Germinal Vesicle Oocytes Research Articles

The impact of serum and follicular fluid secreted frizzled-related protein-5 on ICSI outcome in Iraqi infertile women with different body mass index

Introduction and Aim: Adipose tissue adipokines and cytokines impede oocyte maturation and endometrial epithelial receptivity. Secreted frizzled-related protein 5 (Sfrp5), a novel antagonist adipokine secreted by adipocytes, may have anti-inflammatory and insulin-sensitizing actions, but data is conflicting. This study examined serum and follicular fluid Sfrp5 levels in Iraqi infertile women with varying BMIs undergoing IVF/ICSI, as well as ICSI outcomes. Materials and Methods: This prospective cross-sectional study comprised 90 infertile females aged 18–45 who underwent IVF/ICSI at the High Institute of Infertility Diagnosis and Assisted Reproductive Technologies Centre in Baghdad, Iraq. Participants received antagonist ovarian hyperstimulation. All individuals' blood fasting sugar, serum fasting insulin, serum, and follicular fluid SFRP5 were tested on oocyte retrieval day. HOMA-IR was computed. Oocyte maturity and embryo morphology were categorized. Serum B-hCG was measured 14 days after embryo transfer. Results: A notable disparity was observed in the levels of serum and follicular Sfrp5, total oocyte count, mean metaphase II (MII), grade I, and pregnancy rate (p<0.05) among the participants categorized as normal weight, overweight, and obese. A notable disparity was observed in the average germinal vesicles (GV), grade III (GIII) embryos, and HOMA-IR (p < 0.05) among patients with varying body mass index (BMI) values. The study found a positive correlation between the levels of Sfrp5 in both serum and follicular fluid and the total count of oocytes, MII oocytes, grade I embryos, and pregnancy rate. However, there was a negative correlation between Sfrp5 levels and HOMA-IR, with statistical significance at p<0.05. Conclusion: Increased BMI is associated with lower serum and follicular fluid Sfrp5, reduced total oocyte count, reduced metaphase II oocyte count, more germinal vesicle oocytes, reduced embryo quality and lower pregnancy rate. Serum and follicular fluid Sfrp5 levels are negatively correlated with HOMA-IR but positively associated with ICSI outcome.

The Effect of Choline Salt Addition to Trehalose Solution for Long-Term Storage of Dried and Viable Nuclei from Fully Grown Oocytes.

Although drying techniques are exciting alternatives to cryopreservation, it remains challenging to maintain tightly controlled temperatures and humidity levels during storage of dried products. The objective of this study was to determine if the addition of choline acetate to trehalose solution could enable a wider range of storage conditions for preservation of nuclei from fully grown oocytes, by allowing temporary humidity excursions (>44% relative humidity) that may lead to crystallization of trehalose and loss of DNA integrity. Using domestic cat germinal vesicle oocytes as a model, we characterized the recovery as well as the integrity of samples after microwave-assisted dehydration. Exposure to choline acetate alone did not impair the germinal vesicle's DNA integrity and only had a negative impact on the chromatin configuration. Choline acetate addition enabled us to reach lower moisture contents after 25 min of microwave-assisted drying. Sample recovery after rehydration was also better in the presence of choline acetate. The integrity of the germinal vesicle's DNA was not affected, while the chromatin configuration was impaired by the presence of choline acetate during dehydration. Importantly, choline acetate addition helped to maintain an amorphous state (absence of detrimental crystallization) during excursion from ideal humidity conditions.

Chromatin Morphology in Human Germinal Vesicle Oocytes and Their Competence to Mature in Stimulated Cycles.

