Mature Oocytes Research Articles

A Decade of Oocyte Cryopreservation: New Horizons in Patients Accessing Care.

Objective: Utilization of fertility preservation treatments has increased since the American Society for Reproductive Medicine lifted the "experimental" label for oocyte cryopreservation in 2012. This study characterizes changes in insurance coverage, clinical outcomes, and live birth probabilities over a span of a decade (2012-2022) in patients who underwent planned oocyte cryopreservation. Methods: Retrospective analysis of planned oocyte cryopreservation cycles using vitrification from 2012 to 2022. Medically indicated cycles were excluded. Age, anti-mullerian hormone (AMH), number of mature oocytes vitrified, and insurance coverage were evaluated by year of procedure. Comparative statistics were performed using Kruskal-Wallis and chi-square analysis. Linear regression models and Cochran-Armitage trend test were performed to determine the relationships between each variable and time. Result(s): A total of 4,544 planned oocyte cryopreservation cycles were included. Mean age at egg retrieval decreased significantly over time (37.9 ± 2.9 years versus 34.9 ± 3.3, p < 0.0001). Mature oocytes frozen per cycle rose significantly over time (10.7 ± 7.4 in 2012 versus 13.3 ± 8.6 in 2022, p ≤ 0.0001). Cycles with insurance coverage significantly increased, 0% covered in 2012 versus 46.9% covered in 2022 (p ≤ 0.0001). Conclusions: Since 2012, patient age at time of egg freezing has decreased, coinciding with a mean increase in AMH and number of mature oocytes frozen per cycle. Younger participation in extending fertility is likely driven by a boost in social awareness regarding reproductive aging, cryopreservation technologies, and improved access to treatment. Modern oocyte cryopreservation includes more access to insurance coverage, shown by nearly half of current cycles benefiting from plan support. Shifts in patient demographics and insurance coverage, paired with updates to stimulation protocols that optimize oocyte yield, are expected to improve the overall prognosis and future fertility of patients who utilize thawed oocytes.

Optimal aspiration pressure of suction pump for oocyte retrieval in infertile patients undergoing in vitro fertilization.

The oocyte retrieval is a critical step in assisted reproductive technologies, including in vitro fertilization and fertility preservation. Despite evolving techniques, the optimal aspiration pressure during retrieval remains debatable, with limited in vivo human studies. Existing studies, primarily in vitro and on animals, suggest that inappropriate aspiration pressures can impair oocyte quality. This study aims to compares the effects of two different aspiration pressures, 120mmHg and 150mmHg, on oocyte recovery, damage, and subsequent embryo development and pregnancy outcomes in infertile women undergoing transvaginal ultrasound-guided oocyte retrieval. This retrospective study analyzed data from 891 women at Seoul National University Hospital between May 2018 and August 2023. A total of 400 cycles were included, with 202 at 120 mmHg and 198 at 150 mmHg aspiration pressures. The primary outcomes were the number of retrieved, matured, fractured oocytes, embryos, and good-grade embryos. Pregnancy outcomes were evaluated by comparing the clinical pregnancy rates and live birth rates. Univariate and multivariate logistic regression analyses were conducted to evaluate the relationship between aspiration pressure, clinical pregnancy, and live birth rates. There was statistically significant difference in the number of retrieved oocytes and mature oocytes between the 120 mmHg group and the 150 mmHg group (6.3±5.2 vs. 7.7±6.7, p = 0.018; 4.4±3.7 vs. 5.6±5.3, p = 0.011). The number of embryos and good grade embryos also differed significantly (3.3±3.1 vs. 4.2±3.9, p = 0.011; 1.0±1.6 vs. 1.5±2.6, p = 0.031). However, there were no significant differences in clinical pregnancy and live birth rates between the two groups in multivariate logistic regression analysis (adjusted OR = 0.725, p = 0.519; adjusted OR = 0.370, p = 0.170). Increasing the aspiration pressure to 150mmHg led to a higher yield of oocytes and embryos than 120mmHg, without any negative impact on oocyte quality or live birth rates. These findings provide valuable insights for clinical decision-making in infertility treatments, suggesting that 150mmHg may be a more effective pressure for oocyte retrieval in in vitro fertilization and embryo transfer.

