Germinal Vesicle Oocytes Research Articles

How the Oocyte Nucleolus Is Turned into a Karyosphere: The Role of Heterochromatin and Structural Proteins.

Oocyte meiotic maturation includes large-scale chromatin remodeling as well as cytoskeleton and nuclear envelope rearrangements. This review addresses the dynamics of key cytoskeletal proteins (tubulin, actin, vimentin, and cytokeratins) and nuclear envelope proteins (lamin A/C, lamin B, and the nucleoporin Nup160) in parallel with chromatin reorganization in maturing mouse oocytes. A major feature of this reorganization is the concentration of heterochromatin into a spherical perinucleolar rim called surrounded nucleolus or karyosphere. In early germinal vesicle (GV) oocytes with non-surrounded nucleolus (without karyosphere), lamins and Nup160 are at the nuclear envelope while cytoplasmic cytoskeletal proteins are outside the nucleus. At the beginning of karyosphere formation, lamins and Nup160 follow the heterochromatin relocation assembling a new spherical structure in the GV. In late GV oocytes with surrounded nucleolus (fully formed karyosphere), the nuclear envelope gradually loses its integrity and cytoplasmic cytoskeletal proteins enter the nucleus. At germinal vesicle breakdown, lamin B occupies the karyosphere interior while all the other proteins stay at the karyosphere border or connect to chromatin. In metaphase oocytes, lamin A/C surrounds the spindle, Nup160 localizes to its poles, actin and lamin B are attached to the spindle fibers, and cytoplasmic intermediate filaments associate with both the spindle fibers and the metaphase chromosomes.

Maternal total sleep deprivation causes oxidative stress and mitochondrial dysfunction in oocytes associated with fertility decline in mice.

Previous studies have shown sleep deprivation is increasingly reported as one of the causes of female infertility. However, how and by what relevant mechanisms it affects female fertility remains unclear. In this study, female mice underwent 72 hours of total sleep deprivation (TSD) caused by rotating wheel or 2 different controls: a stationary wheel, or forced movement at night. Even though, there was no significant difference in the number of eggs ovulated by the TSD mice compared to the control groups. Overall levels of estrogen and FSH were lower throughout the estrus cycle. A total of 42 genes showed significant differential expression in GV oocytes after TSD by RNA sequencing (RNA-Seq). These included genes were enriched in gene ontology terms of mitochondrial protein complex, oxidoreductase activity, cell division, cell cycle G1/S phase transition, as well as others. The increased concentrations of reactive oxygen species (ROS) in germinal vesicle (GV) and metaphase II (MII) oocytes from TSD mice were observed, which might be induced by impaired mitochondrial function caused by TSD. The GV oocytes displayed increased mitochondrial DNA (mtDNA) copy number and a significant transient increase in inner mitochondrial membrane potential (Δψm) from the TSD mice probably due to compensatory effect. In contrast, MII oocytes in the TSD group showed a decrease in the mtDNA copy number and a lower Δψm compared with the controls. Furthermore, abnormal distribution of mitochondria in the GV and MII oocytes was also observed in TSD mice, suggesting mitochondrial dysfunction. In addition, abnormal spindle and abnormal arrangement of chromosomes in MII oocytes were markedly increased in the TSD mice compared with the control mice. In conclusion, our results suggest that TSD significantly alters the oocyte transcriptome, contributing to oxidative stress and disrupted mitochondrial function, which then resulted in oocyte defects and impaired early embryo development in female mice.

Comprehensive atlas of mitochondrial distribution and dynamics during oocyte maturation in mouse models

