Aged Oocytes Research Articles

Effects of putrescine on the quality and epigenetic modification of mouse oocytes during in vitro maturation.

Low ovarian putrescine levels and decreased peak values following luteinising hormone peaks are related to poor oocyte quantity and quality in ageing women. To investigate the effects of putrescine supplementation in in vitro maturation (IVM) medium on oocyte quality and epigenetic modification. Germinal vesicle oocytes retrieved from the ovaries of 8-week-old and 9-month-old mice were divided into four groups (the young, young+difluoromethylornithine (DFMO), ageing and ageing+putrescine groups) and cultured in IVM medium with or without 1mM putrescine or DFMO for 16h. The first polar body extrusion (PBE), cleavage and embryonic development were evaluated. Spindles, chromosomes, mitochondria and reactive oxygen species (ROS) were measured. The expression levels of SIRT1, H3K9ac, H3K9me2, H3K9me3, and 5mC levels were evaluated. Sirt1 and imprinted genes were detected. The PBE was higher in the ageing+putrescine group than in the ageing group. Putrescine increased the total and inner cell mass cell numbers of blastocysts in ageing oocytes. Putrescine decreased aberrant spindles and chromosome aneuploidy, increased the mitochondrial membrane potential and decreased ROS levels. Putrescine increased SIRT1 expression and attenuated the upregulation of H3K9ac levels in ageing oocytes. Putrescine did not affect 5mC, H3K9me2 or H3K9me3 levels or imprinted gene expression. Putrescine supplementation during IVM improved the maturation and quality of ageing oocytes and promoted embryonic development by decreasing ROS generation, maintaining mitochondrial and spindle function and correcting aberrant epigenetic modification. Putrescine shows application potential for human-assisted reproduction, especially for IVM of oocytes from ageing women.

Follicular fluid 8-Hydroxy-2-Deoxyguanosine (8-OHdG) as biomarker for oxidative stress in intracytoplasmic sperm injection.

Oxidative stress (OS) is a situation that occurs as a result of un-equilibrium between reactive oxygen species (ROS) formation and the antioxidant defence system. 8-hydroxy-2-deoxyguanosine (8-OHdG), an oxidized form of deoxyguanosine, is found in higher levels in aging oocytes. In this cross-sectional study, we aimed to evaluate the effect of 8-OHdG on the intracytoplasmic sperm injection (ICSI). Follicular fluid (FF) samples were taken after removing the oocyte for later analysis of 8-OHdG. The couples participants were categorized according to the cause of subfertility into three groups (female factor, malefactor, and unexplained infertility). Further division according to pregnancy state was done to evaluate the precise role of 8-OHdG on pregnancy state and ICSI outcome. Result : This study showed that the 8-OHdG levels significantly higher in non-pregnant women (p<0.05). Correlation study showed that 8-OHdG level in follicular fluid is negatively correlating with the number of retrieved oocytes, metaphase two oocytes (MII), fertilized oocytes (2PN = two pronuclei), cleaved zygotes, good quality embryos, and the difference was statistically significant (p<0.05). Conclusion: The study revealed that the 8-OHdG level in follicular fluid negatively influences ICSI outcome and it is higher in non-pregnant women. J. Med. Invest. 69 : 112-116, February, 2022.

Coenzyme Q10 Supplement Rescues Postovulatory Oocyte Aging by Regulating SIRT4 Expression

Coenzyme Q10 Supplement Rescues Postovulatory Oocyte Aging by Regulating SIRT4 Expression

Increased WIP1 Expression With Aging Suppresses the Capacity of Oocytes to Respond to and Repair DNA Damage.

If fertilization does not occur for a prolonged time after ovulation, oocytes undergo a time-dependent deterioration in quality in vivo and in vitro, referred to as postovulatory aging. The DNA damage response is thought to decline with aging, but little is known about how mammalian oocytes respond to the DNA damage during in vitro postovulatory aging. Here we show that increased WIP1 during in vitro postovulatory aging suppresses the capacity of oocytes to respond to and repair DNA damage. During in vitro aging, oocytes progressively lost their capacity to respond to DNA double-strand breaks, which corresponded with an increase in WIP1 expression. Increased WIP1 impaired the amplification of γ-H2AX signaling, which reduced the DNA repair capacity. WIP1 inhibition restored the DNA repair capacity, which prevented deterioration in oocyte quality and improved the fertilization and developmental competence of aged oocytes. Importantly, WIP1 was also found to be high in maternally aged oocytes, and WIP1 inhibition enhanced the DNA repair capacity of maternally aged oocytes. Therefore, our results demonstrate that increased WIP1 is responsible for the age-related decline in DNA repair capacity in oocytes, and WIP1 inhibition could restore DNA repair capacity in aged oocytes.

