Oocyte Developmental Potential Research Articles

High-altitude hypoxia exposure alters follicular metabolome and oocyte developmental potential in women.

To explore the effects of high-altitude hypoxia on the microenvironment of oocyte development and fertilization potential, we compared the metabolomic patterns of follicular fluid from women living in different altitude areas and traced their oocyte maturation and subsequent development. A total of 315 clinical cases were collected and divided into three groups according to their residence altitudes: 138 cases in low-altitude (< 2300m) group, 100 cases in middle-altitude (2300-2800m) group and 77 cases in high-altitude (> 2800m) group. The clinical outcomes were statistically estimated, including hormonal level, oocyte maturation, in vitro fertilization, and embryo development. Meanwhile, a metabolomic analysis was performed on the follicular fluid of women from different groups using ultra-high-performance liquid chromatography and high-resolution mass spectrometry and differential metabolites were analyzed through the KEGG pathway. The clinical data indicated that the physical condition and reproductive hormone secretion were similar among different groups. Although personalized gonadotropin-releasing hormone strategies were applied, the numbers of antral follicles and obtained oocytes were not impacted by the residence altitude change. In in vitro culture, the maturing rate, fertility rate and cleavage rate of high-altitude group were compared with the other groups. However, the rates of high-quality embryo, formative blastocyst, and available blastocyst were gradually decreased with the rise of residence altitude. Metabolome analysis identified 1193 metabolites in female follicular fluid. Differential analysis indicated that metabolic components in follicular fluid were remarkably changed with the elevation of residence altitude. These differential metabolites were closely related with amino acid metabolism, protein digestion and absorption, oocyte meiosis and steroid biosynthesis. The residence altitude alters the microenvironment of follicular fluid, which could damage the oocyte developmental potential. This study provides diagnostic basis and therapeutic targets for research on female oocyte and embryo development.

Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca2+, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca2+, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca2+ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca2+ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.

CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform invitro maturation, invitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the invitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

Decoding molecular features of bovine oocyte fate during antral follicle growth via single-cell multi-omics analysis†.

Antral follicle size is a useful predictive marker of the competency of enclosed oocytes for yielding an embryo following in vitro maturation and fertilization. However, the molecular mechanisms underpinning oocyte developmental potential during bovine antral follicle growth are still unclear. Here, we used a modified single-cell multi-omics approach to analyze the transcriptome, DNA methylome, and chromatin accessibility in parallel for oocytes and cumulus cells collected from bovine antral follicles of different sizes. Transcriptome profiling identified three types of oocytes (small, medium, and large) that underwent different developmental trajectories, with large oocytes exhibiting the largest average follicle size and characteristics resembling metaphase-II oocytes. Differential expression analysis and real-time polymerase chain reaction assay showed that most replication-dependent histone genes were highly expressed in large oocytes. The joint analysis of multi-omics data revealed that the transcription of 20 differentially expressed genes in large oocytes was associated with both DNA methylation and chromatin accessibility. In addition, oocyte-cumulus interaction analysis showed that inflammation, DNA damage, and p53 signaling pathways were active in small oocytes, which had the smallest average follicle sizes. We further confirmed that p53 pathway inhibition in the in vitro maturation experiments using oocytes obtained from small antral follicles could improve the quality of oocytes and increased the blastocyte rate after in vitro fertilization and culture. Our work provides new insights into the intricate orchestration of bovine oocyte fate determination during antral folliculogenesis, which is instrumental for optimizing in vitro maturation techniques to optimize oocyte quality.

P-230 Use of a novel in vivo biosensor to rapidly determine oocyte developmental competence

Abstract Study question Can a novel in vivo biosensor (IVOS) rapidly determine oocyte developmental competence? Summary answer Data collected with the IVOS biosensor can be used to determine oocyte developmental competence, observed as blastocyst development after in vitro embryo production. What is known already Methods allowing for rapid, non-invasive assessment of oocyte quality are limited. Subjective morphology is used for gamete selection, but cannot discern between cells with subcellular dysfunction. Metabolism is an accurate method to distinguish cellular quality, but current methods are not rapid, require highly artificial environments, or lack appropriate sensitivity. Extracellular indicators associated with metabolic output may correlate to inherent oocyte developmental potential and serve as a biomarker. A published in vivo biosensor (IVOS) which analyses REDOX homeostasis, an indicator of subcellular function, may be optimised to use at the cellular level to assess oocyte quality. Study design, size, duration Experiments were performed in South Australia using abattoir derived cumulus-oocytes complexes (COC) aspirated from ewe ovaries for in vitro embryo production. COCs were group matured then maintained individually after IVOS analysis to permit identification of oocyte measurements to embryo developmental endpoint. Methodology optimisation and experiments were performed in June 2023 and October 2023, respectively. A holding pipette was modified to permit compatibility with the IVOS system using a proprietary method. Participants/materials, setting, methods Matured COCs were denuded to the corona radiata and placed into an air-buffered medium for IVOS analysis. The holding pipette suctioned COCs prior to excitation (488nm) of extracellular molecules at the COC surface. Emission wavelengths (515-520nm) were retrieved for ten consecutive replicates. COCs were subsequently co-incubated with frozen-thawed ram semen. Presumptive zygotes were cleaned of cumulus cells and loose spermatozoa, cultured for seven days, and stained using Hoechst H33342 to obtain total cell number. Main results and the role of chance Embryos were graded into three categories: 1) did not display a blastocoel cavity (not blastocyst); 2) embryo displaying a blastocoel and total cell count below 50 (early blastocyst); and 3) embryo displaying a blastocoel with a total cell count above 50 (blastocyst). Standard deviation calculations for technical replicates of each COC (n = 32) were used to form an equation capable of distinguishing between groups based on developmental endpoint reached. From this equation, IVOS measurements were lower in oocytes reaching the early blastocyst stage compared to those which reached the blastocyst stage (P &amp;lt; 0.001) or did not reach the blastocyst stage (P = 0.042). Data from oocytes that became full blastocysts were higher than those which did not become blastocysts (P = 0.031). Limitations, reasons for caution The technology used in this study has been tested on sheep oocytes using blastocyst development as a measure of developmental competence. Further research is required to determine if it is applicable to other species or reproductive outcomes such as pregnancy and live birth. Wider implications of the findings This study provides a new technology and optimised methodology to improve the selection of high-quality oocytes among gametes graded homogeneously using subjective morphology. It can be utilised with existing assisted reproductive technologies to possibly increase the success rate of these procedures. Trial registration number N/A

