Integrated analyses of the single-cell ATAC-seq and RNA-seq reveal the epigenetic landscape of human ovarian aging

Studying the molecular mechanisms of ovarian aging is crucial for understanding the age-related fertility issues in females. Recently, a single-cell transcriptomic roadmap of ovarian aging based on non-human primates revealed the molecular signatures of the oocytes at different developmental stages. Herein, we present the first epigenetic landscape of human ovarian aging, through an integrated analysis of the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and single-cell RNA-seq. We depicted the transcriptional profiles and chromatin accessibility of the ovarian tissues isolated from old (n=4) and young (n=2) donors. The unsupervised clustering of data revealed seven distinct cell populations in the ovarian tissues and six subtypes of oocytes, which could be distinguished by age difference. Further analysis of the scATAC-seq data from the young and old oocytes revealed that the interaction between the Notch signaling pathway and AP-1 family transcription factors may crucially determine oocyte aging. Finally, a machine-learning algorithm was applied to calculate the optimal model based on the single-cell dataset for predicting oocyte aging, which exhibited excellent accuracy with a cross-validated area under the receiver operating characteristics score of 0.99. In summary, this study provides a comprehensive understanding of human ovarian aging at both the transcriptomic and epigenetic levels, based on an integrated analysis of large-scale single-cell datasets. We believe our results will shed light on the discovery of potential therapeutic targets or diagnostic markers for age-related ovarian disorders.

Integrated analyses of the single-cell ATAC-seq and RNA-seq reveal the epigenetic landscape of human ovarian aging

Studying the molecular mechanisms of ovarian aging is crucial for understanding the age-related fertility issues in females. Recently, a single-cell transcriptomic roadmap of ovarian aging based on non-human primates revealed the molecular signatures of the oocytes at different developmental stages. Herein, we present the first epigenetic landscape of human ovarian aging, through an integrated analysis of the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and single-cell RNA-seq. We depicted the transcriptional profiles and chromatin accessibility of the ovarian tissues isolated from old (n=4) and young (n=2) donors. The unsupervised clustering of data revealed seven distinct cell populations in the ovarian tissues and six subtypes of oocytes, which could be distinguished by age difference. Further analysis of the scATAC-seq data from the young and old oocytes revealed that the interaction between the Notch signaling pathway and AP-1 family transcription factors may crucially determine oocyte aging. Finally, a machine-learning algorithm was applied to calculate the optimal model based on the single-cell dataset for predicting oocyte aging, which exhibited excellent accuracy with a cross-validated area under the receiver operating characteristics score of 0.99. In summary, this study provides a comprehensive understanding of human ovarian aging at both the transcriptomic and epigenetic levels, based on an integrated analysis of large-scale single-cell datasets. We believe our results will shed light on the discovery of potential therapeutic targets or diagnostic markers for age-related ovarian disorders.

Autologous mitochondria transport via transzonal filopodia rejuvenates aged oocytes by UC-MSCs derived granulosa cells-oocyte aggregation

Mitochondria transfer can rescue oocyte aging-related infertility. However, heterologous techniques are suspended due to heteroplasmy. Regarding autologous approaches, the donor source and manipulating procedures require further optimization. Here we propose a strategy using umbilical cord mesenchymal stem cells (UC-MSCs) as mitochondria donor cells and employing intercellular mitochondria transport as the transfer method. We cryopreserved UC-MSCs of the female pup. When the female aged, its UC-MSCs were induced into granulosa cells (iGCs). The zona-weakened GV oocytes were aggregated with autologous iGCs into iGC-oocyte complexes. After cultivation in GDF9-containing media, mitochondria migrated from iGCs into the GV oocyte via transzonal filopodia. The maturation rate, quality, and developmental potential of these oocytes were substantially increased. Furthermore, the birth rate after embryo transfer has been improved. This approach utilized noninvasive procedures to collect mitochondria donor cells and optimized mitochondria transfer manipulations, so may represent a promising advance towards the improvement of aging-related infertility.

Nicotinamide Adenine Nucleotide—The Fountain of Youth to Prevent Oocyte Aging?

According to the U.S. Special Operations Command (SOCOM), new clinical trials of an anti-aging oral treatment using nicotinamide adenine nucleotide are planned for 2022. All over the globe, the discovery of the fountain of youth is still a great goal to reach, not only among aging researchers, since people desire to stay longer healthy and feel young when reaching old age. Since the 1960s, women delaying pregnancy to pursue higher educational levels and a career path has contributed to drastically diminished overall female fertility rates (e.g., number of born offspring/woman). Consequently, a growing number of advanced-aged women depend on assisted reproductive technologies (ART) to become pregnant. In 2019, the Society for Assisted Reproductive Technology reported 293672 cycles for oocyte retrieval. This change of demographics influenced women’s age of having their first child, which has increased significantly. However, their reproductive tract shows hallmarks of aging very early in life without an effective preventive treatment. Therefore, we will present whether NAD+ could help to prevent oocyte aging.

Anti-Aging Effect of Urolithin A on Bovine Oocytes In Vitro

Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells’ (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP) and developmental competence of physiologically mature (22 h) and in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes (NFE2L2, NQO1, and mt-DN5) and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on the nuclear maturation progression, MMP, developmental competence and gene expression levels. UA treatment during in vitro maturation enhanced (p < 0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p < 0.05) of UA on physiological maturation, MMP and embryonic development was also identified. UA also interfered on the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.