BackgroundOocytes, the largest cells in mammals, harbor numerous mitochondria within their cytoplasm. These highly dynamic organelles are crucial for providing energy resources and serving as central regulators during oogenesis. Mitochondrial dynamics ensure proper energy distribution for various cellular processes involved in oocyte maturation. Previous studies have used alterations in mitochondrial distribution as a biomarker to assess the oocyte health. However, there are discrepancies between studies regarding mitochondrial distribution profiles in healthy oocytes. Consequently, a comprehensive mitochondrial distribution profile in oocytes during maturation has not been fully characterized. Additionally, there is a lack of objective, quantitative methods to evaluate alterations in mitochondrial distribution profiles in oocytes.MethodsThis study aims to provide an in-depth overview of mitochondrial distribution profiles in mouse oocytes at different maturation stages: germinal vesicle (GV) stage, metaphase I (MI), and mature metaphase II (MII). Freshly collected mouse GV, MI and MII oocytes were stained with MitoTracker Red. Confocal microscopy was used to obtain images of mitochondrial distribution profiles in these oocytes. Using the Imaris software, we reconstructed three-dimensional (3D) surface renderings of each oocyte and quantitatively illustrated the mitochondrial distribution profiles.ResultsAt the GV stage, mitochondria in oocytes were evenly distributed throughout the ooplasm. As oocytes progressed to MI and MII stages, mitochondria aggregated and formed clusters, the mean size of mitochondrial clusters and the proportions of clustered mitochondria increased along with the maturation of oocytes.ConclusionsOur findings reveal that mitochondria in mouse oocytes are highly dynamic, undergoing significant reorganizations during oocyte maturation. We for the first time provided comprehensive mitochondrial distribution profiles in mouse oocytes at the GV, MI and MII stages. These mitochondrial distribution profiles were further quantitatively evaluated. Our methods provide an objective and standardized approach for evaluating alterations in mitochondrial dynamics, which can be used as biomarkers to monitor oocyte conditions during maturation.

D4Z4 Hypomethylation in Human Germ Cells.

Expression of the double homeobox 4 (DUX4) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3-22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal (PEG3 and GTL2) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.

Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes Improved The Aged Mouse IVM Oocytes Quality.

During assisted reproductive technology (ART) treatment, the aged women, especially those over 35years old, have fewer mature oocytes and poorer quality of the oocytes comparing with the young women. In vitro maturation (IVM) technology facilitates the usage of immature oocytes, which is clinically important for the aged women. However, the maturation rate is low for the oocytes from the aged women. Human umbilical cord mesenchymal stem cells derived exosomes (HUCMSCs-exosomes), as important mediators of intercellular communication, have been widely used to restore ovarian function and improve female fertility. In this study, we isolated HUCMSCs-exosomes and collected the immature germinal vesicle oocytes from the naturally aged mouse model. And we added these HUCMSCs-exosomes to the conventional IVM culture system. The effects of HUCMSCs-exosomes on IVM oocytes were observed and analyzed from multiple aspects including maturation rate, spindle morphology, mitochondria function, and development potential. We found the quality of oocytes was improved by HUCMSCs-exosomes. Based on the results, we propose that HUCMSCs-exosomes may provide a novel and cell free strategy in the improvement of the IVM in elderly infertile women in the future.

Mature oocyte found during ovarian tissue cryopreservation in an early adolescent female.

A 15-year-old female with Hodgkin's lymphoma underwent ovarian tissue cryopreservation for preserving fertility in Reproductive Department of Sir Run Run Shaw Hospital, Zhejiang University School of Medical after receiving one course of chemotherapy. During the ovarian tissue cryopreservation, one MⅡmature oocyte and three germinal vesicle oocytes were found. The three immature oocytes underwent in vitro maturation but failed. Ultimately, one mature oocyte and 12 ovarian cortex slices were cryopreserved using vitrification. This case indicates that for patients with established gonadal axis feedback, ovarian tissue cryopreservation may not be the only method for fertility preservation. It is advisable to consider ovarian stimulation and oocyte retrieval for oocyte cryopreservation. Alternatively, for individuals in the ovulation phase of their menstrual cycle, attempting oocyte retrieval before ovarian tissue cryopreservation to obtain mature oocytes from the natural cycle, followed by oocyte cryopreservation, may enhance the likelihood of successful fertility preservation.

The regulatory roles of Smad2/3 protein and SMURF2 gene expression in granulosa cells of germinal vesicle and metaphase II oocytes in polycystic ovarian syndrome: A case-control study.

The impaired functions of granulosa cells (GCs) in the delayed development and immaturity of oocytes have been reported in polycystic ovary syndrome (PCOs). Even with ovarian stimulation, a large number of oocytes in these patients are still in the stage germinal vesicle (GV). The levels of Smad2/3, phosphorylated Smad2/3 (P-Smad2/3), the expression of SARA, Smad4, and SMURF2 genes in the GCs surrounding metaphase II (MII) or GV oocytes in PCOs women were investigated. GCs of MII and GV oocytes were isolated from 38 women with PCOs and the expression levels of SARA, Smad4, and SMURF2 in surrounding GCs of MII and GV oocytes were determined using reverse-transcription polymerase chain reaction. Also, Smad2/3 and P-Smad2/3 proteins were determined using western blotting. The expression level of SMURF2 was significantly higher in GCs surrounding GV oocytes compared with that of GCs encompassing MII oocytes (p 0.001). At the same time, no significant differences were observed in SARA and Smad4 expression levels in GCs surrounding GV and MII oocytes. A lower level of P-Smad2/3 was also found in GCs GV oocytes compared with GCs of MII oocytes (p 0.001). It seems that P-Smad2/3 plays a role in oocyte development, and the downregulation of this protein is associated with a defect in the maturation of GV oocytes. On the other hand, the upregulation of the SMURF2 gene also affects the growth process of GCs and the maturation of GV oocytes.

Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 μM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

Improved low-invasive mRNA electroporation method into immature mouse oocytes visualizes protein dynamics during development†.

One of the major causes of oocyte quality deterioration, chromosome segregation abnormalities manifest mainly during meiosis I, which occurs before and during ovulation. However, currently, there is a technical limitation in the introduction of mRNA into premature oocytes without impairing embryonic developmental ability. In this study, we established a low-invasive electroporation (EP) method to introduce mRNA into pre-ovulatory, germinal vesicle (GV) mouse oocytes in an easier manner than the traditional microinjection method. The EP method with an optimized impedance value resulted in the efficient introduction of mRNAs encoding enhanced green fluorescent protein (EGFP) into the GV oocytes surrounded by cumulus cells at a survival rate of 95.0%. Furthermore, the introduction of histone H2B-EGFP mRNA into the GV oocytes labeled most of the oocytes without affecting the blastocyst development rate, indicating the feasibility of the visualization of oocyte chromosomal dynamics that enable us to assay chromosomal integrity in oocyte maturation and cell count in embryonic development. The establishment of this EP method offers extensive assays to select pre-implantation embryos and enables the surveying of essential factors for mammalian oocyte quality determination.

O-005 The in vitro maturation rate of human oocytes is enhanced following uptake of extracellular vesicles derived from mature follicles

Abstract Study question Could follicular fluid derived extracellular vesicles (ffEVs) impact human oocyte in vitro maturation (IVM)? Summary answer Supplementation of IVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by &amp;gt; 20%, inducing changes in oocyte protein profile and organelle distribution. What is known already IVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent aggressive controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid could facilitate oocyte maturation. The uptake of ffEVs by bovine, equine and feline oocytes, but not human, has been demonstrated. Study design, size, duration Women undergoing transvaginal oocyte retrieval after COS (n = 83) at University Hospital Zurich donated follicular fluid (n = 54 single follicles) and/or immature GV oocytes (n = 95). We aimed to: a) define differences in the protein cargo of ffEVs derived from follicles containing mature oocytes (MII-ffEVs, n = 10) versus (vs.) immature oocytes (GV-ffEVS, n = 5; metaphase I MI-ffEVs, n = 5), b) demonstrate the capacity of GV oocytes to uptake mature MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methods ffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n = 15) was assessed by confocal microscopy. GVs were cultured for rescue-IVM in a timelapse incubator with MII-ffEVs (n = 45 GVs) or without (n = 40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance with transmission electron microscopy (TEM). Main results and the role of chance Successful isolation of ffEVs was confirmed by TEM and Western blotting analysis for characteristic protein markers of EVs (TSG101 and CD63). Mass spectrometry identified a total of 1340 proteins across ffEVs samples. Statistical analysis revealed differentially abundant proteins (DAPs) in ffEVs: 61 DAPs in the comparison MII-ffEVs vs. GV-ffEVs, 15 DAPs in MII-ffEVs vs. MI-ffEVs and 14 DAPs in MI-ffEVs vs GV-ffEVs (FDR&amp;lt;0.2). Among the 61 DAPs, 7 proteins in higher abundance in MII-ffEVs (&amp;gt;log2 fold change) were related to oocyte maturation, lipid accumulation, calcium binding and the coagulation cascade (e.g. FABP5, S100A9, F12, SERPINA10). Confocal microscopy confirmed the uptake of MII-ffEVs by GV oocytes after 24 hours of co-incubation. Supplementation of IVM culture with MII-ffEVs for 48 hours significantly increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% mature oocytes respectively; P-value=0.0372). Single oocyte proteomics of IVM-matured oocytes revealed 59 DAPs (FDR 0.1) between ffEVs-supplemented and control groups. Among them, 37 DAPs were in higher abundance in ffEVs-supplemented mature oocytes (&amp;gt;log2 FC) including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). TEM revealed differences in oocyte organelle distribution and appearance, particularly of endoplasmic reticulum (RE) and RE-mitochondria complexes. Limitations, reasons for caution This study utilised immature oocytes from stimulated cycles, therefore these results should be interpreted within the context of rescue-IVM potential. Wider implications of the findings Our findings pave the way for further mechanistic studies on the role of ffEVs in supporting oocyte maturation and inspire the development of clinical IVM supplements as well as novel complementary regimes for COS based on ffEVs or their associated specific cargo. Trial registration number N/A

