Reproductive medical providers' behaviors, considerations, and plans for fertility treatments during the COVID-19 pandemic in Japan: A nationwide web-based survey.

PURPOSE: This study was conducted to investigate how the COVID-19 pandemic has impacted reproductive medical providers' behaviors and considerations, including their concerns regarding the necessity of fertility treatments. METHODS: A web-based questionnaire was distributed to Japan Society of Fertilization and Implantation (JSFI) members from May 18 through May 31, 2020 to survey their professional behaviors and concerns during the COVID-19 pandemic. RESULTS: Most survey participants reported a decrease in the number of patients and a decrease in their workload. Most also believe that the use of fertility treatments will return to the pre-pandemic levels after the COVID-19 pandemic ends. Additionally, more than half of the participants reported that they consider fertility treatment neither necessary nor unnecessary during the COVID-19 pandemic. CONCLUSIONS: At the institute where reproductive medical providers worked in Japan, the number of outpatients and the working time tended to decrease during the COVID-19 pandemic. However, amid fears of infection during the COVID-19 pandemic, the reproductive medical providers working at fertility institutes in Japan have remained engaged in their work with a sense of mission and hope.

Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Evaluation of antioxidant status and oxidative stress markers in follicular fluid for human in vitro fertilization outcome.

PURPOSE: Antioxidant status and oxidative stress markers in human follicular fluid (FF) surrounding oocytes may be related to the outcomes of in vitro fertilization and embryo transfer (IVF-ET). Therefore, we herein examined the relationship between antioxidant status and oxidative stress markers in FF and the outcomes of IVF-ET. METHODS: One hundred and seventeen infertile women were included in this study. FF was obtained from mature follicles at the time of oocyte retrieval. The total antioxidant capacity (TAC) and total glutathione (GSH), vitamin C, and 8- hydroxy-2'-deoxyguanosine (8-OHdG) concentrations were measured. RESULTS: Total GSH levels were lower in patients who had a low fertilization rate after intracytoplasmic sperm injection (ICSI). In addition, 8-OHdG levels were higher in patients who had a low fertilization rate after ICSI and low rate of good quality blastocysts. Total GSH activity was lower in patients with endometriosis. No significant differences were noted in pregnancy outcomes. CONCLUSIONS: Total GSH and 8-OHdG in human FF may be potential markers for fertilization in ART. Also, our findings may suggest that oxidative stress in women with infertility is associated with endometriosis.

Peroxiredoxin as a functional endogenous antioxidant enzyme in pronuclei of mouse zygotes.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development.

Maternal RNA/protein degradation and zygotic genome activation (ZGA), occurring during maternal-to-zygotic transition (MZT), are the first essential events for the development of pre-implantation embryos. Previously, we have shown the importance of the ubiquitin-proteasome system (UPS) for initiation of minor ZGA at the 1-cell stage of mouse embryos. However, little is known about the mechanism of involvement of the UPS-degraded maternal proteins in ZGA. In this study, we investigated the effect of inhibiting maternal protein degradation by the reversible proteasome inhibitor, MG132, on post-implantation development and ZGA regulation during early cleavage stages. Our study revealed that zygotic transcription by RNA polymerase II (Pol II) at the 1-cell stage was delayed and the full-term development was affected by transient proteasome inhibition during 1 to 9 h post-insemination (hpi). Furthermore, we found that the transient inhibition of proteasome activity at the 2-cell stage delayed the onset of transcription of some major ZGA genes. These results support the model hypothesizing the requirement of sequential degradation of maternal proteins by UPS for the proper onset of ZGA and normal progression of MZT in early mouse embryos.

Histone H3 Methylated at Arginine 17 Is Essential for Reprogramming the Paternal Genome in Zygotes.