The search for simple morphological predictors of oocyte quality is an important task for assisted reproduction technologies (ARTs). One such predictor may be the morphology of the oocyte nucleus, called the germinal vesicle (GV), including the level of chromatin aggregation around the atypical nucleolus (ANu)-a peculiar nuclear organelle, formerly referred to as the nucleolus-like body. A prospective cohort study allowed distinguishing three classes of GV oocytes among 135 oocytes retrieved from 64 patients: with a non-surrounded ANu and rare chromatin blocks in the nucleoplasm (Class A), with a complete peri-ANu heterochromatic rim assembling all chromatin (Class C), and intermediate variants (Class B). Comparison of the chromatin state and the ability of oocytes to complete meiosis allowed us to conclude that Class B and C oocytes are more capable of resuming meiosis in vitro and completing the first meiotic division, while Class A oocytes can resume maturation but often stop their development either at metaphase I (MI arrest) or before the onset of GV breakdown (GVBD arrest). In addition, oocytes with a low chromatin condensation demonstrated a high level of aneuploidy during the resumption of meiosis. Considering that the degree of chromatin condensation/compaction can be determined in vivo under a light microscope, this characteristic of the GV can be considered a promising criterion for selecting the best-quality GV oocytes in IVM rescue programs.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

Single-cell multi-omics profiling reveals key regulatory mechanisms that poise germinal vesicle oocytes for maturation in pigs.

The molecular mechanisms controlling the transition from meiotic arrest to meiotic resumption in mammalian oocytes have not been fully elucidated. Single-cell omics technology provides a new opportunity to decipher the early molecular events of oocyte growth in mammals. Here we focused on analyzing oocytes that were collected from antral follicles in different diameters of porcine pubertal ovaries, and used single-cell M&T-seq technology to analyze the nuclear DNA methylome and cytoplasmic transcriptome in parallel for 62 oocytes. 10× Genomics single-cell transcriptomic analyses were also performed to explore the bi-directional cell-cell communications within antral follicles. A new pipeline, methyConcerto, was developed to specifically and comprehensively characterize the methylation profile and allele-specific methylation events for a single-cell methylome. We characterized the gene expressions and DNA methylations of individual oocyte in porcine antral follicle, and both active and inactive gene's bodies displayed high methylation levels, thereby enabled defining two distinct types of oocytes. Although the methylation levels of Type II were higher than that of Type I, Type II contained nearly two times more of cytoplasmic transcripts than Type I. Moreover, the imprinting methylation patterns of Type II were more dramatically divergent than Type I, and the gene expressions and DNA methylations of Type II were more similar with that of MII oocytes. The crosstalk between granulosa cells and Type II oocytes was active, and these observations revealed that Type II was more poised for maturation. We further confirmed Insulin Receptor Substrate-1 in insulin signaling pathway is a key regulator on maturation by in vitro maturation experiments. Our study provides new insights into the regulatory mechanisms between meiotic arrest and meiotic resumption in mammalian oocytes. We also provide a new analytical package for future single-cell methylomics study.

Chromatin Configuration in Diplotene Mouse and Human Oocytes during the Period of Transcriptional Activity Extinction.

In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.

Changing clinical significance of oocyte maturity grades with advancing female age advances precision medicine in IVF

In current IVF practice, metaphase-2 (M2) oocytes are considered most efficient in producing good quality embryos. Maximizing their number at all ages is standard clinical practice, while immature germinal vesicle (GV) oocytes are mostly automatically discarded. We present preliminary evidence that oocyte maturity grades with advancing age significantly change in their abilities to produce good quality embryos, with M2 oocytes significantly declining, GV oocytes improving, and M1 oocytes staying the same. These data contradict the over-40-year-old dogma that oocyte grades functionally do not change with advancing age, supporting potential changes to current IVF practice: (1) Stimulation protocols and timing of oocyte retrieval can be adjusted to a patient's age and ovarian function. (2) In older and younger women with prematurely aging ovaries, GV oocytes may no longer be automatically discarded. (3) In some infertile women, rescue invitro maturation of immature oocytes may delay the need for third-party egg donation.

Melatonin Improves Turbot Oocyte Meiotic Maturation and Antioxidant Capacity, Inhibits Apoptosis-Related Genes mRNAs In Vitro.