Rapamycin Increases the Development Competence of Yak (Bos grunniens) Oocytes by Promoting Autophagy via Upregulating 17β-Estradiol and HIF-1α During In Vitro Maturation

High-quality oocyte production strategies play an important role in animal-assisted reproductive biotechnologies, and rapamycin (Rap) has been commonly used to increase the development potential of mammalian oocytes. The purpose of this study is to evaluate the effects and possible molecular mechanisms of rap on the maturation of yak oocytes. Different concentrations of Rap were supplemented during in vitro maturation (IVM) of yak oocytes. The maturation rates of oocytes and development rates of parthenogenetically activated embryos were assessed. The levels of 17β-estradiol (E2) were detected via ELISA, and the expression of autophagy-related factors, steroidogenic enzymes, and HIF-1α was detected via qRT-PCR, western blotting, and fluorescence microscopy, respectively. In addition, the impacts of E2 and HIF-1α on Rap-mediated oocyte autophagy were investigated by investigating the activities of E2 and HIF-1α. Our results showed that 0.1 nM Rap substantially enhanced the developmental ability of yak oocytes. In this group, the levels of E2, CYP19A1, CYP17A1, and autophagy-related factors were also significantly increased, and the expression of ATG5 and BECN1 in subsequent embryos was also increased. Further analysis revealed that Rap tends to enhance the development competence of yak oocytes and that the levels of autophagy-related factors are reduced when the activity of E2 or HIF-1α is inhibited. Furthermore, the levels of E2, CYP19A1, and CYP17A1 were downregulated when the activity of HIF-1α was inhibited, and the levels of HIF-1α were also significantly reduced by the estrogen receptor antagonist G15. Nevertheless, the levels of CYP11A1 mRNA in mature yak COCs were not significantly different among these groups, a phenomenon which implies that the levels of E2 were not correlated with the CYP11A1 content in yak COCs. There was an increasing tendency for the development competence of yak oocytes at the optimum concentration of Rap during IVM. The potential underlying mechanism is that Rap can activate autophagy and upregulate the levels of E2 and HIF-1α in mature oocytes. Additionally, the levels of both E2 and HIF-1α are regulated by each other and involve Rap-regulated autophagy in oocytes.

Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection

The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT, using a microinjection mixture with a concentration of 10 ng/μL Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1–5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding.

A randomized controlled trial comparing embryo vitrification with slush nitrogen to liquid nitrogen in women undergoing frozen embryo transfer: embryology and clinical outcomes.

Does the use of slush nitrogen (SN) for embryo vitrification improve embryo transfer outcomes compared to liquid nitrogen (LN)? SN is a safe method for embryo preservation and significantly improves post-warming survival rates during repeated vitrification-warming cycles; however, after a single freeze-thaw cycle, pregnancy outcomes are not improved when embryos are vitrified with SN compared to LN. SN is a combination of solid and LN, with a temperature lower than regular LN, and it is an alternative to conventional LN in achieving a faster cooling speed. Studies have shown that SN improves survival in non-human embryos and human oocytes. However, it is unknown whether the use of SN reduces blastocyst damage in humans during vitrification-as indicated by increased survival across multiple vitrification-warming cycles-or whether it enhances pregnancy outcomes in a single vitrification-warming cycle. Following the pre-clinical trial assessing embryo survival after repeated freeze-thaw cycles using SN and LN on 50 donated embryos per group, a randomized controlled trial was performed, where 253 patients were enrolled between September 2020 and January 2022, and 245 underwent an IVF stimulation, which resulted in at least one blastocyst for cryopreservation. Of those, 121 were allocated to the SN (study), and 124 were allocated to the LN (control) group. Randomization occurred on the day of blastocyst biopsy using a computer-generated block schema. Groups were assigned via opaque envelopes, opened by the embryologist on vitrification day. The patient, physician, and clinical team were blinded to the intervention. All couples with female aged between 18 and 42 years old undergoing IVF stimulation at one university-affiliated infertility center, with plan for preimplantation genetic testing for aneuploidy and subsequent single, frozen embryo transfer (FET) were eligible for inclusion in this study. The pre-clinical trial demonstrated significant improvements in blastocyst survival, with the SN group achieving a mean of 7.5 survived vitrification-warming cycles (range: 3-22), significantly surpassing the mean of 3.0 cycles (range: 0-10) in the LN group (P < 0.0001). Following the pre-clinical trial, 223 patients randomized to SN or LN underwent single FET. Baseline characteristics were similar between groups, as were embryology outcomes, including the number of oocytes retrieved, mature oocytes, fertilization rate, and total blastocysts biopsied. No significant differences were observed between the two groups in pregnancy rate, clinical pregnancy rate, sustained implantation rate, or miscarriage rate (P = 0.16, 0.80, 0.49, and 0.74, respectively, using Student's t-test). A futility analysis indicated no value in continuing recruitment and therefore the study was closed. Neonatal or birth outcomes were not assessed. Termination of the study based on futility analysis precludes a conclusion of equivalence between SN and LN. This study demonstrates that SN is a safe alternative to traditional LN for vitrification; however, it did not demonstrate improvements in the reproductive potential of vitrified embryos. The project was funded by the Foundation for Embryonic Competence. NCT04496284. 3 August 2020. 5 September 2020.