Effects of intracytoplasmic sperm injection timing and fertilization methods on the development of bovine spindle transferred embryos

Cytoplasmic replacement by spindle transfer (ST) technique can be applied to improve the developmental competence of poor qualitied or aged oocytes. In cattle, ST technology has not been well established for producing embryos and the calves successfully using intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). The objective of this study was to develop a novel procedure for producing bovine ST embryos, which could be fundamental to applying ST technology in other mammals. In the present study, the efficacies of performing ICSI before (ICSI-ST) or after (ST-ICSI) and IVF on the development of ST bovine embryos were investigated. Results indicated that the blastocyst rate of ST embryos produced by ICSI-ST (24.7%) was higher (P < 0.05) than that produced by ST-ICSI (5.9%). On the other hand, ST-IVF had the highest fertilization rate (97.3%), polyspermy rate (24.7%), and lowest blastocyst rate (22.7%) when compared to denuded oocytes (DO), zona cut oocytes (ZC), and cumulus-oocyte complexes (COCs)-IVF groups. Finally, the in vitro development rates of ICSI-ST (24.5%) and ST-IVF (25.2%) were not significantly different (P > 0.05). However, the pregnancy rate (46.7%) and birth rate (33.3%) of ST-IVF were higher (P < 0.05) than those of ICSI-ST (6.3% and 0%, respectively). The percentage of mitochondrial DNA (mtDNA) heteroplasmy derived from donor karyoplasts of the 5 claves was between 2% and 18%. Taken together, performing ICSI prior to ST can improve the embryonic development of ST bovine embryos. Moreover, using IVF, instead of ICSI, for ST oocyte fertilization dramatically increased the pregnancy rate and birth rate of ST calves, in which mtDNA heteroplasmy derived from donor karyoplasts exists.

Melatonin improves the quality of maternally aged oocytes by maintaining intercellular communication and antioxidant metabolite supply

In mammalian ovaries, oocytes are physically coupled to somatic granulosa cells, and this coupling is crucial for the growth and development of competent oocytes as it mediates the transfer of metabolic support molecules. However, aging-mediated dysregulation in communication between the oocytes and granulosa cells affects the oocyte quality. In the present study, we examined the defected germline-soma communication and reduced mRNA levels encoding key structural components of transzonal projections (TZPs) in maternally aged oocytes. Oral administration of melatonin to aged mice substantially increased TZPs and maintained the cumulus cells-oocyte communication, which played a central role in the production of adequate oocyte ATP levels and reducing the accumulation of reactive oxygen species (ROS), apoptosis, DNA damage, endoplasmic reticulum (ER) stress and spindle/chromosomal defects. This beneficial effect of melatonin was inhibited by carbenoxolone (CBX), a gap junctional uncoupler, which disrupts bidirectional communications between oocyte and somatic cells. Simultaneously, melatonin significantly increased the mRNA and protein levels corresponding to genes associated with TZPs and prevented TZP retraction in in vitro-cultured cumulus-oocyte complex (COCs). Furthermore, we infused melatonin and CBX into the COCs in vitro culture system and monitored the levels of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) in cumulus cells and oocytes. Notably, COCs treated with melatonin demonstrated improved NADPH and GSH levels. Of note, CBX was capable of reducing NADPH and GSH levels, aggravated the ROS accumulation and ER stress. Collectively, our data demonstrate the role of melatonin in preventing age-associated germline-soma communication defects, aiding the relay of antioxidant metabolic molecules for the maintenance of oocyte quality from cumulus cells, which have important potential for improving deficient phenotypes of maternally aged oocytes and the treatment of woman infertility.

Effects of post-ovulatory aging on centromeric cohesin protection in murine MII oocytes.