High-quality eggs are essential for the sustainability of commercial aquaculture production. Melatonin is a potent candidate for regulating the growth and maturation of oocytes. Therefore, research on the effect of melatonin on marine fish oocytes in vitro has been conducted. The present study successfully established a culture system of turbot (Scophthalmus maximus) oocytes in vitro and investigated the effect of melatonin on oocyte meiotic maturation, antioxidant capacity, and the expression of apoptosis-related genes. The cultures showed that turbot Scophthalmus maximus late-vitellogenic denuded oocytes, with diameters of 0.5-0.7 mm, had a low spontaneous maturation rate and exhibited a sensitive response to 17α, 20β-dihydroxyprogesterone (DHP) treatment in vitro. Melatonin increased by four times the rate of oocyte germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. The mRNA of melatonin receptor 1 (mtnr1) was significantly upregulated in the oocyte and follicle after treatment with melatonin (4.3 × 10-9 M) for 24 h in vitro, whereas melatonin receptor 2 (mtnr2) and melatonin receptor 3 (mtnr3) remained unchanged. In addition, melatonin significantly increased the activities of catalase, glutathione peroxidase, and superoxide dismutase, as well as the levels of glutathione, while decreasing the levels of malondialdehyde and reactive oxygen species (ROS) levels in turbot oocytes and follicles cultures in vitro. p53, caspase3, and bax mRNAs were significantly downregulated in oocytes and follicles, whereas bcl2 mRNAs were significantly upregulated. In conclusion, the use of turbot late-vitellogenesis oocytes (0.5-0.7 mm) is suitable for establishing a culture system in vitro. Melatonin promotes oocyte meiotic maturation and antioxidative capacity and inhibits apoptosis via the p53-bax-bcl2 and caspase-dependent pathways, which have important potential to improve the maturation and quality of oocytes.

Growth hormone reduces aneuploidy and improves oocytes quality by JAK2-MAPK3/1 pathway in aged mice

BackgroundThe global delay in women’s reproductive age has raised concerns about age-related infertility. The decline in oocyte quality is a limiting factor of female fertility, yet there are currently no strategies to preserve oocyte quality in aged women. Here, we investigated the effects of growth hormone (GH) supplementation on aneuploidy of aged oocytes.MethodsFor the in vivo experiments, the aged mice (8-month-old) were intraperitoneally injected with GH daily for 8 weeks. For the in vitro experiments, germinal vesicle oocytes from aged mice were treated with GH during oocyte maturation. The impacts of GH on ovarian reserve before superovulation was evaluated. Oocytes were retrieved to assess oocyte quality, aneuploidy and developmental potential characteristics. Quantitative proteomics analysis was applied to investigate the potential targets of GH in aged oocytes.ResultsIn this study, we demonstrated that GH supplementation in vivo not only alleviated the decline in oocyte number caused by aging, but also improved the quality and developmental potential of aged oocytes. Strikingly, we discovered that GH supplementation reduced aneuploidy in aged oocytes. Mechanically, in addition to improving mitochondrial function, our proteomic analysis indicated that the MAPK3/1 pathway may be involved in the reduction in aneuploidy of aged oocytes, as confirmed both in vivo and in vitro. In addition, JAK2 may also act as a mediator in how GH regulates MAPK3/1.ConclusionsIn conclusion, our research reveals that GH supplementation protects oocytes against aging-related aneuploidy and enhances the quality of aged oocytes, which has clinical significance for aged women undergoing assisted reproduction technology.Graphical

Effects of meiotic stage-specific oocyte vitrification on mouse oocyte quality and developmental competence.