Cumulus cells and the TNF-alpha signaling facilitate aging of ovine oocytes.

Post-maturation oocyte aging (PMOA) is known to significantly impair the developmental potential of oocytes; however, comprehensive studies on ovine PMOA remain limited. In mice, cumulus cells (CCs) accelerate oocyte aging by releasing cytokines, but the roles of CCs and cytokines in PMOA of domestic animals are poorly understood. This study aimed to elucidate the involvement of CCs and tumor necrosis factor (TNF)-α in the PMOA of ovine oocytes. Our findings reveal that PMOA significantly reduced blastocyst rates and the expression of development-promoting genes, while increasing oocyte degeneration and activation rates, along with expression of development-inhibiting genes, compared to newly matured oocytes. These detrimental effects were more pronounced in oocytes aged as cumulus-oocyte complexes than as cumulus-denuded oocytes. Additionally, PMOA led to increased apoptotic rates, TNF-α production, and TNF receptor 1 (TNFR1) expression in CCs, coupled with a significant reduction in the expression of anti-apoptotic genes. Mature oocytes expressed TNFR1, with levels decreasing significantly during PMOA. Importantly, the addition of the TNF-α antagonist Etanercept to the aging medium markedly improved parthenogenetic embryo development and the expression of competence-related genes, while mitigating CC apoptosis during PMOA of COCs. In conclusion, PMOA compromises developmental potential while heightening oocyte degeneration and activation sensitivity in ovine oocytes. Cumulus cells exacerbate PMOA through increased TNF-α signaling activity, highlighting the potential of TNF-α antagonists as therapeutic agents to counteract the deleterious effects of PMOA.

Controlled ovarian hyperstimulation with or without letrozole for fertility preservation in breast cancer patients: study protocol for a randomised controlled trial

IntroductionMultimodal anticancer therapies greatly damage the fertility of breast cancer patients, which raises urgent demand for fertility preservation. The standard options for fertility preservation are oocyte and embryo cryopreservation; both...

Effects of Chlorogenic Acid on In Vitro Maturation and Vitrification Cryopreservation of Sheep Oocytes.

Chlorogenic acid (CGA) has strong antioxidant properties. In order to improve the low maturation rate and poor vitrification freezing effect of sheep oocytes caused by oxidative stress. In this study, oocytes from 200 2-3-year-old Kazakh sheep were collected, and different concentrations of CGA were added to the maturation medium and vitrification freezing solution to study the effects of CGA on the maturation rate, cleavage rate, blastocyst rate, reactive oxygen species (ROS) and glutathione (GSH) levels, mitochondrial membrane potential, and the expression levels of oxidation and apoptosis-related genes in sheep oocytes. The results showed that adding 40 μmol/L CGA to the oocyte in vitro maturation solution significantly increased the maturation rate of oocytes, adding 50 μmol/L CGA to the vitrification cryopreservative solution significantly increased the cleavage and blastocyst rates of mature oocytes activated by parthenogenetic activation after freezing. During in vitro maturation and vitrification freezing in sheep oocytes, CGA significantly reduced the level of ROS and the expression of apoptosis-related genes (Caspase-3 and Bax/Bcl-2), and significantly increased the level of glutathione (GSH), mitochondrial membrane potential, and the expression of antioxidant and anti-apoptosis-related genes (SOD-2 and GPX-3). In addition, CGA significantly increased the expression of the anti-apoptotic gene (AKT) and anti-stress gene (FOXO) during vitrification freezing of sheep oocytes. In conclusion, 40 μmol/L CGA improves the maturation rate of sheep oocytes, and 50 μmol/L CGA improves the quality of parthenogenetic activation embryos after vitrification freezing of mature oocytes in sheep. These results provide a basis for the production of sheep in vitro embryos and the establishment of a germplasm resource bank.

Study of the feasibility of polar body transfer combined with preimplantation genetic testing for blocking the intergenerational transmission of mitochondrial genetic diseases