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p &amp;lt; 0.001), number of litters per female (4.33 vs. 7.50, p &amp;lt; 0.001), and number of pups per female (13.00 vs. 40.67, p &amp;lt; 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p &amp;lt; 0.05), ovulated oocytes (9.81 vs. 20.91, p &amp;lt; 0.001) and 2-cell embryos (3.50 vs. 10.17, p &amp;lt; 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p &amp;lt; 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p &amp;lt; 0.05) and polar body extrusion (42.76% vs. 81.87%, p &amp;lt; 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p &amp;lt; 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p &amp;lt; 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p &amp;lt; 0.001) and the ratio (62.50% vs. 18.67%, p &amp;lt; 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

P-171 Mitochondrial complex I, a key component of the mitochondrial respiratory chain, is necessary for maintaining chromosomal stability during the final stages of oocyte maturation

Abstract Study question What is the role of mitochondrial complex I during the final stages of human oocyte maturation? Summary answer Mitochondrial complex I is necessary for the cytoplasmic maturation of germinal vesicle (GV) oocytes and the subsequent organization of the meiotic spindle. What is known already Mitochondria play a pivotal role in oogenesis. Primordial human oocytes are characterized by an abundance of immature mitochondria, which exhibit low activity and a diminished capacity to produce adenosine triphosphate (ATP). During oocyte recruitment, mitochondrial fusion and fission play an essential role, contributing to oocyte maturation and oocyte developmental competence. Furthermore, an increase in ATP levels during polar body extrusion has been associated with higher fertilization rates, suggesting a reliance on mitochondria as ATP suppliers during oocyte maturation. However, the precise role of mitochondria during the final stages of human oocyte maturation is yet to be understood. Study design, size, duration At total of 99 immature oocytes, including 56 GVs and 40 metaphase I (MI) oocytes, were included in the study. These were obtained from 40 oocyte donation cycles, performed at a single IVF centre between August 2023 and September 2023. Participants/materials, setting, methods Immature denuded oocytes were cultured in G2TM PLUS medium to reach nuclear maturation (rescue in vitro maturation; rIVM). rIVM was performed in the absence (0.1% DMSO, control) or presence of 0.5 µM Rotenone (an inhibitor of mitochondrial complex I) or 10 µM FCCP (an uncoupler of mitochondrial oxidative phosphorylation). To assess mitochondrial and nuclear status, we performed immunofluorescence analyses for TOMM20 and the meiotic spindle (DNA and TUBA). Main results and the role of chance After 40 hours of culture, 64.3% of control GV oocytes progressed to the MII stage, 7.1% reached MI, while 21.4% arrested, and 7.1% degenerated. Control MI oocytes showed a 90% rIVM rate after 24 hours, with none arresting and 10% degenerating. Different mitochondrial inhibitors had varied effects on GV and MI oocytes. FCCP resulted in the degeneration of all treated oocytes. When treated with rotenone, 87.5% of GVs advanced to the MI stage, after which they arrested, but no signs of degeneration were observed. Notably, 100% of MIs and MIIs derived from GVs subjected to rotenone treatment presented with misaligned chromosomes, compared to 33.3% in the control group. When starting from MIs, the rIVM rate decreased to 41.7% following rotenone treatment, with no signs of degeneration. Here, misalignments were observed in 40% of the rIVM-MII oocytes and 43% of those that arrested, compared to 33.3% in controls. These results suggest that both GV and MI oocytes have adequate complex I activity, essential for proper nuclear maturation at the MII stage. We further highlight the importance of complex I activity, especially in GVs, for processes such as polar body extrusion during oocyte maturation. Limitations, reasons for caution Rescue in vitro maturation poses a challenge due to its limited effectiveness in achieving sufficient MII developmental competence. Consequently, mitochondrial inhibitors may have unforeseen effects on mitochondria and spindle formation. The specific role of complex I should be confirmed by employing other complex I inhibitors not related to rotenone. Wider implications of the findings Our results suggest that complex I activity, a key component of the mitochondrial respiratory chain, plays a crucial role in spindle organization during oocyte maturation. This relationship between mitochondrial function and chromosomal stability in oocytes suggests that interventions targeting the mitochondrial respiratory chain may be promising for enhancing oocyte quality. Trial registration number Not applicable