P–155 Oocyte recovery 39 hours (from 39h to 41h) after administration of follicular maturation trigger does not affect clinical results

Study question What is the clinical outcome of oocytes recovered after 39 hours from ovulation inducing drug administration? Summary answer Oocytes obtained after 39 hours from follicular maturation triggering are equally viable to those obtained at the standard time of 36 hrs. What is known already In the clinical setting of ART, ovum pick-up (OPU) is generally performed around 36 hours after the administration of ovulation inducing drugs (OID). However, there are cases where OPU cannot be performed at this time often due to long operating lists. As the time elapsed between the administration of ovulation inducing drugs and OPU becomes longer, there is a concern about time-related oocyte aging. Nevertheless, there are few reports of clinical results of OPU after 36 hours from OID. Study design, size, duration We conducted a review of 1187 cycles and 1951 patients in which OPU and embryo transfer was performed in 2017–2018. All cycles underwent a ‘freeze-all’ of embryos and the transfer cycle was in the thawed embryo transfer cycle for all cases. Participants/materials, setting, methods The time from the administration of OID to the end of OPU was divided into 36h group and over 39h group and the MII and normal fertilization rate of oocytes obtained from OPU after ovarian stimulation were compared. After confirmation of fertilization, the D3 good-quality embryo and the D5 and 6 good-quality blastocyst rates of embryos that continued to be cultured and the pregnancy and miscarriage rates of cleavage-stage embryos and blastocyst transfers were compared. Main results and the role of chance The MII rate in the 36h and &gt;39h groups was 78.1% vs. 80.0%, and the normal fertilization rate was 77.9% vs. 78.1% (ICSI) and 65.4% vs. 67.6% (Conventional-IVF). The D3 good-quality embryo rate (good-quality embryos are embryos with less than 5% fragmentation in 7–9 cells and compaction with more than 50% adhesion between split spheres) was 21.8% vs. 25.3%, the D5 good-quality blastocyst rate (at least 3BB according to Gardner classification) was 33.6% vs. 40.1%, and the D6 good-quality blastocyst rate was 31.1% vs. 37.5%, all of which were not significantly different. The pregnancy rate for cleavage-stage embryo transfer was 26.6% vs. 6.7%, and the miscarriage rate was 25.3% vs. 42.9%, both of which were not significantly different. The pregnancy rate for blastocyst transfer was 45.4% vs. 50.0%, and the miscarriage rate was 22.2% vs. 20.0%, both of which were not significantly different. (The significance difference test was a χ-square test) Limitations, reasons for caution The study was a retrospective study. Wider implications of the findings: Even if OPU is conducted after 36h of the administration of OID, to the extreme range of 39h–41h, oocyte aging does not seem apparent and pregnancy outcomes are similar to the standard time interval of 36 hours. Trial registration number ‘not applicable’

O-158 The effect of circadian rhythm disruption due to constant light on ovarian and oocyte aging through possible relationship between PER2 and mTOR signaling pathway

Study question Does circadian rhythm disruption by constant light affect the ovarian morphology and function, and cause ovarian and oocyte aging through possible relationship between PER2 and mTOR? Summary answer We demonstrated that circadian rhythm disruption by light may cause ovarian and oocyte aging. What is known already Circadian rhythm regulates multiple physiological processes and PER2 is one of the core circadian rhythm components. Changes in light conditions may cause circadian rhythm disruptions. Light exposure at night may cause attenuation in PER2 mRNA and protein levels. Circadian rhythm disruptions are thought to be associated with reproductive diseases. mTOR signaling pathway functions in folliculogenesis and oocyte maturation in ovary. Also, it is associated with ovarian and oocyte aging. Study design, size, duration A total of 32 female Balb/c mice which enter estrous cycle were used in the study. Mice were randomly assigned to one of two groups as 12:12h L:D and 12:12h L:L. During the experiment, 12:12h L:D (control group) was housed in a 12:12h light:dark cycle and 12:12h L:L (experiment group) was housed in a constant light conditions 12:12h light:light for 1 week. Participants/materials, setting, methods We housed 12:12h L:D group in standard lightening conditions and 12:12h L:L group in constant light for one week. We performed food intake and body weight change analysis. We evaluated ovarian morphology, follicle counting analysis. We evaluated ZP3 and nitrotyrosine (NTY) expression for oocyte aging markers. We performed western blot for PER2, mTOR, p-mTOR, p70 S6K, p-p70 S6K, and Caspase-3 protein levels. Main results and the role of chance We demonstrated that circadian rhythm disruption caused alteration in their food intake and decrease in primordial follicle numbers and increase in atretic follicles (p &lt; 0.05). It caused increase in oxidative stress and decrease in ZP3 expression in oocytes (p &lt; 0.05). We showed decreased protein levels of PER2, mTOR, p-mTOR and p70 S6K (p &lt; 0.05). Limitations, reasons for caution The explanation of molecular mechanism underlying the relationship between circadian rhythm disruptions by light and ovarian function may lead the usage of circadian rhythm-based or light-based therapies currently using to treat some diseases on female reproductive system related diseases. Wider implications of the findings We conclude that constant light may reduce follicle reserve, cause follicles to go rapidly atresia and disrupt the oocyte quality, thus it may be a risk factor for female reproductive diseases such as premature ovarian insufficiency and early menopause. Trial registration number not applicable