To assess the feasibility of first polar body transfer (PB1T) combined with preimplantation mitochondrial genetic testing for blocking the transmission of a pathogenic mitochondrial DNA 8993T>G mutation. A Chinese family affected with Leigh syndrome which had attended the Reproductive Medicine Centre of the First Affiliated Hospital of Anhui Medical University in September 2021 was selected as the study subject. Controlled ovarian hyperstimulation was carried out for the proband after completing the detection of the mitochondrial DNA 8993T>G mutation load among the pedigree members. Mature MII oocytes were inseminated by intracytoplasmic sperm injection (ICSI), cultured in vitro for 5 to 6 days to the blastocyst stage, and trophoblastocytes were obtained by microbiopsy. Mitochondrial DNA testing (PGT-MT) and chromosomal aneuploidy (PGT-A) analyses were carried out after whole-genome amplification, and the embryos with zero mutation load were selected for transfer. Amniotic fluid and umbilical cord blood samples were collected during middle pregnancy and after birth respectively for mitochondrial DNA testing to verify the reliability of embryo screening. As an attempt, PB1 with good morphology of MII oocytes was selected for transfer into the enucleated oocytoplasm from healthy donors, followed by ICSI fertilization, blastocyst culture and PGT of embryos using the same procedure. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (No. 2021zhyx-B12). An antagonist protocol was used for ovarian stimulation, and a total of 19 oocytes were obtained, of which 14 MII were fertilized by ICSI, and 2 had developed into blastocysts. PGT-MT was carried out on biopsied trophoblastocytes, in which the mitochondrial DNA 8993T>G mutation load was not detected in one embryo, the other was 100% mutated, and the mutation loads of the remaining unfertilized eggs and developmentally arrested embryos ranged from 0% ~ 100%, presenting a clear biased distribution. With fully informed consent, one PGT-MT zero mutation load blastocyst was transferred and clinical pregnancy was achieved. Mitochondrial DNA and chromosomal testing of amniotic fluid cells during middle pregnancy had revealed no abnormalities. The proband had delivered a healthy boy through Caesarean section at 39+5 weeks of gestation, and no mutation was detected in the cord blood sample. Five well-formed PBs from 14 eggs were selected for PB1 transfer, followed by ICSI and culture, and two of the reconstituted embryos had formed blastocysts, with none of the above mutations detected in the biopsied samples. The PGT-MT technology can help families affected with mitochondrial diseases to have healthy offspring. PB1 transfer in combination with ICSI and PGT-MT holds the promise of turning waste into treasure and providing an alternative means of fertility for such families.

Oocyte maturation defect in women undergoing IVF: contributing factors and effects on mature sibling oocyte outcomes.

This study aimed to identify demographic and clinical factors associated with low maturation rates and to investigate if the rate of immature oocytes impacts the outcomes of mature sibling oocytes. Women undergoing their first IVF-ICSI cycle between 2018 and 2022 at a fertility clinic were included. Cycles were classified into five groups according to the proportion of Metaphase II stage oocytes (MII): Null (0% MII, n = 46), Poor (1-25% MII, n = 44), Low (26-50% MII, n = 453), Acceptable (51-75% MII, n = 1641), and Optimal (76-100% MII, n = 2642). Demographic characteristics and clinical outcomes were compared between the five groups. In patients with a Null/Poor maturation rate, subsequent cycle outcomes were also evaluated. A total of 4826 cycles were included in the study; 69,909 oocytes were recovered, and 53,065 were MIIs (75.9%). The Null group was older, had lower levels of anti-Müllerian hormone (AMH), needed more gonadotropins and days of stimulation, had higher follicle stimulating hormone (FSH) levels on day 3, and had less follicles > 15mm on the day of trigger. When the outcomes of mature oocytes were compared, fertilization, usable blastocyst, aneuploidy, and life birth rates were comparable among groups. A binary logistic regression model using number of oocytes, paternal age, and trigger type with live birth rate endpoint found no differences between the categories and the base line Poor category. When patients whose maturation rate was Null/Poor, 42 (47.0%) carried out a second cycle; the maturation rate increased (56.9 ± 31.5 vs. 11.6 ± 11.2%, P < 0.0001). Our data suggest that poor responders are more likely to have low rates of oocyte maturation. The proportion of immature oocytes does not impact the outcomes of mature sibling oocytes. In patients with Null/Poor maturation in their first cycle, the subsequent cycle is often associated with improved maturation rates.

Development of a novel calculator to predict gonadotropin dose and oocyte yield in oocyte cryopreservation cycles.

To develop a predictive model for estimating the total dose of gonadotropins and the number mature oocytes in planned oocyte cryopreservation cycles. In this retrospective study, oocyte cryopreservation cycles recorded in the Society for Assisted Reproductive Technology Clinic Outcome Reporting System Database from 2013 to 2018 were analyzed. Bivariate copula additive models for location, scale, and shape were performed to create a predictive model for estimating total dose of gonadotropins and number of mature oocytes. A total of 15,806 oocyte cryopreservation cycles between 2013 and 2018 were included in the analysis. The average age of participants was 35.4years, the mean duration of stimulation was 11.8days, and the average number of mature oocytes retrieved was 11.8. The treatment dose increased with age, FSH levels, BMI ≥ 35kg/m2, smoking, and history of diminished ovarian reserve, while it decreased with increasing AMH, ovulatory disorder, and BMI < 25kg/m2. The number of mature oocytes retrieved was positively correlated with AMH and negatively correlated with age, FSH levels, Asian race, and diminished ovarian reserve. With a maximum gonadotropin dosage of 450IU per day for 12 ± 3days of stimulation and a tolerance level of six mature oocytes, the predictive model achieved 70% accuracy. An interactive version of the equation was created as an online tool. We developed a predictive model to estimate the total treatment dosage and the number of mature oocytes. This calculator, utilizing objective patient variables, can assist in patient counseling prior to planned oocyte cryopreservation cycles.