At fertilization, the paternal genome undergoes extensive reprogramming through protamine-histone exchange and active DNA demethylation, but only a few maternal factors have been defined in these processes. We identified maternal Mettl23 as a protein arginine methyltransferase (PRMT), which most likely catalyzes the asymmetric dimethylation of histone H3R17 (H3R17me2a), as indicated by in vitro assays and treatment with TBBD, an H3R17 PRMT inhibitor. Maternal histone H3.3, which is essential for paternal nucleosomal assembly, is unable to be incorporated into the male pronucleus when it lacks R17me2a. Mettl23 interacts with Tet3, a 5mC-oxidizing enzyme responsible for active DNA demethylation, by binding to another maternal factor, GSE (gonad-specific expression). Depletion of Mettl23 from oocytes resulted in impaired accumulation of GSE, Tet3, and 5hmC in the male pronucleus, suggesting that Mettl23 may recruit GSE-Tet3 to chromatin. Our findings establish H3R17me2a and its catalyzing enzyme Mettl23 as key regulators of paternal genome reprogramming.

CYP2C76 deficiency is embryonic lethal in cynomolgus macaques: The potential role of CYP2C76 in early embryogenesis.

Cynomolgus macaques are an important primate species for drug metabolism studies; however cynomolgus CYP2C76, an important drug-metabolizing enzyme, accounts for drug metabolism differences to humans, so that CYP2C76-null animals might show drug-metabolizing properties more similar to humans. In this study, attempts were made to produce CYP2C76-null animals by assisted reproduction technology. Oocytes and sperm collected from the heterozygotes for the null allele (c.449TG > A) were subjected to intracytoplasmic sperm injection, and the embryos produced were cultured in vitro through the blastocyst stage. Preimplantation genetic diagnosis using a biopsied portion of the blastocyst revealed that none of the 32 blastocysts analyzed were homozygotes. In contrast, 2 of the 20 embryos analyzed were homozygotes at the 8-cell stage, indicating that CYP2C76-null embryos most likely stop developing between the 8-cell and blastocyst stage. By polymerase chain reaction, expression of CYP2C76 mRNA was detected in oocytes and blastocysts, but not in 2-, 4-, 8-, or 16/32-cell stage embryos. Metabolic assays showed that CYP2C76 metabolized progesterone. These results indicated that CYP2C76 null was likely embryonic lethal, suggesting its potential role during early embryogenesis in cynomolgus macaques.

Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms.

Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy. The correlation among ultrastructural head abnormality in sperms from the upper layer of swim-up, fertilization in in vitro fertilization, and pregnancy after embryo transfer was also investigated. Furthermore, sperms with DNA fragmentation in the samples processed via a combination of DGC and swim-up was assessed in a sperm chromatin structure assay. Ultrastructural abnormalities in sperm heads and tails in the upper layer after swim-up following DGC was the lowest among the three groups. Sperms with nuclear vacuoles were the most difficult to eliminate using a combination of DGC and swim-up in all types of head abnormalities. A negative correlation was confirmed between the fertilization rates of intracytoplasmic sperm injection and head abnormality of sperms obtained from the upper layer of the swim-up following DGC. Sperms with DNA fragmentation were effectively decreased using the combination of two techniques. In conclusion, the combination of DGC and swim-up effectively decreased the number of sperms with ultrastructural abnormalities both in the head and in the tail. However, sperms with ultrastructural abnormalities that cannot be completely decreased using a combination of DGC and swim-up may impair fertilization in some cases of intracytoplasmic sperm injection.

Development of a new device for artificial insemination in cynomolgus macaques.

In cynomolgus macaques, an important animal species for biomedical research, efficient reproduction has been hampered partly due to the difficulties of artificial insemination (AI) using straw tubes developed for humans or farm animals, because cynomolgus macaques have a complex cervical canal structure. In this study, taking into consideration the unique structure of the macaque cervical canal, we developed a novel device for AI, comprised of a syringe and an outer cylinder. At 24 and 48 h after using this device to inject semen into one female, viable sperm were observed in the oviduct where the sperm meets the oocytes. We then attempted AI using this new device on 10 females that were at pre-ovulation, and pregnancy was successful in three animals (30% pregnancy rate). These results show that the newly developed device can be used for AI in cynomolgus macaques.

Cryopreservation of a small number of human sperm using enzymatically fabricated, hollow hyaluronan microcapsules handled by conventional ICSI procedures.