Acquisition of germinal vesicle (GV) stage oocytes for fertility preservation (FP) offers several benefits over in vivo matured oocyte cryopreservation following ovarian stimulation, particularly for cancer patients necessitating immediate treatment. Two FP approaches for GV oocytes are available: vitrification before in vitro maturation (IVM) at the GV stage (GV-VI) or post-IVM at the metaphase II (MII) stage (MII-VI). The optimal method remains to be determined. In this study, mouse oocytes were collected without hormonal stimulation and vitrified either at the GV stage or the MII stage following IVM; non-vitrified in vitro matured MII oocytes served as the control (CON). The oocyte quality and developmental competence were assessed to obtain a better method for immediate FP. No significant differences in IVM and survival rates were observed among the three groups. Nevertheless, GV-VI oocytes exhibited inferior quality, including abnormal spindle arrangement, mitochondrial dysfunction, and early apoptosis, compared to MII-VI and CON oocytes. Oocyte vitrification at the GV stage impacted maternal mRNA degradation during IVM. In addition, the GV-VI group demonstrated significantly lower embryonic developmental competence relative to the MII-VI group. RNA sequencing of 2-cell stage embryos revealed abnormal minor zygotic genome activation in the GV-VI group. Vitrification at the GV stage compromised oocyte quality and reduced developmental competence. Consequently, compared to the GV stage, oocyte vitrification at the MII stage after IVM is more suitable for patients who require immediate FP.

O-205 Poor mitochondrial metabolism impairs meiosis and contributes to reduced oocyte maturation rates in patients with advanced maternal age

Abstract Study question Is there a relationship between poor mitochondrial metabolism and impaired meiotic progression of germinal vesicle (GVs) oocytes in women of advanced maternal age (AMA)? Summary answer Poor mitochondrial metabolism impairs meiotic progression in human oocytes, contributing to a lower oocyte maturation capacity in women of AMA. What is known already High aneuploidy rates and impaired metabolism are characteristic features of AMA oocytes. Studies in mouse suggest that successful meiotic maturation and metabolic competency may be functionally linked, however this relationship has not been clearly established in human. Metabolic dynamics can be visualized by indirect measurements through mitochondrial staining and quantified more directly using Fluorescence Lifetime Imaging Microscopy (FLIM). This live-imaging approach can generate metabolic timelapse profiles of oocytes throughout meiosis. We performed an extensive characterization of oocyte metabolism in maturing GVs, obtained from both young and AMA patients, to establish the importance of mitochondrial metabolism during meiosis. Study design, size, duration A total of 401 GVs from young (≤30 years, n = 264) and AMA (>37 years, n = 137) women were included in the study. GVs were matured in vitro in G2-plus medium for 30 hours. Maturation was determined by the presence of an extruded polar body. Oocytes were used for FLIM (n = 175) and immunofluorescence (IF) analysis (n = 226). Further, mitochondrial metabolism loss of function studies (n = 64) were performed by treating young GVs with 1μM Trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 minutes. Participants/materials, setting, methods The proteins Dihydrolipoamide-S-Acetyltransferase (D-LAT) and Translocase-of-outer mitochondrial-membrane (TOMM20) were analysed in young and AMA oocytes by IF (Arbitrary-Unit, AU) to assess mitochondrial activity and localization, respectively. Fluorescence mean intensities were quantified with ImageJ and compared by t-test; maturation rates were compared by Chi-squared test. FLIM comprehensive metabolism (NADH; FAD+) were taken at GV stage. Different FLIM parameters (fluorescence intensity, fraction bound, short/long lifetime) were evaluated individually and combined into the Redox ratio (NADH intensity/FAD+ intensity). Main results and the role of chance Mitochondrial staining showed a common pattern in young and AMA GV oocytes, with a uniform localization of mitochondria in the ooplasm (TOMM20) and a subcortical localization of active organelles (D-LAT). These patterns were confirmed by in-live FLIM analysis. The total mitochondria abundance was comparable between AMA and young GV oocytes (intensity of 61674±24322 AU in young, 32186±33414 AU in AMA, p = 0.195), however, active mitochondria were diminished in AMA compared to young GVs both by IF and FLIM (intensity of 78614±58534 AU in young, 12517±10187 AU in AMA, p = 0.003; Redox ratio in young 2e+00±0.15, in AMA 1e+00 ± 0.16, p = 2.969e-05). Notably, young oocytes matured at a significantly higher rate (86.3%; 63/73) than AMA oocytes (62.3%; 38/61; p = 0.002). Moreover, FLIM imaging revealed that GVs with a higher metabolism were more likely to complete meiosis to MII (Redox ratio 2e+00±0.17 in GVs matured to MII, 1e+00±0.18 in non-matured, p = 1.054e-06). Following treatment with FCCP, young GVs showed a similar phenotype to AMA oocytes, with a significant decrease in mitochondrial activity (intensity of 78614±58534 IU in untreated and 11554±16131 IU in treated GVs, p = 0.019), and an associated drop-in maturation rate. Ultimately, only 39.5% (17/43) of the young treated GVs were able to accomplish meiotic maturation. Limitations, reasons for caution Maturation rates were assessed by the presence of an extruded PB and variations in spindle assembly timings may have been overlooked. The quantification of mitochondrial activity in loss of function studies was assessed only by IF staining. Wider implications of the findings Our findings demonstrate the presence of a functional link between oocyte metabolism and impaired meiosis, which may contribute to the decline of oocyte quality with age. Given that live-metabolic imaging is able to discern GVs that progress through meiosis, this approach may represent a valuable non-invasive tool in oocyte selection. Trial registration number not applicable