P-501 Superovulation alters telomere length and telomerase expression in mouse oocytes, possibly by changing estradiol and progesterone levels

Abstract Study question How does the single and repeated superovulation affect the telomerase components expression and telomere length in mouse GV and MII oocytes? Summary answer Superovulation significantly altered the expression of the telomerase components Tert and Terc genes, TERT protein levels, and telomere length in the GV and MII oocytes. What is known already Assisted reproductive technologies (ARTs) are commonly used to treat infertility problems. One of the basic ART steps is controlled ovarian stimulation, also known as superovulation. Superovulation can lead to several adverse effects, such as increased oxidative stress, decreased oocyte and embryo quality, and mitochondrial dysfunction. These effects of superovulation largely overlap with those that causing telomere shortening. Study design, size, duration The effect of superovulation on the telomerase expression and telomere length of GV and MII oocytes is not fully addressed yet. In this study, we investigated the potential effects of single and repeated superovulation on telomerase expression and telomere length in mouse oocytes at germinal vesicle (GV) and metaphase II (MII) stages. In addition, we measured the estradiol and progesterone as well as oxidant and antioxidant capacity in serum samples of the oocytes-collected mice. Participants/materials, setting, methods Four groups of female Balb/C mice were formed: Control (C; n = 12), superovulated once (1S; n = 12), superovulated three times (3S; n = 12), and superovulated five times (5S; n = 12) at one-week intervals. The Tert and Terc gene expression, TERT protein levels, and telomere length in the mouse oocytes were assessed by RT-qPCR, immunofluorescence, and q-PCR, respectively. The hormone levels were analyzed by ELISA, and oxidant-antioxidant capacities were measured. All the ovarian follicles were counted in the H&amp;E stained-sections. Main results and the role of chance We found that superovulation (1S, 3S, and 5S) significantly altered expression of the telomerase components Tert and Terc genes, TERT protein levels, and telomere length in the GV and MII oocytes (P&amp;lt;0.05). While estradiol levels in GV oocyte-collected and progesterone levels of GV and MII oocyte-collected mice were higher in the 1S, 3S, and 5S groups compared with the C group (P&amp;lt;0.01), the estradiol levels in MII oocyte-collected mice decreased progressively from C to 5S groups (P&amp;lt;0.01). However, no significant changes were found in the total oxidant status (TOS), antioxidant status (TAS), and oxidative stress index (OSI) between the groups. Also, primordial and atretic follicle numbers in the ovaries of 3S and 5S groups were significantly decreased compared to the control and 1S groups (P&amp;lt;0.05). Limitations, reasons for caution The limitation of this study is that we could not evaluate telomerase activity and oxidative stress levels in the mouse oocytes. Wider implications of the findings All these results suggest that altered telomerase expression and telomere length in GV and MII oocytes, possibly due to changed estradiol and progesterone levels, may be associated with adverse effects of superovulation on oocyte maturation and even preimplantation embryo development. Trial registration number not applicaple

P-141 Elevating human oocyte vitrification and warming to a new level: fast, efficient, and effective

