adolescents, who gave a mean age of 42.6 years compared to university students who asserted a mean age of 35.9 years (p<0.001). Overall, the lowest rate of correct answers was found with respect to emergency contraception (30.7%), while knowledge of contraceptive methods was comparatively high (99.2%). Substantial lack of knowledge of reproductive health issues exists, with differences found between pupils and university students, between genders, and according to field of study. Future health and fertility awareness programs at school and university should focus on gender-specific aspects to prevent unplanned pregnancies, sexually transmitted diseases, and childlessness.

Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. N/A.

The Importance of Natural Antioxidants in Female Reproduction.

Oxidative stress (OS) has an important role in female reproduction, whether it is ovulation, endometrium decidualization, menstruation, oocyte fertilization, or development andimplantation of an embryo in the uterus. The menstrual cycle is regulated by the physiological concentration of reactive forms of oxygen and nitrogen as redox signal molecules, which trigger and regulate the length of individual phases of the menstrual cycle. It has been suggested that the decline in female fertility is modulated by pathological OS. The pathological excess of OS compared to antioxidants triggers many disorders of female reproduction which could lead to gynecological diseases and to infertility. Therefore, antioxidants are crucial for proper female reproductive function. They play a part in the metabolism of oocytes; in endometrium maturation via the activation of antioxidant signaling pathways Nrf2 and NF-κB; and in the hormonal regulation of vascular action. Antioxidants can directly scavenge radicals and act as a cofactor of highly valuable enzymes of cell differentiation and development, or enhance the activity of antioxidant enzymes. Compensation for low levels of antioxidants through their supplementation can improve fertility. This review considers the role of selected vitamins, flavonoids, peptides, and trace elements with antioxidant effects in female reproduction mechanisms.

Assessing knowledge regarding fertility and attitude and intentions towards future parenthood among undergraduate medical students in Karachi

This study aimed to assess the knowledge regarding female fertility, infertility treatments and the attitudes regarding parenthood of medical students in Pakistan. Delayed childbirth among medical trainees due to extended years of medical education and training puts this population at a higher risk for involuntary childlessness later in life due to age-related decline in female fertility. A knowledge, attitude and practice study was carried out among medical students in Karachi in July 2021 using the English version of the Swedish Fertility Awareness questionnaire, which has been used in similar studies. Most participants wished to have children at some point in the future. However, a majority of students did not have sufficient knowledge regarding age-related decline in female fertility and overestimated the efficacy of infertility treatments. The results of this study indicate that despite planning to have children and placing great importance on parenthood, medical students severely overestimate female fertility and plan to start having children at an age at which female fecundity has begun to decline. These findings highlight a need for better provisions regarding fertility knowledge in the curriculum for medical students, as they are an at-risk group for involuntary childlessness due to age-related fertility decline.

Clinical study of traditional Chinese medicine in the treatment of decreased ovarian reserve function

The decline of ovarian reserve function refers to the decline of ovarian reproductive function and endocrine function, which is reflected by the decline of the quantity and quality of ovarian follicles. The occurrence of this disease directly leads to the decline of female fertility and the lack of sex hormones, and further can develop into premature ovarian failure. The effect of traditional Chinese medicine in the treatment of this disease is quite significant. We will make a brief introduction to the understanding and treatment of this disease in traditional Chinese medicine, so that our later research can be more specific and serve the majority of patients.

Epigenetic clocks provide clues to the mystery of uterine ageing.