Association of pentachlorophenol in urine and follicular fluid with ovarian reserve and reproductive outcomes among women undergoing in vitro fertilization based on a prospective cohort study.

Pentachlorophenol (PCP), a persistent organic pollutant, has endocrine disrupting properties and there may be a link between its exposure and reproductive outcomes. In this study, we assessed the relationship of PCP exposure levels with ovarian reserve markers and reproductive health outcomes in women (N = 656) undergoing in vitro fertilization (IVF). PCP concentrations were determined in urine (n = 1,968; repeated measures) and follicular fluid samples (n = 603). Generalized linear models or generalized estimating equations were used to analyze adjusted association between PCP exposure and selected outcomes (ovarian reserve and IVF outcomes among the women). The median concentration of PCP in the follicular fluid was (1.38 ng/mL) significantly higher compared with that in the urine (SG-adjusted: 0.79 ng/mL). We observed that the urinary PCP concentrations were significantly associated with increased estradiol levels (-12.4%; 95% CI: 0.76, 25.4%) but decreased total oocyte yield (-8.35%; 95% CI: -9.64, -7.04%), mature oocytes (-12.0%; 95% CI: -13.4, -10.6%), and fertilization proportion (-2.98%; 95% CI: -5.51, -0.39%). Moreover, there were significant associations of follicular fluid PCP concentrations with declines in total oocyte yield (-10.6%; 95% CI: -11.9, -9.26%), mature oocytes (-10.6%; 95% CI: -12.0, -9.09%), and proportions of fertilization (-3.75%; 95% CI: -6.39, -1.03%), blastocyst formation (-8.01%; 95% CI: -16.6, -0.37%), and usable blastocysts (-13.9%; 95% CI: -23.6, -3.03%). Our results revealed that exposure to PCP was related with impaired reproductive outcomes of IVF, while additional research is needed to confirm the findings and clarify the underlying mechanisms.

Polycystic ovary syndrome and morphokinetic embryonic development: A case-control study evaluating 791 embryos.

To investigate the association between Polycystic Ovary Syndrome (PCOS) and the rate of embryo development, using time-lapse monitoring systems (TLM), compared to a control group of women with mechanical (tubal) factor infertility. A retrospective case-control study conducted in a university affiliated IVF unit. Women with PCOS undergoing in-vitro fertilization (IVF) treatments and those with non-PCOS controls with tubal factor infertility only. Development morphokinetic milestones were compared and analysis of covariance for time to distinct cell number as well as logistic mixed models to determine predictors for embryos over the 75th percentile was performed. Not applicable. Embryo development morphokinetic parameters in women with and without PCOS undergoing IVF treatments. The study included 791 embryos from 115 women, 364 embryos from 52 women with PCOS and 427 embryos from 63 women with non-PCOS controls with tubal factor infertility. The PCOS group was 4 years younger (30.07±6.03 vs. 34.08±4.84 years, p=0.002) and had higher number of oocytes retrieved (16.00 vs. 11.00, p<0.001), mature oocytes (11.00 vs. 7.00,<0.001) and fertilized oocytes (8.00 vs. 5.00, p<0.001). The PCOS and control groups demonstrated comparable clinical pregnancy rates (55.8% vs. 32.1%,p=0.053), miscarriage rate (12.5% vs. 11.8%, p=0.994) and live birth rate (48,8% vs. 31.2%, p=0.089). Morphokinetic parameters were comparable between the groups. While age was associated with later time to 5 and 8 discrete cells and start of blastulation (tSB), PCOS was only associated with later tSB, including tSB>75th percentile. This study demonstrated comparable IVF outcomes in women with PCOS and non-PCOS controls. An analysis of TLM data from these patients showed no evidence that PCOS negatively affects embryonic development rate in women undergoing IVF cycles.