P-118 Are cumulus cells required during in vitro maturation of human oocytes at the germinal vesicle stage? a systematic review

Abstract Study question Does the presence of cumulus cells affect the in vitro maturation (IVM) rate of human oocytes at the germinal vesicle (GV) stage obtained in vivo? Summary answer The oocyte maturation and fertilization rates are higher in the presence of cumulus cells (CC) comparing with their absence in the culture system. What is known already According to the ESHRE guidelines IVM is currently not considered experimental, but nevertheless it is not very often used in reproductive medicine clinics. Accumulated data of more than 30 years of existence of IVM method can present to us how effective is the level of oocytes maturation in vitro obtained by aspiration follicles in vivo. Some studies reported that oocytes were matured after denudation, and in others they are cultured in cumulus oocyte complexes or in the presence of CC. Therefore, a comparison of oocyte maturation at the GV stage with and without CC was performed in this study. Study design, size, duration We searched Medline and Embase up to January 10, 2023. Full texts of classical articles, clinical studies, clinical trials, clinical trial protocols, comparative studies, controlled clinical trials, multicentre studies, randomized controlled trials were included Participants/materials, setting, methods Participants were women undergoing infertility treatment with IVM. All oocytes from the included studies were obtained by follicular aspiration in vivo. Main results and the role of chance A preliminary screening of the word combination “human oocyte in vitro maturation” yielded 355 results, from which 42 were original publications with full-text. Selected papers included a full description of the stimulation protocol, maturation medium, and the group of immature oocytes consisted of gametes only at the GV stage. A total of 24,649 GV oocytes were examined for IVM rate. The average of oocyte maturity was 61.5 ± 10.7%. However, the distribution of oocyte maturation data depending on the presence or absents of CC in the culture media. The maturation rate was higher in oocytes with cumulus cells than in denuded oocytes without cumulus (64.7% vs 53.5%, respectively, p˂0.05). The fertilization rate had the same tendency (69.2% vs. 59.5%, p˂0.05). However, there was no significant difference between the groups in embryo development up to the cleavage stage (74.9% vs 76.5%, p > 0.05). Unfortunately, there are very only a few studies that provide data on the more advanced stages of embryo development as a blastocyst, which does not allow a large sample to be made for any IVM method. Limitations, reasons for caution This study does not take into account the influence of stimulation protocols and the composition of the maturation medium, which may in turn also affect the results of oocyte IVM. Wider implications of the findings The obtained data can be used for comparison with new maturation protocols that will be aimed at increasing the maturation rates, in addition, these data are important for comparing the efficacy of maturation obtained from ovaries outside the body for the purpose of preserving fertility. Trial registration number not applicable

O-206 Growth hormone supplementation reduces aneuploidy and improves the quality of aged oocytes by activating the JAK2-ERK1/2 pathway