PurposePost‐ovulatory aging causes a high frequency of aneuploidy during meiosis II in mouse oocytes, irrespective of maternal age. In this study, we evaluated the effects of post‐ovulatory oocyte aging on the protection of chromosomal cohesion involved in aneuploidy and verified the relationship between PP2A or SGO2 expression and the phosphorylation level of REC8 in oocytes.MethodsMurine ovulated oocytes were incubated for 6 or 12 h in vitro after collection and denoted as the aged group. The oocytes examined immediately after collection were used as the control group. Immunofluorescent staining was used to detect the localization of PP2A, SGO2, BUB1, AURORA B, and MAD2 in the chromosomal centromere. Immunoblotting was used to quantify the expression of proteins describe above and REC8 in the oocytes.ResultsPP2A expression involved in the de‐phosphorylation of REC8 decreased over time in oocytes, suggesting a deficiency in PP2A in centromeres. This indicated an increase in the level of phosphorylated REC8, which destabilizes centromeric cohesion in oocytes. In contrast, SGO2 expression was significantly high in aged oocytes.ConclusionsThe findings show that post‐ovulatory aging destabilizes the centromeric cohesin protection in oocytes and can cause aneuploidy, which is often observed in aged oocytes during meiosis II.

Sorbitol accumulation decreases oocyte quality in aged mice by altering the intracellular redox balance.

Sorbitol is a product of glucose metabolism through the polyol pathway. Many studies have demonstrated that excessive sorbitol can disrupt the intracellular redox balance. However, we still know very little about the impact of excessive intracellular sorbitol on oocyte quality, oocyte maturation, and embryo developmental potential. This study explored whether intracellular sorbitol accumulates in the oocytes of aged mice during in vitro maturation (IVM) and what roles sorbitol plays in oocyte development and maturation. Our results showed that sorbitol levels were significantly higher in in vitro-matured oocytes from aged mice than in oocytes from young mice (14.08 ± 3.78 vs. 0.23 ± 0.04 ng/oocyte). The expression of aldose reductase (AR) mRNA was significantly higher in the in vitro-cultured oocytes from 9-month-old mice than prior to culture. To decrease the excessive intracellular sorbitol in oocytes from aged mice, sorbinil, a specific inhibitor of aldose reductase, was supplemented in IVM medium, and the sorbitol level was significantly decreased (14.08 ± 3.78 vs. 0.48 ± 0.19 ng/oocyte). Our results indicated that the percentage of oocytes with first polar body extrusion (PBE) was significantly higher in the sorbinil group than in the aged group (82.4% ± 7.2% vs. 66.1% ± 6.9%), and the content of sorbitol was drastically increased in the aged group. The ROS fluorescence intensity in the sorbinil group was drastically lower than that in the aged group, while the GSH fluorescence intensity was significantly higher. Interestingly, SOD1 was upregulated in the sorbinil group. The present study suggests that excessive sorbitol accumulation is induced during IVM in aged mouse oocytes, which negatively influences oocyte quality by altering the intracellular redox balance. Inhibition of sorbitol accumulation may be a potential method to improve the nuclear maturation of aged oocytes.

4,4’-dimethoxychalcone increases resistance of mouse oocytes to postovulatory aging in vitro

Research questionDoes 4,4’-dimethoxychalcone (DMC), a natural antioxidant compound, effectively improve the quality of postovulatory ageing (POA) oocytes? DesignFreshly ovulated MII oocytes were cultured in 10-µM DMC for 12 h in vitro. Reactive oxygen species (ROS) production, apoptosis rate, mitochondrial distribution, formation of the actin cap, and fertilization and development potential of POA oocytes, were studied. The change of autophagy level was detected, and the autophagy inhibitor 3-methyladenine (3-MA) was used to establish the relationship between DMC and autophagy. ResultsDMC supplementation eliminated the accumulated ROS (P < 0.0001) and ROS dependent 4 hydroxynonenal products (P = 0.0399), and decreased apoptosis (P = 0.0033), reduced abnormal mitochondrion distribution (P = 0.0280), improved mitochondrial membrane potential (P = 0.0135) and restored the formation of the actin cap (P = 0.0487), thus improving fertilization ability (P = 0.0156) and developmental potential (P = 0.0130) in POA oocytes. The role autophagy plays in the effects of DMC supplementation was investigated. The immunofluorescence results showed that POA leads to the accumulation of SQSTM1/p62 (P = 0.0083) but DMC supplementation could eliminate this (P < 0.0001). The western blot result of p62 protein was similar to the immunofluorescence results of the POA group (P = 0.0441) and DMC supplementation group (P = 0.0154). After inhibiting autophagy by 3-MA, the DMC supplementation group could no longer eliminate the accumulation of ROS (P = 0.1704). ConclusionsDMC supplementation activates autophagy to protect oocytes from postovulatory ageing. This approach can feasibly improve the reproductive outcome of ART.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Resveratrol Hinders Postovulatory Aging by Modulating Oxidative Stress in Porcine Oocytes.