Rising maternal ages and age-related fertility decline are a global challenge for modern reproductive medicine. Clinicians and researchers pay specific attention to ovarian ageing and hormonal insufficiency in this regard. However, uterine ageing is often left out of the picture, with the majority of reproductive clinicians being close to unanimous on the absence of age-related functional decline in the uterine tissues. Therefore, most existing techniques to treat an age-related decline in implantation rates are based primarily on hormonal supplementation and oocyte donation. Solving the issue of uterine ageing might lead to an adjustment to these methods. A focus on uterine ageing and the possibility of slowing it emerged with the development of the information theory of ageing, which identifies genomic instability and erosion of the epigenetic landscape as important drivers of age-related decline in the functionality of most cells and tissues. Age-related smoothing of this landscape and a decline in tissue function can be assessed by measuring the ticking of epigenetic clocks. Within this review, we explore whether the uterus experiences age-related alterations using this elegant approach. We analyse existing data on epigenetic clocks in the endometrium, highlight approaches to improve the accuracy of the clocks in this cycling tissue, speculate on the endometrial pathologies whose progression might be predicted by the altered speed of epigenetic clocks and discuss the possibilities of slowing down the ticking of these clocks. Data for this review were identified by searches of Medline, PubMed and Google Scholar. References from relevant articles using the search terms 'ageing', 'maternal age', 'female reproduction', 'uterus', 'endometrium', 'implantation', 'decidualization', 'epigenetic clock', 'biological age', 'DNA methylation', 'fertility' and 'infertility' were selected. A total of 95 articles published in English between 1985 and 2022 were included, six of which describe the use of the epigenetic clock to evaluate uterine/endometrium ageing. Application of the Horvath and DNAm PhenoAge epigenetic clocks demonstrated a poor correlation with chronological age in the endometrium. Several approaches were suggested to enhance the predictive power of epigenetic clocks for the endometrium. The first was to increase the number of samples in the training dataset, as for the Zang clock, or to use more sophisticated clock-building algorithms, as for the AltumAge clock. The second method is to adjust the clocks according to the dynamic nature of the endometrium. Using either approach revealed a strong correlation with chronological age in the endometrium, providing solid evidence for age-related functional decline in this tissue. Furthermore, age acceleration/deceleration, as estimated by epigenetic clocks, might be a promising tool to predict or to gain insights into the origin of various endometrial pathologies, including recurrent implantation failure, cancer and endometriosis. Finally, there are several strategies to slow down or even reverse epigenetic clocks that might be applied to reduce the risk of age-related uterine impairments. The uterine factor should be considered, along with ovarian issues, to correct for the decline in female fertility with age. Epigenetic clocks can be tested to gain a deeper understanding of various endometrial disorders.

RF22 | PMON08 The effects of Bisphenol A on miR-21 expression and miR-21 mediated apoptosis in bovine granulosa cells.

Abstract BPA, a widespread endocrine-disrupting compound (EDC), plays a significant role in the decline of female fertility. Granulosa cells are susceptible to EDCs and are important for oocyte competency. This research focuses on epigenetic mechanisms in granulosa cells that are disrupted by BPA, with a specific focus on microRNA-21(miR-21), which is crucial for oocyte maturation and primarily recognized as an antiapoptotic regulator. miR-21 targets the apoptotic genes PDCD4 and PTEN and limits their expression, resulting in an antiapoptotic effect. Previous studies in our laboratory demonstrate that BPA induces apoptosis and simultaneously increases miR-21 expression, leading to our hypothesis that BPA induces apoptosis in a miR-21 independent manner in bovine granulosa cells.To test this hypothesis, in vitro cultured granulosa cells were transfected with miR-21 inhibitor or scramble with or without BPA, followed by knockdown validation and apoptotic measurements. Using flow cytometry, transfection efficiencies were measured in time- and dose-dependent experiments, showing optimal knock-down greater than 95% at 0.5µM for 12 hrs. miR-21 expression measured by qPCR showed a significant decrease within inhibited groups (p=0.0008, n=3). Although PDCD4 and PTEN mRNA expression remained unaffected, PDCD4 protein expression is inhibited cells was significantly increased (p=0.0233, n=4), suggesting a miR-21 role in PDCD4 translation. PTEN protein was unaffected by miR-21 inhibition.In further experiments, transfected cells were exposed to BPA, and apoptosis was examined. Using Annexin V/PI staining, a significant decrease in healthy cells and an increase in early apoptotic cells within inhibited untreated groups was shown (p=0.0395 and 0.0193, n=3). This finding is likely due to antiapoptotic gene inhibition. BPA exposure resulted in significantly decreased healthy cells, increased late apoptotic cells (p=0.0047 and 0.0145, n=3), with no changes in necrotic cells. Furthermore, no differences were found between inhibited and non-inhibited groups, suggesting that BPA-induced apoptosis is miR-21 independent.Additionally, three proapoptotic (BAX, BAD, Casp-9) and two antiapoptotic (Bcl-2 and HSP70) genes were quantified by qPCR. Unexpectedly, BPA increased the expression of all genes at the mRNA level (p= 0.0021, 0.0283, 0.0143, 0.025, 0.0145, n=4). Quantification of protein levels will reveal whether this change is functional. Moreover, no differences were found between inhibited and non-inhibited groups, suggesting these changes are likely miR-21 independent. Lastly, initiator caspase 9 activity was measured after 6, 12, 24, 48, 72 hrs of BPA exposure, as caspases are primary effectors of apoptosis. 6 and 12hrs timepoints showed no changes; however, at 24 and 48 hrs significantly increased caspase activity was detected (p=0.0087 and 0.0014, n=4). At 72hrs, BPA exposure was lethal, resulting in insufficient cell numbers for measurements.Overall, this research focuses on effects of EDCs on apoptotic regulation within granulosa cells and aims to uncover epigenetic consequences of BPA's mechanism of action, ultimately affecting oocyte competency and fertility. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