Abstract Study question Does growth hormone (GH) supplementation improves the quality of oocytes in aged mice via reducing aneuploidy? Summary answer Daily injections of GH for 8 weeks reduces aneuploidy by activating JAK2-ERK1/2 pathway and improves the quality of oocyte in aged mice. What is known already Aging-induced low-quality oocytes, which are mainly caused by aneuploidy, decrease female fertility. However, few treatments are available to improve the poor quality of oocytes in aged women. Age-related decline in GH levels may be related to the insufficient reproductive potential in aged women. Several studies have reported that GH improves assisted reproductive technology outcomes in poor ovarian response patients, although its role is still controversial. In animal models, GH supplementation improved the oocyte quality by enhancing mitochondrial function. However, whether GH reduces the aneuploidy rate in aged oocytes, and if so the underlying mechanisms, are unclear. Study design, size, duration The aged (8-month-old) C57BL/6J female mice were used in the study. For the in vivo experiments, the aged mice were intraperitoneally injected with GH (GenSci, Changchun, China; 1.6 mg/kg body weight) or an equivalent volume of normal saline for 8 weeks; For the in vitro experiments, germinal vesicle (GV) oocytes from aged mice were treated with GH (200 ng/mL) or PBS in M16 medium. Participants/materials, setting, methods We counted oocytes by immunohistochemistry to assess ovarian reserve. The expression of growth hormone receptor and mitochondrial genes were measured by quantitative real-time PCR. Time–lapse incubator was utilized to record the developmental potential of early embryos. Immunofluorescence was performed to assess parameters of oocyte quality (mitochondrial functions, spindle/chromosome defects, and DNA damage). Chromosome spread and DNA sequencing were used to analyze aneuploidy rate. We performed quantitative proteomics analysis to identify the potential targets of GH. Main results and the role of chance Firstly, we found that GH promoted GHR expression in aged oocytes (30.64±1.70 vs 21.68±1.08, P < 0.001). GH ameliorated aging-induced decline in ovarian reserve of aged mice, with increased ovarian index (0.040%±0.0031 vs 0.023%±0.0019, P < 0.01), and number of antral follicles (8.67±1.2 vs 3.33±0.88, P < 0.05) compared to the controls. In addition, GH improved the quality of aged oocytes, representing restored mitochondrial functions (P < 0.05), decreased DNA damage (23.37±1.76 vs 38.35±2.52, P < 0.001) . As expected, GH promoted the fertilization rate (39.9%±1.10 vs 14.4%±0.62, P < 0.001) and early embryo development (P < 0.01) of aged oocytes. Of note, GH recovered the spindle/chromosome defects (42.6%±2.12 vs 63.9%±1.83, P < 0.01, and reduced aneuploidy rate (20.9%±1.61 vs 39.3%±1.84, P < 0.01) in aged oocytes. GH activated ERK1/2 expression to decrease aneuploidy rate in aged oocytes. In addition, JAK2 might be involved in the regulation of GH on ERK1/2 expression in aged oocytes. Altogether, GH restored age-related decline in oocyte number, and decreased the occurrence rate of aneuploidy by activating JAK2-ERK1/2 pathway, thereby improving the quality of aged oocytes. Limitations, reasons for caution The molecular action mechanism of GH to regulate aneuploidy remains to be determined. Besides, future work should be extended to human oocyte to determine whether this mechanism is conserved between mice and humans Wider implications of the findings Our work expounds a theoretical basis for application of GH to improve the fertility of aged women. Besides, the results also feed new ideas for the prevention and treatment of oocyte quality decline in assisted reproductive technology. Trial registration number not applicable