Postovulatory aging of the mammalian oocytes causes deterioration of oocytes through several factors including oxidative stress. Keeping that in mind, we aimed to investigate the potential of a well-known antioxidant, resveratrol (RV), to evaluate the adverse effects of postovulatory aging in porcine oocytes. After in vitro maturation (IVM), a group of (25–30) oocytes (in three replicates) were exposed to 0, 1, 2, and 4 μmol/L of RV, respectively. The results revealed that the first polar body (PB1) extrusion rate of the oocytes significantly increased when the RV concentration reached up to 2 μmol/L (p < 0.05). Considering optimum RV concentration of 2 μmol/L, the potential of RV was evaluated in oocytes aged for 24 and 48 h. We used fluorescence microscopy to detect the relative level of reactive oxygen species (ROS), while GHS contents were measured through the enzymatic method. Our results revealed that aged groups (24 h and 48 h) treated with RV (2 μmol/L) showed higher (p < 0.05) ROS fluorescence intensity than the control group, but lower (p < 0.05) than untreated aged groups. The GSH content in untreated aged groups (24 h and 48 h) was lower (p < 0.05) than RV-treated groups, but both groups showed higher levels than the control. Similarly, the relative expression of the genes involved in antioxidant activity (CAT, GPXGSH-Px, and SOD1) in RV-treated groups was lower (p < 0.05) as compared to the control group but higher than that of untreated aged groups. Moreover, the relative mRNA expression of caspase-3 and Bax in RV-treated groups was higher (p < 0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RV-treated group was significantly lower than control but higher than untreated aged groups. Taken together, our findings revealed that the RV can increase the expression of antioxidant genes by decreasing the level of ROS, and its potent antiapoptotic effects resisted against the decline in mitochondrial membrane potential in aged oocytes.

The role of mitophagy during oocyte aging in human, mouse, and Drosophila: implications for oocyte quality and mitochondrial disease.

There is a worldwide trend for women to have their first pregnancy later in life. However, as oocyte quality declines with maternal aging, this trend leads to an increase in subfertility. The cellular mechanisms underlying this decline in oocyte competence are poorly understood. Oocyte mitochondria are the subcellular organelles that supply the energy that drives early embryogenesis, and thus their quality is critical for successful conception. Mitochondria contain their own DNA (mtDNA) and mutations in mtDNA cause mitochondrial diseases with severe symptoms, such as neurodegeneration and heart disease. Since mitochondrial function declines in tissues as humans age accompanied by an accumulation of mtDNA mutations, mtDNA is implicated as a cause of declining oocyte quality in older mothers. While this mutation load could be caused by declining accuracy of the mitochondrial replisome, age-related decline in mitochondrial quality control likely contributes, however knowledge is lacking. Mitophagy, a cellular process which specifically targets and recycles damaged mitochondria may be involved, but studies are scarce. And although assisted reproductive technologies can help older mothers, how these techniques affect the mechanisms that regulate mitochondrial and oocyte quality have not been studied. With the long-term goal of understanding the molecular mechanisms that control mitochondrial quality in the oocyte, model systems including Drosophila and mouse as well as human oocytes have been used. In this review, we explore the contribution of mitophagy to oocyte quality and the need for further systematic investigation in oocytes during maternal aging using different systems.Lay summaryMitochondria are small parts of cells called organelles that generate the chemical energy needed for life. Hundreds of thousands of mitochondria in the developing eggs of the mother support the initial growth and development of the fertilized egg. However, due to increasingly diminished function over time, mitochondria generate less energy as we age, posing real problems for older women considering pregnancy. It is possible that this declining energy could be responsible for declining fertility as women age. Energy may decline because mitochondria fail and the cell’s way of keeping them healthy become less efficient as we age. This review summarizes what is known about mitochondrial quality control in developing eggs as they age. In the future, understanding how the best mitochondria are selected and maintained in the egg, and hence the future baby, may enable older women with or without mitochondrial problems, to have healthy children.

CDC26 is a key factor in human oocyte aging.