Acupuncture Increased the Number of Retrieved Oocytes in a Mouse Model of POR: The Involvement of DNA Methylation in the Oocytes

Background: Poor ovarian response (POR) reduces the success rate of in vitro fertilization mainly because of fewer oocytes retrieved. Acupuncture (Ac) therapy can improve the number of retrieved oocytes in the controlled ovarian stimulation program. The role of Ac in the corresponding epigenetic mechanism of POR has not been studied. Objective: This study was conducted to determine the effect of Ac on the number of retrieved oocytes and its role in DNA methylation in a mouse model of POR. Methods:: Forty C57BL/6N female mice with normal estrous cycles were randomly classified into 4 groups of 10 each: control (Con) group, Ac-Con group, POR group, and Ac-POR group. Mice in POR and Ac-POR groups received a gastric gavage of Tripterygium wilfordii polyglycoside suspension of 50 mg/kg-1 once a day for 14 consecutive days. Ac was applied at “Shenting” (DU 24), “Guanyuan” (CV 4), “Zusanli” (ST 36), and “Shenshu” (BL 23) in the Ac-POR group for 10 min per session, once a day for 14 consecutive days. All four groups were stimulated with pregnant mare serum gonadotropin and human chorionic gonadotropin, and the number of retrieved oocytes and proportion of mature oocytes were recorded. The DNA methylation level in a single mouse oocyte in each group was analyzed using single-cell genome-wide bisulfite sequencing (scBSseq), and key pathways were identified using GO and KEGG enrichment analyses. Results: A dissecting microscope revealed that the Ac therapy improved the number of retrieved oocytes compared with the POR group (p &lt; 0.05). ScBS-seq showed that there was no significant change in global DNA methylation levels between the POR model and control group mice. However, differences were primarily observed in the differentially methylated regions (DMRs) of each chromosome, and Ac decreased global DNA methylation. DMR analysis identified 13 genes that may be associated with Ac treatment. Cdk5rap2 and Igf1r, which mediate germ cell apoptosis, growth, and development, maybe most closely related to the Ac treatment of POR. KEGG analysis revealed that differentially expressed genes were mainly enriched in Wnt, GnRH, and calcium signaling pathways. The genes were closely related to the regulation of POR via Ac. Conclusion: The results suggest that DNA methylation in oocytes is related to the development of POR and that the role of Ac in affecting DNA methylation in oocytes is associated with the Wnt, GnRH, and calcium signaling pathways as well as Cdk5rap2 and Igf1r in POR mice.

Superovulation treatment of immature female rabbits increases the number of ovulated oocytes that can in vitro develop into blastocytes.

To clarify the efficiency of superovulation in immature female rabbits, immature female rabbits were superovulated with pregnant mare serum gonadotropin, and the number of recovered oocytes, their maturity, and their ability to develop into blastocysts under in vitro fertilization and culture were examined in this study. More than 80 oocytes were recovered from 12-14-week-old immature female rabbits. In particular, an average of more than 100 oocytes were recovered from 13-week-old immature female rabbits. The number of oocytes in immature female rabbits was significantly-approximately 4 times-higher than in mature female rabbits. To compare oocyte maturity, oocytes from immature and mature female rabbits were compared by brilliant cresyl blue staining and mitochondrial distribution analysis. The proportion of mature oocytes detected by brilliant cresyl blue staining was higher in oocytes from mature female rabbits than in oocytes from immature female rabbits, but there were no significant differences in active mitochondrial distribution, fertilization rate, or blastocyst development rate. These results indicate that superovulation with immature female rabbits may be a useful technique for the collection of many oocytes.

MiRNA-125a regulates porcine oocyte maturation in vitro by targeting ADAR.

Follicular fluid extracellular vesicles are beneficial for in vitro oocyte maturation and development; however, their effect on the expression profiles of oocyte microRNAs (miRNAs) and the roles of related miRNAs are unknown. In this study, we aimed to investigate miRNA expression in mature oocytes cultured in follicular fluid extracellular vesicles and the effect of miRNA-125a (miR-125a) on oocyte maturation. The expression profiles of the miRNAs were determined by microRNA sequencing, followed by target gene prediction analysis. We transfected miR-125a mimics and an miR-125a inhibitor to evaluate the effect of modulated miRNA-125a on cumulus expansion, oocyte maturation rate, changes in cytoplasmic maturation-related indicators, and changes in the expression of oocyte maturation-related, cumulus expansion-related, and predicted target genes. We found that miR-125a overexpression decreased the levels of cumulus expansion-related, oocyte maturation-related, and predicted target genes, adenosine deaminase RNA specific (ADAR), and lipid droplet number, and it increased the percentage of oocytes with abnormal cortical granule distribution. Inhibiting miR-125a increased the expression levels of oocyte maturation-related and target genes, number of lipid droplets, and endoplasmic reticulum function, and it decreased lipid droplet size. Mitochondrial membrane potential and reactive oxygen species levels were not significantly different between groups. In conclusion, our results suggest that extracellular vesicles may improve oocyte quality by modulating ADAR through regulating miR-125a.

Activation of Proteolysis During Oocyte In Vitro Maturation.