P-650 Follicular extracellular vesicles of older women inhibit oocyte maturation

Abstract Study question Whether follicular extracellular vesicles of older women interfere with the quality of oocytes. Summary answer OLD-EVs induced the oxidative stress in the oocytes and inhibited oocyte maturation by increasing the abnormal mitochondria distribution and abnormal spindle rates. What is known already Ovary is a critical regulator of female fertility and the source of oocytes. With the modern tendency of delaying childbearing, ovarian aging has become an age-related disease. Ovarian aging is generally characterized by a gradual decrease in both the quantity and quality of oocytes. The pregnancy outcomes of older women were far from satisfying with higher abortion rates, birth defects rates, and lower live birth rates even after the assistance of IVF. However, the detailed mechanisms of decreased oocyte quality of aging women have not been clarified. Study design, size, duration Extracellular vesicles (EVs) were isolated from follicular fluid of older women (aged 40-50, n = 7, OLD-EVs) and younger women (aged 25-30, n = 6, YOUNG-EVs) via ultracentrifugation. Germinal vesicle (GV) oocytes from female ICR mice were co-cultured with OLD-EVs, YOUNG-EVs, or PBS (blank control), respectively. GV breakdown (GVBD) rate and maturation rate were calculated at two-hour and fourteen-hour of co-culture, respectively. Besides, oocyte mitochondria distribution, meiosis spindle morphology, and oxidative status were assessed in different groups. Participants/materials, setting, methods EVs were determined by western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence labeled EVs were used to visualize internalization by oocytes. Oocytes mitochondria and mitosis spindles were stained with fluorescence, and abnormal mitochondria rate or abnormal spindle rate was calculated. Reactive oxygen species (ROS) level was detected in the differently treated oocytes. Moreover, the expression of CAT, GSS, and SOD was determined in the oocytes using quantitative reverse transcription polymerase chain reaction. Main results and the role of chance Both OLD-EVs and YOUNG-EVs are bilayered vesicles, ranging from 100 to 150 nm and enriched in Alix, TSG101, and CD9. EVs could be internalized by oocytes within one hour. After coculture, GVBD rate was similar among the three groups; whereas maturation rate was significantly decreased in the OLD-EV group compared with YOUNG-EV group or PBS group. In addition, the abnormal mitochondria distribution rate or abnormal spindle rate were significantly increased in the OLD-EV group compared with PBS or YOUNG-EV group. The ROS level was increased in the PCOS-EV group compared with YOUNG-EV group, and the expression of CAT, GSS, and SOD was increased in the OLD-EV-treated oocytes. Limitations, reasons for caution Our study did not identify the contents of OLD-EVs and YOUNG-EVs, and the molecular mechanisms of dysregulations induced by OLD-EVs need further researches to investigate. Wider implications of the findings This work confirmed that EV-conducted cellular communication played an important role in oocyte maturation. The dysregulation of oocytes induced by OLD-EVs might be related to the poor oocyte quality of aging women, which provide a novel target to improve pregnancy outcomes of these patients. Trial registration number not applicable

P-710 Single-cell proteomic analysis of human oocytes reveals a decreased abundance of meiosis and proteostasis regulators with advanced maternal age