Abstract Study question Due to the rising trend for oocyte cryopreservation and increasing demand, there is an urgency to make oocyte vitrification and warming protocols more efficient. Summary answer Oocyte vitrification and warming can be done successfully in 4 minutes, reducing the time of commonly used current protocols by up to 25 minutes. What is known already Vitrification and warming of oocytes is widely used in many IVF clinics worldwide and routinely utilized as a dominant technique for fertility preservation and freezing of donor as well as autologous oocytes in many clinical settings. Outcome data using cryopreserved oocytes is comparable to fresh oocytes. However, vitrification and warming of human oocytes can be time consuming. Usually, vitrification of oocytes is done in about 11-16 minutes, and warming requires another 11 minutes. Also, it is a common practice to load not more than 2 oocytes per carrier at time of vitrification. Study design, size, duration From November 2022 to November 2023, 504 germinal vesicles (GV) and 207 metaphase I (MI) oocytes, which did not convert into mature oocytes at day of oocyte retrieval, were vitrified in Vit Kit Freeze (FujiFilm Irvine Scientific, USA), and RapidVit Oocyte (Vitrolife, Sweden). Their survival rates were compared, and as an additional viability indicator, post-warming developmental capabilities were evaluated after 24 and 48hrs of culture in Global High Protein (LifeGlobal, USA). Participants/materials, setting, methods A group of 4 GV or MI oocytes was vitrified at room temperature for 1 minute in Equilibration solution (ES) and 1 minute in Vitrification solution (VS), loaded on a S-Cryolock (Biotech, USA) and then plunged into liquid nitrogen. Warming was done at 37ºC in 0.5M sucrose for 1 minute and Wash solution (WS) for another minute. Post warming, GV and MI oocytes were cultured for developmental evaluation. Statistical significance was defined as P &amp;lt; 0.05. Main results and the role of chance There was no statistically significant difference in the survival rate of GV oocytes between Irvine (209/219; 95.4%), and Vitrolife media (270/285; 94.7%; P = 0.72). The survival of MI oocytes was also comparable and not significantly different between both media; Irvine: 103/106; 97.2%, vs Vitrolife: 96/101; 95.0%; P = 0.43). Because of the similar survival rates of oocytes in both groups, for the developmental potential and conversion rates, oocytes of both groups were combined. Of the 504 GV, 479 survived (95.0%) and after 24hrs of culture, 441 converted to MI (92.1%). After a culture of 48hrs, 56.5% (249/441) converted to metaphase II (MII) oocytes. Combining the vitrified and warmed MI oocytes from both groups, 96.1% (199/207) survived the warming, and after 24hrs of culture, 168 (84.4%) converted to MII oocytes. Vitrification and warming of oocytes at different developmental stages can be done with a short exposure time to hypertonic (ES/VS) and hypotonic (0.5M sucrose) solutions in a total of 4 minutes. Limitations, reasons for caution Immature oocytes were used as study material to evaluate a new protocol with significantly reduced vitrification and warming times. At this moment we cannot provide data on fertilization, embryo development, and blastocyst formation. Wider implications of the findings Oocytes are exposed to less suboptimal temperatures during dehydration and rehydration and help to maintain their developmental potential. Loading 4 oocytes per carrier combined with the decrease in time resulted in increased efficiency. Warming in 0.5M instead of 1M sucrose contributes positively to survival and allows earlier resumption of functionality. Trial registration number not applicable

P-526 Identification of mouse cumulus cell-specific miRNAs and their role in the acquisition of the oocyte developmental competence

Abstract Study question Do cumulus cells (CCs), during mouse germinal vesicle (GV)-to-metaphase II (MII) transition, produce miRNAs with a role in the acquisition of the oocyte developmental competence? Summary answer We identified a group of 74 miRNAs with a known role in key processes of oocytes maturation and developmental competence. What is known already We recently developed a co-culture platform whereby CC-free, denuded, GV oocytes (DOs) were matured to MII upon a feeder layer (FL) of CCs derived either from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) oocytes. When cultured upon FL-SN-CCs, DOs displayed a developmental competence equal to that showed by control cumulus-enclosed oocytes (COCs). Instead, when cultured upon FL-NSN-CCs, all DOs arrested at the 2-cell stage. We found that CCs released into the medium large amounts of extracellular vesicles (EVs), whose content analysis by NGS led to the identification of candidate miRNAs. Study design, size, duration Five-eight-week-old CD1 mouse females (Charles River) were injected with 10 I.U. Pregnant Mare Serum Gonadotropin (PMSG). After 48 hr, the ovaries were isolated and their surface punctured with a thin sterile needle to collect fully-grown antral oocytes which were separated from the surrounding CCs. Eight-ten DOs were cultured upon FL-SN-CCs or FL-NSN-CCs for 15 hr in 50 µl exosome-free alpha-MEM Glutamax medium for EVs production. Experiments were performed in triplicates. Participants/materials, setting, methods EVs were collected and processed for EVs characterisation by Amnis-Imaging-flow-cytometer and transmission electron microscopy (TEM), or for RNA extraction (miRNeasy Micro Kit, Qiagen), libraries production (Small RNA-Seq Library Prep kit, Lexogen) and NGS (Illumina Genome Analyzer and the NextSeq 500/550 High Output v2.5). To identify miRNA’s target genes, we used “mirPath v.3” server of the DIANA tools website (https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath). Then, STRING and KEGG were used to highlight the pathways in which these genes are involved. Main results and the role of chance Imaging flow cytometry recorded an amount of released EVs in both FL-SN-CCs or FL-NSN-CCs in the mean range of 5-5.9 x 10(8) particles/ml (p &amp;gt; 0.05), with &amp;gt;96% positive to CD9 exosome surface marker. TEM analysis showed an EVs size in diameter comprised between 18.5-54.9 nm. By comparing FL-SN-CCs vs. FL-NSN-CCs, NGS of EVs’ miRNAs content displayed 74 differentially expressed miRNAs, 43 up- and 31 down-regulated. Of them, 7 resulted involved in the regulation of 71 target genes with a known function in meiosis resumption (N = 24), follicle growth (N = 23), fertilisation (N = 1) and in the acquisition of the oocyte’s developmental competence (N = 23). Limitations, reasons for caution We used an in vitro platform to mimic the cumulus-oocyte-complex environment and, thus, the artificial set-up may differ from that of the natural condition. Also, our hypothesis on the role of these miRNAs, needs to be further confirmed by functional assays (e.g., miRNA inactivation). Wider implications of the findings Overall, our results indicate CC-derived miRNAs as candidates of the CC-to-oocyte bidirectional communication and suggest a group of miRNAs as potential regulative molecules in the acquisition of the oocyte developmental competence. Trial registration number NOT APPLICABLE