In vitro maturation (IVM) is a form of assisted reproductive technology (ART) applied to obtain mature oocytes in culture. Decline in IVM success rates by age has led consideration of novel approaches based on cellular dynamics. Our aim was to achieve proteostasis in old bovine oocytes from 13 to 16-year-old bovine with a lower potential for fertilization. Lysosomal activation was achieved through increasing concentrations of proton pump activators PIP2 (0.1, 0.5, 1, and 5 μM), PMA (0.1, 1, 10, and 50 μM), and DOG (0.1, 1, 10, and 50 μM) at 6, 12, 18, and 24 h of IVM in old bovine oocytes. Morphological analysis was performed and IVM rates were determined. DQ-Red BSA was applied to live oocytes to determine proteolytic activation while lysosome density was determined by Lysotracker probe. Protein carbonylation was detected through oxyblot analysis. Polar body extrusion (PBE), through which a haploid nonfunctional polar body is released in the perivitelline space after completion of the first meiotic division, was observed in PIP2-0.1 μM, -0.5μM-6h; PIP2-5μM-12h; PMA-0.1μM-18h; PIP2-0.1μM, -0.5μM-24h groups. Oocyte diameter was the highest in DOG-1μM-6h, PMA-0.1μM-12h, PIP2-1μM-18h, and PIP2-0.5μM-24h groups. Morphological scores of oocytes were higher in young and old control groups. PIP2, PMA, and DOG affected oocyte quality positively after 6 h of IVM yielding in oocyte scores similar to the control group oocytes. However, they had a negative impact on the oocyte scores in longer periods of IVM, except for lower doses PMA (0.1 and 1 μM) at 12 h and PIP2 (0.5 μM) and PMA (0.1 μM) at 18 h, which were able to maintain the scores relatively closer to the control oocytes. Proteolytic activation was achieved in all groups at 6 h of culture. At all other time points PIP2 and PMA groups showed a better response to proteolytic activation. Lysosome density was increased in PIP2-5μM-6h; PIP2-0.1μM, -1μM-12h; PIP2-1μM, -5μM-18h as well as PMA-0.1μM-6h; PMA-1μM, -10μM-12h; PMA-1μM-18h; DOG-50μM-6h and DOG-0.1μM-12h. Protein carbonylation was the lowest in PIP2-0.1 μM groups at 12, 18, and 24 h. This study suggests that proton pump activators PIP2 and PMA was found to have a positive impact on IVM in terms of both morphological scores and proteolytic activation in a time and dose dependant manner.

The interplay between mitochondrial DNA genotypes, female infertility, ovarian response, and mutagenesis in oocytes.

Is there an association between different mitochondrial DNA (mtDNA) genotypes and female infertility or ovarian response, and is the appearance of variants in the oocytes favored by medically assisted reproduction (MAR) techniques? Ovarian response was negatively associated with global non-synonymous protein-coding homoplasmic variants but positively associated with haplogroup K; the number of oocytes retrieved in a cycle correlates with the number of heteroplasmic variants in the oocytes, principally with variants located in the hypervariable (HV) region and rRNA loci, as well as non-synonymous protein-coding variants. Several genes have been shown to be positively associated with infertility, and there is growing concern that MAR may facilitate the transmission of these harmful variants to offspring, thereby passing on infertility. The potential role of mtDNA variants in these two perspectives remains poorly understood. This cohort study included 261 oocytes from 132 women (mean age: 32 ± 4 years) undergoing ovarian stimulation between 2019 and 2020 at an academic center. The oocyte mtDNA genotypes were examined for associations with the women's fertility characteristics. The mtDNA of the oocytes underwent deep sequencing, and the mtDNA genotypes were compared between infertile and fertile groups using Fisher's exact test. The impact of the mtDNA genotype on anti-Müllerian hormone (AMH) levels and the number of (mature) oocytes retrieved was assessed using the Mann-Whitney U test for univariate analysis and logistic regression for multivariate analysis. Additionally, we examined the associations of oocyte maturation stage, infertility status, number of ovarian stimulation units, and number of oocytes retrieved with the type and load of heteroplasmic variants using univariate analysis and Poisson or linear regression analysis. Neither homoplasmic mtDNA variants nor haplogroups in the oocytes were associated with infertility status or with AMH levels. Conversely, when the relationship between the number of oocytes retrieved and different mtDNA genotypes was examined, a positive association was observed between the number of metaphase (MII) oocytes (P = 0.005) and haplogroup K. Furthermore, the presence of global non-synonymous homoplasmic variants in the protein-coding region was significantly associated with a reduced number of total oocytes and MII oocytes retrieved (P < 0.001 for both). Regarding the type and load of heteroplasmic variants in the different regions, there were no significant associations according to maturation stage of the oocyte or to fertility status; however, the number of oocytes retrieved correlated positively with the total number of heteroplasmic variants, and specifically with non-synonymous protein-coding, HV and rRNA variants (P < 0.001 for all). The current work is constrained by its retrospective design and single-center approach, potentially limiting the generalizability of our findings. The small sample size for specific types of infertility restricts this aspect of the findings. This work suggests that mitochondrial genetics may have an impact on ovarian response and corroborates previous findings indicating that the size of the oocyte cohort after stimulation correlates with the presence of potentially deleterious variants in the oocyte. Future epidemiological and functional studies based on the results of the current study will provide valuable insights to address gaps in knowledge to assess any prospective risks for MAR-conceived offspring. This work was supported by the Research Foundation Flanders (FWO, Grant numbers 1506617N and 1506717N to C.S.), by the Fonds Wetenschappelijk Fonds, Willy Gepts Research Foundation of Universitair Ziekenhuis Brussel (Grant numbers WFWG14-15, WFWG16-43, and WFWG19-19 to C.S.), and by the Methusalem Grant of the Vrije Universiteit Brussel (to K.S.). M.R. and E.C.d.D. were supported predoctoral fellowships by the FWO, Grant numbers 1133622N and 1S73521N, respectively. The authors declare no conflict of interests. N/A.