BackgroundSmokers directly inhale mainstream cigarette smoke, which contains numerous known and potential toxic substances, and thus, smoking is expected to have broad harmful effects that cause tissue injury and dysfunction. Interestingly, many studies have suggested that the recent decline in female fertility and increased rate of spontaneous abortion could be associated with increased smoking rates. Indeed, women that smoked for 10 years or more were reported to have a ~ 20% higher infertility rate than women that had never smoked. However, the reasons for the underlying harmful aspects of smoking on female fertility remain a matter of debate. Importantly, a previous study revealed that resident endometrial stem cell deficiency significantly limits the cyclic regeneration potential of endometrium, which, in turn, decreases successful pregnancy outcomes. In this context, we postulated that exposure to mainstream cigarette smoke extracts might decrease female fertility by inhibiting the functions of resident endometrial stem cells.MethodsWe investigated whether cigarette mainstream smoke exposure directly inhibits various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, pluripotency, and differentiation capacity in vitro. Next, we determined whether SERPINB2 mediates cigarette smoke-induced suppressive effects on various tissue regeneration-associated functions by depleting SERPINB2 expression with specific shRNA targeting SERPINB2. Mice were injected intraperitoneally with low (0.5 mg/kg) or high (1 mg/kg) doses of cigarette smoke extract (10 times for two weeks), and endometrial stem cells were then isolated from mice uterine tissues.ResultsWe found that exposure to cigarette smoke extracts remarkably suppressed various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, multilineage differentiation ability, and pluripotency in vitro and in vivo by activating the SERPINB2 gene. Indeed, cigarette smoke-induced inhibitory effects on various endometrial stem cell functions were significantly abolished by SERPINB2 knockdown.ConclusionsThese findings provide valuable information on the harmful effects of cigarette smoking on resident endometrial stem cells and hopefully will facilitate the developments of promising therapeutic strategies for subfertile or infertile women that smoke cigarettes.

Age-related fertility decline and elective oocyte cryopreservation: Knowledge, attitudes and practices in a pilot study of general practitioners.

Increasing numbers of women are experiencing age-related infertility. General practitioners (GPs) are ideally placed to provide initial age-related fertility counselling and fertility specialist referral. The aim of this pilot study was to document doctors' knowledge, attitudes and practices relating to proactive fertility counselling, ovarian reserve testing and oocyte cryopreservation. Seventy-two GPs and general practice registrars were recruited through social media and completed an online survey. Knowledge gaps were shown relating toage-related female fertility decline, ovarian reserve testing and elective oocyte cryopreservation. GPs were more likely to discuss reproductive planning with women aged 35-44 years, compared with younger women. The majority of participants agreed fertility counselling should be provided by GPs; however, barriers identified include limited time, knowledge and concern for causing patient distress. Providing GPs with fertility-focused education and resources may enable sensitive, accurate and timely counselling to improve reproductive outcomes.