PURPOSE: We investigated whether enzymatically fabricated hyaluronan (HA) microcapsules were feasible for use in the cryopreservation of a small number of sperm. METHODS: HA microcapsules were fabricated using a system of water-immiscible fluid under laminar flow. Three sperm were injected into a hollow HA microcapsule using a micromanipulator. Capsules containing injected sperm were incubated in a freezing medium composed of sucrose as the cryoprotectant and then placed in a Cryotop® device and plunged into liquid nitrogen. After thawing, the capsule was degraded by hyaluronidase, and the recovery rate of sperm and their motility were investigated. RESULTS: The HA microcapsule measuring 200 µm in diameter and with a 30-µm thick membrane was handled using a conventional intracytoplasmic sperm injection (ICSI) system, and the procedure involved the injection of sperm into the capsule. The HA microcapsules containing sperm were cryopreserved in a Cryotop® device and decomposed by the addition of hyaluronidase. The recovery rate of sperm after cryopreservation and degradation of HA microcapsules was sufficient for use in clinical practice (90 %). CONCLUSIONS: Hollow HA microcapsules can be used for the cryopreservation of a small number of sperm without producing adverse effects on sperm quality.

Production of somatic cell nuclear transfer embryos using in vitro-grown and in vitro-matured oocytes in rabbits.

We examined growing oocytes collected from follicles remaining in superovulated rabbit ovaries, that were grown (in vitro growth, IVG) and matured (in vitro maturation, IVM) in vitro. We produced somatic cell nuclear transfer (SCNT) embryos using the mature oocytes and examined whether these embryos have the ability to develop to the blastocyst stage. In addition, we examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), on the developmental competence of SCNT embryos derived from IVG-IVM oocytes. After growth for 7 days and maturation for 14-16 h in vitro, the growing oocytes reached the metaphase II stage (51.4%). After SCNT, these reconstructed embryos reached the blastocyst stage (20%). Furthermore, the rate of development to the blastocyst stage and the number of cells in the blastocysts in SCNT embryos derived from IVG-IVM oocytes were significantly higher for TSA-treated embryos compared with TSA-untreated embryos (40.6 versus 21.4% and 353.1 ± 59.1 versus 202.5 ± 54.6, P < 0.05). These results indicate that rabbit SCNT embryos using IVG-IVM oocytes have the developmental competence to reach the blastocyst stage.

Possible role of ZPAC, zygote-specific proteasome assembly chaperone, during spermatogenesis in the mouse.

In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly chaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit α4/PSMA7 in the adult mouse testis. ZPAC and α4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of α4 persisted until step 12. We then examined the expression profile of ZPAC and α4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of α4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.

Oral melatonin supplementation improves oocyte and embryo quality in women undergoing in vitro fertilization-embryo transfer.

The aim of this study was to evaluate the efficacy of oral melatonin supplementation on oocyte and embryo quality in patients in an assisted reproductive technologies program. All patients were treated for at least 2 weeks with melatonin (3 mg/day). To evaluate the cumulative effect of melatonin supplementation, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. There were no significant differences in maturation rates (p = 0.50), blastocyst rates (p = 0.75), and the rate of good quality blastocysts (p = 0.59) between the first and second cycles. The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (69.3 versus 77.5%). Being limited to patients with a low fertilization rate in the first cycle (<60%), the fertilization rate dramatically increased after melatonin treatment (35.1 versus 68.2%). The rate of good quality embryos also increased (48.0 versus 65.6%). An important finding in our study was that oral melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred due to a reduction in oxidative damage.

Utilization of water chestnut for reclamation of water environment and control of cyanobacterial blooms.

Overgrowth of water chestnut (Trapa spp.) is a regional problem throughout Asia and North America because of waterway blockage and water fouling upon decomposition. In the present study, we investigated the potential of water chestnut to control cyanobacterial blooms, via a high content of phenolic compounds. In addition, we assessed the impact of biomass harvesting and crude extract application on nutrient balance. We showed that the floating parts of water chestnut contained high concentrations of total phenolics (89.2 mg g(-1) dry weight) and exhibited strong antioxidant activity (1.31 mmol g(-1) dry weight). Methanol-extracted phenolics inhibited growth of Microcystis aeruginosa; the half maximal effective concentration (EC50) of the extracted phenolics was 5.8 mg L(-1), which was obtained from only 103 mg L(-1) of dry biomass (the floating and submerged parts). However, the crude extracts also added important quantities of nitrogen, phosphorus, and potassium (1.49, 1.05, and 16.3 mg g(-1), respectively; extracted dry biomass weight basis); therefore, in practice, nutrient removal before and/or after the extraction is essential. On the other hand, biomass harvesting enables recovery of nitrogen, phosphorus, and potassium from the water environment (23.1, 2.9, and 18.7 mg g(-1), respectively; dry biomass weight basis). Our findings indicate that water chestnut contains high concentrations of phenolics and exhibits strong antioxidant activity. Utilization of these resources, including nutrients, will contribute to reclamation of the water environment, and also to disposal of wet biomass.