Ruvbl1 silencing affects reproduction of the corn planthopper, Peregrinus maidis.

Ruvbl1 (also known as TIP49, Pontin) encodes an ATPase of the AAA+ protein superfamily involved in several cellular functions, including chromatin remodeling, control of transcription, and cellular development (motility, growth, and proliferation). While its role has been well established in model organisms including vertebrates and invertebrates (e.g. mice, Xenopus and Drosophila), putative functions of Ruvbl1 in non-model insect pests have not been addressed. To exploit Ruvbl1 as a potential target gene for applications in insect control, we used an in-vivo RNA interference (RNAi) approach to evaluate the effect of Ruvbl1 silencing on the physiology of the corn planthopper, Peregrinus maidis. Silencing of P. maidis Ruvbl1 (PmRuvbl1) was correlated with visible morphology changes in female individuals with significant increases in body mass observed at 8 and 12 days after double strand RNA (dsRNA) injection. Ovary function was significantly affected in adult females with PmRuvbl1 silenced, with no mature oocytes observed at 8 and 12 days after gene silencing. Whereas no significant difference in egg laying was observed 4 days after dsRNA injection, significantly fewer eggs were laid in plants at 8 and 12 days after dsRNA treatment. Furthermore, dramatic reductions in egg hatching were observed at all time points after PmRuvbl1 silencing, compared to dsGFP-injected controls. These results extend Ruvbl1 functions as a putative regulator of P. maidis reproduction and demonstrate the potential of Ruvbl1 to be further exploited as a target for developing new technologies (e.g. RNA interference, CRISPR-mediated control) for insect control.

Beeswax Alcohol (BWA, Raydel®) Improved Blood Oxidative Variables and Ameliorated Severe Damage of Zebrafish Kidneys, Testes, and Ovaries Impaired by 24-Week Consumption of a High-Cholesterol and High-Galactose Diet: A Comparative Analysis with Coenzyme Q10.

The present study describes the comparative effect of 24-week supplementation of beeswax alcohol (BWA, Raydel®, 0.5% and 1.0%, wt/wt) and coenzyme Q10 (CoQ10, 0.5% and 1.0%, wt/wt) on plasma oxidative variables and the prevention of organ injury in adult zebrafish subjected to a high-cholesterol (HC, 4%, wt/wt) and -D-galactose (Gal, 30%, wt/wt) diet. Adult zebrafish were fed various HC+Gal diets enriched with either BWA or CoQ10. After 24 weeks of dietary intervention, blood and organs were harvested for subsequent biochemical and histological evaluations. The HC+Gal-elevated plasma oxidative variables were reverted by the consumption of BWA, marked by the lowest plasma malondialdehyde (MDA) level and highest sulfhydryl content. The HC+Gal-impaired zebrafish swimming ability (staggering movement) was substantially recovered by BWA, manifested by a ~three-fold (p < 0.001) enhancement in swimming distance and speed. Also, the intake of BWA affected the morphology of HC+Gal-compromised kidney and induced histological changes by mitigating reactive oxygen species (ROS) production and cellular senescence, which was markedly more effective than the results seen in the CoQ10 group. Likewise, BWA proved effective in preventing HC+Gal-induced testis damage, apparent in the 48.3% (p < 0.05) higher spermatozoa and 26.3% (p < 0.01) reduced interstitial space between the seminiferous tubules. BWA substantially prevented HC+Gal-induced ovary damage by suppressing oxidative stress, lipid deposition and senescence, leading to the restoration of mature vitellogenic oocyte counts. BWA demonstrated a greater ability than CoQ10 to enhance plasma antioxidant status, suppress ROS generation, delay organ aging and alleviate HC+Gal-induced adversity in zebrafish.