O-091 From Family Planning to Fertility Planning - ‘FertPlan’ awareness amongst contraceptive healthcare providers and users

Abstract Study question Are women and healthcare professionals seeking or providing contraception aware of age-related decline in female fertility and information support regarding fertility awareness? Summary answer 89.9% of contraceptive users and 71% of providers stated lack of awareness regarding age and fertility emphasising website as the preferred information resource. What is known already The last 50 years has seen an upward trend in the age at which women are giving birth. The birth rate for women aged 35 to 39 has trebled since 1980 and is now at its highest ever level. Delaying childbearing may mean that some women will inevitably leave it too late and become childless involuntarily. Women are also seeking fertility treatment at older ages, however, success rates decrease dramatically with age. It is not clear whether women who delay their fertility are aware of the decline in fertility with age, and whether healthcare professionals discuss fertility planning with women. Study design, size, duration We conducted two independent anonymous questionnaire surveys of reproductive age women seeking contraception and healthcare professionals(HCP) providing contraception in the UK following research ethics approval, to determine their awareness of the age-related decline in fertility, information resource and potential barriers to provision of information. . This was a prospective study design conducted over a year period using an online questionnaire. A total of 249 participants completed the survey. Participants/materials, setting, methods 138 reproductive age women on contraception consented and participated in the study by completing the online questionnaire. Of these, 96.4% were of white origin,1.4% mixed ethnicity, 1.4% Asian origin and 0.7% Black. 111 HCP providing contraceptive advice in family planning clinics or in General practice completed the survey. Of these, 6 were allied healthcare professionals ie specialist nurses. Data was collated and analysed using percentages and descriptive statistics. Main results and the role of chance Of the total 138 female contraceptive users, 87 were aged 30-39 years whilst 35 were 40-45 years. 51.4% of women indicated no concern about their future fertility, whilst the remainder gave roughly uniformly distributed ratings of concern from unconcerned to highly concerned. However, one third of all women (31.1%) believed that age-related fertility decline occurred after 40 years. 89.9% of women felt fertility awareness and education is important. Of the total 111 HCP, ∼one in 10 HCP (11.7%) misconstrued the female age-related decline to begin from 40 years onwards. The contraceptive providers also were misinformed regarding age related decline in IVF success rate with 45% of them citing 40 years or over to be the age that contributed to decline in IVF success. Despite these figures, majority (71.2%) of HCP agreed there was lack of fertility awareness and 33.3% felt fertility education should be commenced as early as school age. 27% HCP always discussed future fertility whilst 33.3% discussed most of the times. The most commonly-stated barriers to providing information were lack of time, self-perceived lack of knowledge, and women not asking. 73.2% of reproductive age contraceptive users and 85.6% HCPs expressed website to be the most preferred option for information resource. Limitations, reasons for caution To the best of our knowledge, this is one of the first studies seeking fertility awareness amongst those practising family planning. As this was not a qualitative interview based study, the findings are open to interpretation and one must exercise caution. Wider implications of the findings This study highlights clear need for fertility education and awareness amongst contraceptive providers and users. By providing resources to inform, we may increase awareness and confidence amongst both groups thereby empowering women to make informed choices about their future fertility by integrating Fertility planning within Family Planning. Trial registration number Not applicable (IRAS 248991 and R&amp;D Ref 8999)

P-401 The impact of the recipients’ age in vitrified/warmed oocyte donation (OD) cycles