Is CDC26 a key factor in human oocyte aging? The lack of CDC26 disrupts the oocytes maturation process, leading to oocyte aging, but these defects could be partially rescued by overexpression of the CDC26 protein. Age-related oocyte aging is the main cause of female fertility decline. In mammalian oocytes, aberrant meiosis can cause chromosomal abnormalities that might lead to infertility and developmental disorders. CDC26 participates in the meiosis process. Differential gene expression in young and old women oocytes were screened by single-cell RNA-seq technology, and the functions of differentially genes were verified on mouse oocytes. Finally, transfection technology was used to evaluate the effect of a differentially expressed gene in rescuing human oocyte from aging. Discarded human oocytes were collected for single-cell RNA-seq, q-PCR and immunocytochemical analyses to screen for and identify differential gene expression. Female KM mice oocytes were collected for IVM of oocytes, q-PCR and immunocytochemical analyses to delineate the relationships between oocyte aging and differential gene expression. Additionally, recombinant lentiviral vectors encoding CDC26 were transfected into the germinal vesicle oocytes of older women, to investigate the effects of the CDC26 gene expression on oocyte development. Many genes were found to be differentially expressed in the oocytes of young versus old patients via RNA-seq technology. CDC26 mRNA and protein levels in aged oocytes were severely decreased, when compared with the levels observed in young oocytes. Moreover, aged oocytes lacking CDC26 were more prone to aneuploidy. These defects in aged oocytes could be partially rescued by overexpression of the CDC26 protein. N/A. Our study delineated key steps in the oocyte aging process by identifying the key role of CDC26 in the progression of oocyte maturation. Future studies are required to address whether other signaling pathways play a role in regulating oocyte maturation via CDC26 and which genes are the direct molecular targets of CDC26. Our results using in vitro systems for both mouse and human oocyte maturation provide a proof of principle that CDC26 may represent a novel therapeutic approach against maternal aging-related spindle and chromosomal abnormalities. This work was supported by grants from the National Natural Science Foundation of China (81571442 and 81170571), the outstanding Talent Project of Shanghai Municipal Commission of Health (XBR2011067) and Clinical Research and Cultivation Project in Shanghai Municipal Hospitals (SHDC12019X32). The authors declare no conflict of interest.

Integrative proteome analysis implicates aberrant RNA splicing in impaired developmental potential of aged mouse oocytes.

Aging has many effects on the female reproductive system, among which decreased oocyte quality and impaired embryo developmental potential are the most important factors affecting female fertility. However, the mechanisms underlying oocyte aging are not yet fully understood. Here, we selected normal reproductively aging female mice and constructed a protein expression profile of metaphase II (MII) oocytes from three age groups. A total of 187 differentially expressed (DE) proteins were identified, and bioinformatics analyses showed that these DE proteins were highly enriched in RNA splicing. Next, RNA‐seq was performed on 2‐cell embryos from these three age groups, and splicing analysis showed that a large number of splicing events and genes were discovered at this stage. Differentially spliced genes (DSGs) in the two reproductively aging groups versus the younger group were enriched in biological processes related to DNA damage repair/response. Binding motif analysis suggested that PUF60 might be one of the core splicing factors causing a decline in DNA repair capacity in the subsequent development of oocytes from reproductively aging mice, and changing the splicing pattern of its potential downstream DSG Cdk9 could partially mimic phenotypes in the reproductively aging groups. Taken together, our study suggested that the abnormal expression of splicing regulation proteins in aged MII oocytes would affect the splicing of nascent RNA after zygotic genome activation in 2‐cell embryos, leading to the production of abnormally spliced transcripts of some key genes associated with DNA damage repair/response, thus affecting the developmental potential of aged oocytes.

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 293,672 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.

DNA damage repair is suppressed in porcine aged oocytes.

This study sought to evaluate DNA damage and repair in porcine postovulatory aged oocytes. The DNA damage response, which was assessed by H2A.X expression, increased in porcine aged oocytes over time. However, the aged oocytes exhibited a significant decrease in the expression of RAD51, which reflects the DNA damage repair capacity. Further experiments suggested that the DNA repair ability was suppressed by the downregulation of genes involved in the homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways. The expression levels of the cell cycle checkpoint genes, CHEK1 and CHEK2, were upregulated in porcine aged oocytes in response to induced DNA damage. Immunofluorescence results revealed that the expression level of H3K79me2 was significantly lower in porcine aged oocytes than in control oocytes. In addition, embryo quality was significantly reduced in aged oocytes, as assessed by measuring the cell proliferation capacity. Our results provide evidence that DNA damage is increased and the DNA repair ability is suppressed in porcine aged oocytes. These findings increase our understanding of the events that occur during postovulatory oocyte aging.