P-550 Monoallelic CCNB2 variants may cause oocyte maturation defects and embryo developmental arrest

Abstract Study question Have all the genes that may cause embryo development arrest and oocyte maturation defects been identified in female patients? Summary answer We identified a variant in CCNB2 in our patient. To our knowledge, this is first time that meiotic arrest can be caused by mutated-CCNB2. What is known already Successful reproduction involves the maturation of gametes, fertilization, and early development of the embryo. Oocyte maturation involves a sequence of both morphological and molecular transformations, progressing from germinal vesicle oocytes to metaphase I oocytes and ultimately to metaphase II oocytes. Any disruption in the process of oocyte maturation can result in infertility. With the widespread use of whole exome sequencing (WES), several genes that influence oocyte maturation at different stages have been recently identified. Study design, size, duration We here report the case of repeated ART failure due to oocyte maturation defect and embryo developmental arrest. Participants/materials, setting, methods A 30-year-old female and 38-year-old male applied to our clinic due to unexplained infertility for 4 years. The first ART attempt resulted in 2 of 13 oocytes with late MII. During ART performed in our center, germinal vesicle and meiotic arrest were observed in all oocytes. No mature oocytes were obtained. Her grandfather’s sister was also infertile. WES and Sanger analysis were performed to detect any mutation in known/candidate genes and show familial segregation Main results and the role of chance WES analysis revealed no mutation in genes that were previously reported as a cause of oocyte maturation defect or early embryo development in human. We do a further analysis of genes highly or specifically expressed in oocytes or expressed at low levels yet previous studies have suggested its role in embryo development. We identified a c.787C&amp;gt;T, p.Arg263* heterozygous nonsense variant in the CCNB2 gene that was extremely rare in both multiple databases and our in-house database. Multiple lines of computational evidence support the deleterious effect of the variant on the gene product. This variant was transmitted from her healthy father. CCNNB2 is not expressed in any cells other than oocytes in human. The activity of the M phase-promoting factor (MPF) is crucial for advancing through both mitosis and meiosis. CCNB2 plays a vital role by contributing to the activity of pre-MPF during prophase, necessary for generating enough MPF to proceed through maturation. Furthermore, CCNB2 is significantly involved in mouse oocyte meiosis, playing a key role in both the G2/M and MI/anaphase I transitions. Limitations, reasons for caution New disease-causing variants in this gene should be demonstrated in larger cohorts with female infertility. In addition, further functional studies should be carried out both in CCNB2-mutated human and animals. Wider implications of the findings This study may provide a better understanding background of female infertility and the failure of the ART process. CCNB2 may have a crucial role in the oocyte maturation process and can be utilized as a female infertility genetic marker. Trial registration number NA