Abstract Study question Does female age affect reproductive outcomes in a vitrified/warmed OD program with fresh embryo transfer? Summary answer Oocyte donation performed below the age of 40 was associated with less favorable reproductive outcomes. What is known already It is well established that oocyte and embryo aneuploidy are mainly responsible for the age-related decline in female fertility. Previous studies evaluating the effect of maternal age on implantation potential in OD cycles generated controversial results. It remains unclear whether female age affects reproductive outcome independent of embryo quality. Study design, size, duration This is a retrospective, single tertiary center, cohort study analyzing a vitrified/warmed OD program over a ten-years period (February 2010-February 2020). More specifically, data from 491 unique oocyte recipients were included who performed a fresh embryo transfer after sharing (with at least two recipients) sibling oocytes from the same donor stimulation cycle. For all treatments ICSI was performed without preimplantation genetic testing. Participants/materials, setting, methods The association between recipient’s age and reproductive outcomes was investigated according to three age categories (&amp;lt;35/35-40/&amp;gt;40 years old). The primary outcome was live birth rate (LBR), while the secondary outcome was biochemical pregnancy rate (BPR). A multivariate regression model was developed adjusting for the following covariates (entered simultaneously): recipient’s BMI, recipient’s endometrial thickness, age of the oocyte donor, number of mature oocytes used for ICSI, embryo transfer stage and number of embryos transferred. Main results and the role of chance Mean age of donors was 28.6 years old(±4.1). Mean starting dose was 192.6 IU(±139.9) and duration of stimulation 11.0 days (±1.7). Mean number of collected cumulus oocyte complexes was 25.6(±9.6) and sibling oocytes were assigned to 2, 3 or more recipients in, respectively, 83.1%, 12.4% and 4.5% of cases. Mean age of recipients was 38.6 years old(±5.1), BMI 24.3 kg/m²(±4.1) and endometrial thickness 8.9mm(±2.1). ICSI was performed on a mean number of 6.5 (±2.0) warmed, intact, mature oocytes with a fertilization rate of 68.2% (±22.5). Cleavage-stage embryos were transferred in 91.0% of the recipients, with a double embryo transfer in 42.6%. 81.3% of embryos were scored as top-quality and a mean of 1.4 (±1.4) surplus embryos were available for additional vitrification. Overall BPR was 44.2%, clinical pregnancy rate 39.3% and LBR 31.0%. Categorizing according to recipient’s age showed a LBR of 24.3%,26.7% and 37.0% for, respectively, the group &amp;lt;35, 35-40 and &amp;gt;40 years old (p = 0.02). The multivariate GEE regression model confirmed the crude data showing that the age was a significant positive predictor of LBR [coefficient (-), 0.01,0.11, for group&amp;lt;35,35-40 and &amp;gt;40 years old, p = 0.05] and BPR [coefficient (-),0.008,0.15, for group &amp;lt;35,35-40 and &amp;gt;40 years old, p = 0.006]. Limitations, reasons for caution Sibling donor oocytes were used, but the retrospective nature of the study remains a reason for caution. Also, the fact that the majority of embryos were transferred at the cleavage stage is a limitation. Wider implications of the findings The observation that recipients below the age of 40 have lower reproductive success rates following oocyte donation deserves further attention. The need for oocyte donation at younger age might be associated with a less performant uterine/endometrial function due to underlying conditions of genetic and/or endocrine nature. Trial registration number B1432020000146

RETRACTED: Parallel bimodal single-cell sequencing of transcriptome and methylome provides molecular and translational insights on oocyte maturation and maternal aging

Advanced maternal aging has become a worldwide public health issue contributing to female fertility decline. To provide a complete landscape of transcriptome and epigenetic changes during oocyte aging and maturation, we applied a parallel bimodal genomics approach to parallel transcriptome and methylome profiles of mouse oocytes at single-cell and single-base resolution. Age-associated gene expression changes were associated with defective spindle assembly and mitochondrial dysfunction. Parallel sequencing data suggested aged-related defects in mRNA degradation and methylome remodelling during oocyte maturation. Differentially methylated region in aged mature oocyte was associated with trimethylation of Histone H3 at Lysine 4. More importantly, RNA expression-based prediction model for assessing maturation and oocytes age. Taken together, our work provides a better understanding of molecular mechanisms during mouse oocyte aging, points a new direction of oocyte quality assessment and paves the way for developing novel treatments to improve oocyte quality.