AUTOPHAGY INDUCTION RESCUES DNA DAMAGE RESPONSE IN MOUSE OOCYTES

To determine the effect of DNA damage on oocyte meiosis, and to investigate whether autophagy plays a role in weakened DNA damage response in young and aged mouse oocytes. Using immunocytochemistry, chemical approaches and time-lapse confocal microscopy, we investigated the effect of increased DNA damage (DNA double-strand breaks, DSBs) on oocyte meiosis I. We also investigated the effect of autophagy induction (using Rapamycin) or inhibition (using Spautin-1) on DNA damage response in: (1) control (DMSO) vs. etoposide-treated oocytes (DNA DSBs inducer) and (2) young (6 W) vs. old (>48 W) oocytes. Finally, we investigated whether autophagy has a protective role against DNA damage or plays a role in DNA damage repair machinery. Student’s t-test (2 groups) or One-way ANOVA followed by Tukey’s post hoc test (multiple groups) were performed to analyze the data. Etoposide treatment (50 μg/ml) in fully-grown germinal vesicle oocytes increased (p<0.01) γ-H2AX (phosphorylated H2AX, a marker of DNA damage) relative to controls. Moreover, we found that DNA damage was higher (p<0.01) in aged oocytes, compared to young oocytes. Importantly, autophagy induction (100nM rapamycin) rescued the DNA damage in both aged oocytes and etoposide-treated oocytes as evidenced by the decrease of γ-H2AX signals, suggesting a role of autophagy in DNA damage response. Further analyses revealed that inducing DNA damage in GV oocytes perturbed meiotic progression and resulted in metaphase I (Met I) chromosome morphology alteration. In contrast to the normal bivalent structure of chromosomes in controls, etoposide-treated oocytes had chromosomes that were abnormally compact (83.4% vs 0% in controls, p<0.05) with clear signs of DNA fragmentation (77.05 % vs 0% in controls, p<0.01). Strikingly, induction of autophagy in etoposide-treated oocytes rescued the aforementioned phenotypes (perturbed meiotic progression, DNA fragmentation and compact chromosome), whereas autophagy inhibition exaggerated these phenotypes, compared to controls. Finally, we found that induction of autophagy ∼3 h after DNA damage induction did not significantly rescue the damaged DNA, indicating that in mouse oocytes, autophagy plays a protective role against DNA damage. Autophagy plays a protective role against DNA damage and its induction rescues DNA damage-induced alterations in oocytes.

CAN THE ENDOMETRIUM BE TOO THICK? AN ANALYSIS OF SINGLE EUPLOID EMBRYO TRANSFER (SEET) CYCLE PREGNANCY OUTCOMES

Estrogen is used to prepare the endometrium prior to a frozen embryo transfer. An endometrial thickness (EMT) ≥7mm on the day of embryo transfer (ET) is associated with increased likelihood of pregnancy. However, few studies have investigated whether there is an upper limit of endometrial thickness at ET that affects implantation potential.1 The objective of this study is to determine whether there is a correlation between increasing EMT and ART cycle outcomes in SEET cycles. The study included patients who underwent an autologous or donor egg SEET cycle from September 2016 to March 2021 with an EMT ≥7mm on cycle day 10-15 after preparation with oral Estradiol. Demographic and cycle characteristics including oocyte age, BMI, AMH, peak E2, P4 prior to ET, day of embryo biopsy, embryo quality and days of estrogen treatment were collected. Primary outcome was ongoing clinical pregnancy rate (OPR). Secondary outcomes included implantation rate (IR), biochemical pregnancy rate, and early pregnancy loss rate. Data was analyzed using student’s t-test, chi-square, and Spearman’s correlation. General estimating equations (GEE) model with exchangeable working correlation structure was used to account for multiple cycles from the same patients. A total of 4,911 SEET cycles were identified and included in the study. Included patients had a mean oocyte age of 35.63, BMI of 24.2, and an average of 18.2 days of oral estradiol prior to ET. Implantation, biochemical pregnancy, early pregnancy loss, and ongoing clinical pregnancy rates were calculated for each mm of endometrial thickness starting at 7mm. Increasing EMT was correlated with IR (r = 0.50, p = <0.0001) and OPR (r = 0.84, p = <0.0001). The highest IR was observed for women with an EMT of 14mm (79%), above which the IR decreased to 71% at 15mm, and 60% at ≥16mm. The highest OPR occurred at an EMT of 15mm (67%), above which it decreased to 47%. However this decrease was not statistically significant after adjusting for oocyte age, BMI, peak E2, P4 prior to transfer, endometrial type, AMH, uterine factor, embryo quality, prior transfer outcomes and embryo biopsy day for both IR (β= 0.27, p = 0.61), and OPR (β= 0.26, p = 0.64). Our study demonstrates a positive correlation between increasing EMT ≥ 7mm and OPR in SEET cycles up to 15mm. Future studies evaluating endometrial vascularity and endometrial metabolic profiles and their association with endometrial thickness may further expand our understanding of endometrial physiology and its impact on clinical outcomes.