Abnormal lysine acetylation with postovulatory oocyte aging.

BACKGROUND: A postovulatory mammalian oocyte decreases developmental potential with in vivo aging in the oviduct or in vitro aging in the culture dish. The mechanism underlying oocyte aging still largely remains an enigma. Accumulating data suggest that the epigenetic alterations such as histone acetylation are also associated with postovulatory aging. OBJECTIVE: To perform a review evaluating a new aspect of oocyte aging in terms of the epigenetic alterations focusing on lysine acetylation. METHODS: In addition to a search of the literature in Pubmed, we introduced our recent published data. RESULTS: Histone acetylation in the mouse oocyte increases during aging, potentially impacting gene regulation in the subsequent embryonic development. Oocyte aging results in increased acetylation of alpha-tubulin, a non-histone protein, and nicotinamide, an inhibitor of class III HDAC, partially prevents some of oocyte aging phenotypes. CONCLUSION: Abnormal regulation of protein acetylation itself is suggested in oocyte aging and could contribute to the aging phenotypes.

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

Good thermally conducting material supports follicle morphologies of porcine ovaries cryopreserved with ultrarapid vitrification.

Effects of supporting materials during vitrification procedure on the morphologies of preantral follicles of pig ovaries were assessed. Ovarian cortical sections of prepubertal pigs were randomly allocated to 5 groups. The sections were vitrified ultrarapidly with 5 different vitrification devices. The sections were put on 4 fine needles (Cryosupport), on a thin copper plate, or on a carbon graphite sheet or were sandwiched between copper plates or between carbon graphite sheets before cooling. The cooling and warming rates with the graphite sheets were significantly higher than those with the copper plates (P<0.05). A total of 3,064 follicles were analyzed following HE staining after vitrification with 5 different devices. The morphologies follicles vitrified on the Cryosupport or on the graphite sheet were well preserved compared with those vitrified on the copper plate or between copper plates (P<0.01). The morphologies of follicles vitrified between copper plates were mostly damaged (P<0.05). Taken together, good thermally conducting material supports follicle morphologies of ovaries cryopreserved with ultrarapid vitrification.

Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos.

Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated α-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and α-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to α-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated α-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including α-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development.

GSE is a maternal factor involved in active DNA demethylation in zygotes.

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.

Nicotinamide: a class III HDACi delays in vitro aging of mouse oocytes.

Postovulatory mammalian oocyte developmental potential decreases with aging in vivo and in vitro. Aging oocytes typically show cellular fragmentation and chromosome scattering with an abnormally shaped spindle over time. Previously, it was shown that histone acetylation in the mouse oocyte increased during aging and that treatment with trichostatin A (TSA), an inhibitor for class I and II histone deacetylases (HDACs), enhanced the acetylation, that is, aging. In this study, we examined the effect of nicotinamide (NAM), an inhibitor for class III HDACs, on in vitro aging of mouse oocytes as well as TSA. We found that treatment with NAM significantly inhibited cellular fragmentation, spindle elongation and astral microtubules up to 48 h of culture. Although presence of TSA partially inhibited cellular fragmentation and spindle elongation up to 36 h of culture, treatment with TSA induced chromosome scattering at 24 h of culture and more severe cellular fragmentation at 48 h of culture. Further, we found that α-tubulin, a nonhistone protein, increased acetylation during aging, suggesting that not only histone but nonhistone protein acetylation may also increase with oocyte aging. Thus, these data indicate that protein acetylation is abnormally regulated in aging oocytes, which are associated with a variety of aging phenotypes, and that class I/II and class III HDACs may play distinct roles in aging oocytes.