Abstract Study question How does maternal age affect the proteomic content of her oocytes during their final meiotic progression? Summary answer Compared to young oocytes, oocytes obtained from women of advanced maternal age (AMA) show a decreased abundance of meiosis and proteostasis regulators. What is known already It is well established that AMA leads to a decline in oocyte number and quality, key limiting factors of female reproductive success. Poor oocyte quality is tied to aneuploidies and alterations in cytoplasmic fitness, however the precise mechanisms that underlie oocyte aging remain unknown. Currently knowledge has been largely restricted to transcriptomic studies with no information on the proteome of oocytes obtained from women of AMA. Single-cell proteomic studies may shed light on these mechanisms, as proteins and (post)-translational modifications regulate events unfolding at the oocyte-to-embryo transition in transcriptionally silent oocytes. Study design, size, duration A total of 67 oocytes, obtained from 52 women undergoing controlled ovarian stimulation cycles between May 2021 and May 2022, were included in the study. We obtained 18 fresh germinal vesicle oocytes (GVs) and 17 vitrified in vivo matured metaphase II oocytes (MIIs) from young women (oocyte donors), and 18 GVs and 14 MIIs from AMA. Participants/materials, setting, methods The oocytes were obtained from oocyte donors (n = 27) and AMA patients (n = 25) with a mean age of 24 ± 3.9 years and 39 ± 1.8 years respectively. Single denuded oocytes were snap frozen and their proteomic profiles were generated using the mass spectometry based method plexDIA . Mann-Whitney U test (fold change >|1.5|, adjusted p-value <0.05), Spearman correlation (rs ≥|0.4|, p-value <0.05) and ggplot were used in R to compare the different groups. Main results and the role of chance A total of 2105 proteins were quantified across oocytes stages and age groups. Most of proteins were present in oocytes independently of their maturation state and related to vital processes, such as cellular respiration and energy metabolism. Fifty-four proteins varied in abundance between immature and mature oocytes, including RS14, YBOX2 and RL10A (translational regulation), CLUS and AHSA1 (post-translational modifications) and proteins involved in meiosis progression (WEE2, AURKA, BUB1B, SPDLY). Notably, we observed a negative correlation between protein levels and maternal age for meiosis regulators (e.g.1433E, CDK1; rs: -0.7 and -0.5), meiotic spindle proteins (e.g. DYL2, CAPZB, ARP3, GCP3; rs: -0.6, -0.6, -0.5, -0.5 respectively) and for 5 proteasome subunits (PRS8, PRS6A, PRS10, PSA6, PSMF1; rs: -0.4 to -0.7). The proteasome is a degradation machinery with multiple roles, including meiosis regulation and proteostasis maintenance. Additional proteins of the proteostasis network were found to decline with age (e.g., TCPH, TCPA; rs: -0.5, -0.5). Loss of proteostasis is a common feature of aging and diseases. We also observed a positive correlation between mitochondrial and stress response proteins and maternal age (e.g. ATP5L, TMX1; rs: 0.6, 0.5). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the proteome. Wider implications of the findings We show that a woman’s age correlates with changes in her oocyte proteome, which may contribute to the deterioration of oocyte quality through dysregulation of meiosis and disturbance of proteostasis. Proteins showing differential expression in oocytes from women of AMA, such as proteasome, may become targets for improving oocyte quality. Trial registration number Not applicable

PATL2 regulates mRNA homeostasis in oocytes by interacting with EIF4E and CPEB1.

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

Expression pattern and subcellular localization of p62/SQSTM 1 during mouse oocyte maturation

Objective: Autophagy is a survival mechanism, and it is initiated by several factors such as oxidative stress. Cells give autophagy response against oxidative stress through Nrf2-Keap1-p62 pathway. p62 or Sequestosome 1 (SQSTM 1) which takes place during autophagy, and it is showed that p62 plays critical role during primordial follicle formation. During oocyte maturation, Germinal Vesicle (GV) oocytes resume meiosis with the beginning of puberty and progress through Metaphase I and Metaphase II stages. Metaphase II (MII) oocytes are ready to be fertilized and until fertilization they are arrested at metaphase II stages. It is known that oxidative stress is increasing during oocyte maturation. However, it is not shown that spatial and temporal expression of p62 throughout oocyte maturation. Thus, the aim of the study was to reveal expression pattern and the subcellular localization of the p62 protein in mouse GV, MI and MII oocytes. Methods: GV oocytes were received from female Balb/C mice at 4 weeks aged and GV oocytes were cultured for 8h to develop into MI oocytes and for 14h to mature into MII oocytes. Then, expression of p62 protein was observed in oocytes at GV, MI and MII maturation stages by using immunofluorescence method. Results: Our results showed that there is a significant increasing p62 expression when GV oocytes are compared to MI and MII oocytes, but there is no difference between MI and MII oocytes. Moreover, we revealed that p62 is localized in cytoplasm of GV oocytes, while it is localized around chromosomes in MI and MII oocytes. Conclusion: In conclusion, our results indicate that further study is mandatory to understand the participation of p62 during meiosis, and the impact of p62 during oocyte maturation.

The developmental potential of mature oocytes derived from rescue in vitro maturation

To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue invitro maturation (IVM) compared with their sibling invivo matured oocytes. Retrospective cohort study. IVF clinic. A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. Invitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.