THE BIOLOGICAL CLOCK IS TICKING- ON OOCYTES AGING AND EPIGENETICS

As we age, shifts in heterochromatin levels change the epigenetic landscape in our cells. One of the consequences of heterochromatin de-regulation may be the de-repression of transposable elements (TE). In this work we examine the epigenetic changes in aged oocytes, and their impact on oocyte quality. Importantly, we examined this effect before the onset of significant aneuploidy.

REDEFINING ANEUPLOIDY RATES IN THE ERA OF MOSAICISM REPORTING: A LONGITUDINAL STUDY OF 24,341 EMBRYOS ANALYZED USING NEXT GENERATION SEQUENCING

Our understanding of the relationship between female age and rate of embryonic aneuploidy has remained fairly consistent over the years.1 The detection of embryo mosaicism in preimplantation genetic testing for aneuploidy (PGT-A) has introduced a third category into the possible options for embryo ploidy. The aim of this study is to determine how reporting of mosaicism in PGT-A alters the ratio of euploid and aneuploid embryos. This study included patients who underwent an IVF cycle at a single academic center with PGT-A from February 2016 through March 2021. Trophectoderm biopsies were analyzed using a modified FAST-SeqS NGS-based PGT method and bioinformatics pipeline. Patients were separated into groups based on availability of mosaic reporting (Group A: January 2020-March 2021, routine reporting included mosaicism; Group B: February 2016-December 2019, routine reporting did not include mosaicism). The proportion of embryos reported as euploid and aneuploid was compared among SART age groups. Embryos with both full aneuploidy and mosaicism were reported as aneuploid. Indeterminate results were excluded. Student’s t-test and chi square were used for analysis, with multivariable logistic regression to adjust for confounders. Bonferroni correction was performed to control for multiple comparisons with a corrected p value of .004. Of the 24,341 embryos from 4,407 patients identified in the study, 12,850 (52.8%) were reported as euploid and 10,450 (42.9%) were reported as aneuploid. The mean oocyte age was 35.2 years. The rate of embryonic euploidy was similar during the era of mosaic reporting compared to the period prior to mosaic reporting (51.8% vs. 53.5%, p=.02) while the rate of aneuploidy decreased by 11.6% (35.1% vs. 46.7%, p<.0001). No differences were seen in euploid rates between the study and control groups when stratified by SART age group. Aneuploidy rates were significantly lower in the era of mosaic reporting among age SART Group A (21.0% vs. 35.4%, p<.0001), Group B (33.0% vs. 43.7%, p<.0001), and Group C (46.7% vs. 56.5%, p<.001) but not Group D (68.3% vs. 71.6%, p=.13) or Group E (76.5% vs. 79.7%, p=.28). Multivariable logistic regression controlling for oocyte age, high quality embryo, and day of biopsy revealed that mosaic reporting was significantly associated with a lower odds of aneuploid reports in Groups A, B, and C but not in D or E (Group A: OR 0.48, 95% CI 0.44-0.53, p<.0001; Group B: OR 0.63, 95% CI 0.56-0.71, p<.0001; Group C: OR 0.69, 95% CI 0.61-0.78, p<.0001). Mosaicism reporting alters the proportions of embryos interpreted as aneuploid, particularly among younger age groups. In older age groups, the predominance of meiotic errors resulting in full aneuploidy leads to a minimal shift in the proportions of embryos reported as euploid or aneuploid.