P-795 Robust and simple in vitro maturation of ovarian tissue oocytes

Abstract Study question Using in-vitro gametogenesis results in mice, can we perform simple, robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? Summary answer In vitro oogenesis in mice reveals 3 phases of oocyte maturation, IVD, IVG, and IVM. In vivo IVD and IVG simplifes a more robust IVM. What is known already For fertility preservation in cancer patients, IVM of GV’s obtained from ovary tissue with live birth has been achieved, but the results are variable, and great effort has gone into trying to make the “right” culture media, and most are given booster shots of gonadotropin with questionable results. It is easy to obtain many GV oocytes from tiny follicles using open cortical dissection. But it still has remained a puzzle how to do robust IVM in the best way. Study design, size, duration A total of 146 females underwent ovary tissue cryopreservation since 1977. The most recent 38 consecutive such patients between 2 and 38 years of age underwent in-vitro maturation of oocytes (IVM) derived from their ovarian tissue. This is a prospective non-randomized pilot study based upon our results with in vitro oogenesis. Participants/materials, setting, methods After ovarian cortex dissection, the spent media is examined for cumulous complexes (CC’s), which were placed in culture with widely varying concentrations of FSH and HCG, and in a variety of different media. Based on now established mechanisms of in vitro oogenesis, we postulated that the specific media for IVM and the concentrations of HCG would not impact results. There was no prior hormone administration. Main results and the role of chance The harvested germinal vesicle oocyte developed into mature metaphase II oocytes with simple culture media just as well as with “IVM media”, and with HCG concentration from 10 mIU/l to 1000 mIU/l. Of the 38 cases, 18 had had previous chemotherapy or were pre-pubertal. None of these had CC’s. Of the 20 who were post-pubertal and had not yet undergone chemotherapy, the number of CC’s averaged 20 per patient. Of a total of 402 GV oocytes, 137 became mature MII’s (34.1%), varying between 20% and 56%. The only case with low maturation rate (9%) occurred when the cumulus was stripped immediately instead of at 24 hours. Thus mature oocytes can robustly and easily be obtained from ovary tissue with simple media and no need for ovarian stimulation. Furthermore, ovarian stimulation and ovulation are not necessary for oocyte maturation. Oocytes that have just exited IVD that have been exposed to less than 7 days of gonadotropin (IVG) will not mature. Those oocytes exposed to more than 11 days of IVG will degenerate. Thus about 35% of oocytes at any given time will have already been properly prepared in vivo for IVM. Limitations, reasons for caution This is an early proof of concept study in humans with only 30 participants. However, it is completely consistent with what we have predicted from in vitro oogenesis in the mouse. Wider implications of the findings Ovary tissue freezing along with robust IVM will at once provide cancer patients with two possibilities for fertility preservation, without having to delay cancer treatment. Four months of in vivo IVD (non-hormonal) followed by 11 days of IVG (FSH) have already prepared oocytes for IVM. Trial registration number not applicable

Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

Assessment of active translation in cumulus-enclosed and denuded oocytes during standard in vitro maturation and early embryo development.

Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes? Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes. Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis. This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months. All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling. Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633). It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation. Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation. This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.

Role of Menstrual Blood Stem Cell-Derived Secretome, Follicular Fluid, and Melatonin in Oocyte Maturation and Embryo Development in Polycystic Ovary Syndrome.

In vitro maturation has been considered an approach to mature oocytes derived from women with polycystic ovary syndrome (PCOS). It is suggested that the IVM of oocytes may benefit from mesenchymal stem cells derived conditioned medium (CM-MSC). The purpose of this study was to determine the efficacy of a cocktail of menstrual blood stem cell (MenSCs)-derived secretome, along with follicular fluid and melatonin, in oocyte maturation and embryo development in PCOS. Four hundred left germinal vesicle oocytes were collected from 100 PCOS patients and randomly divided into four treatment groups: 1) control, 2) secretome, 3) follicular fluid, and 4) melatonin. Oocyte maturation, fertilization rate, and embryo development were monitored, as well as the expression levels of oocyte-secreted factors (GDF9- BMP15), oocyte maturation (MPK3), and apoptosis (BAX- Bcl2). The rate of oocyte maturation increased in all test groups, but only the results for the SEC group were significant (P= 0.032). There were no significant differences in oocyte fertilization and embryo yield among groups. However, the quality of embryos significantly increased in the melatonin group compared to the control. Cytoplasmic maturation was confirmed by the expression of oocyte maturation-related genes using Real-time PCR. Additionally, the expression level of BCL-2 was significantly higher in the SEC-FF-MEL group than in the control group (p ≤ 0.01). Enrichment of IVM media using MenSCs-secretome, particularly along with melatonin, could be an effective strategy to improve oocyte maturation and embryo development in PCOS.