Oocyte developmental potential and embryo production before puberty in cattle.

With the development of in vitro technologies, embryos can be produced using oocytes retrieved directly from the ovaries, i.e., regardless of ovulation. This has allowed the use of different animal categories as oocyte donors, including prepubertal cattle. The advantages of using this strategy to shorten the generation interval and accelerate genetic gain over time were soon recognized, and the first offspring generated using oocytes collected from calves were born in the early 1990s. Nevertheless, embryo production from calves and prepubertal heifers remains a challenge. The oocytes collected before puberty present low in vitro developmental potential, and the subsequent blastocyst rates are consistently lower than those from pubertal females. The acquisition of developmental competence by the oocytes occurs progressively throughout the prepubertal period, which can be subdivided into an early, intermediate, and late prepubertal (or peripubertal) phases, each characterized by different physiological and endocrine features. Therefore, embryo yield increases with age but will only achieve its maximum after puberty. The most common strategy to improve oocyte developmental potential before puberty is the use of gonadotrophic stimulation prior to oocyte retrieval. The results with superstimulation, however, vary among studies, depending on the source, dose, and length of FSH treatment, as well as the age and breed of the donors. The use of calves and prepubertal heifers as oocyte donors should also consider the possible impacts of the oocyte retrieval technique (LOPU or OPU) and the use of exogenous hormones on their subsequent fertility and productive life.

A compact, high-throughput semi-automated embryo vitrification system based on hydrogel

Research questionWhat is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DesignThis comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. ResultsThe Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. ConclusionsThe Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Effect of vitrification on protein O-GlcNAcylation in mouse metaphase II oocytes.

Oocyte vitrification leads to DNA hypomethylation, which results in defect in early embryo development. This study reveals that oocyte vitrification impairs the DNA methylation pattern by influencing protein O-GlcNAcylation. Oocyte vitrification leads to decreased DNA methylation levels, which impairs the quality and the developmental potential of oocytes. However, the underlying molecular mechanism still need to be further revealed. In this study, mouse metaphase II (M II) oocytes were frozen by vitrification technology, while fresh oocytes were used as the control group. The effect of oocyte vitrification on protein O-GlcNAcylation and its impact on the developmental potential of oocytes were elucidated. We found that the protein O-GlcNAcylation levels were significantly increased in vitrified oocytes. Increase of protein O-GlcNAcylation levels in control oocytes by PUGNAc (an O-GlcNAcase inhibitor) decreases blastocyst rate after parthenogenetic activation (20.82% in PUGNAc-treated group; 53.82% in control group, P < 0.05). We also discovered that DNA methylation was disrupted in two-cell embryos derived from vitrified oocytes, resulting in decreased 5mC and increased 5hmC, showing similar phenotypes to that derived from PUGNAc-treated oocytes. In vitrified and PUGNAc-treated oocytes, O-GlcNAcylated TET3 was significantly increased. Notably, by inhibiting protein O-GlcNAcylation in vitrified oocytes using OSMI1 (an O-GlcNAc transferase inhibitor) we restored the DNA methylation in two-cell embryos and ameliorated the developmental defects in early embryo. Thus, elevated protein O-GlcNAcylation in vitrified oocytes is an essential contributor to their declining embryonic developmental potential. Modulation of protein O-GlcNAcylation improves the developmental potential of vitrified oocytes.

FRI382 High Androgen And Progesterone Metabolite Concentrations In Naturally Matured Follicles Are Concurrent With High Serum Anti-Müllerian Hormone Concentrations

Abstract Disclosure: T. Du Toit: None. M. Von Wolff: None. A.C. Swart: None. M. Groessl: None. C.E. Flueck: None. Infertility in women is routinely evaluated with the assessment of circulating steroid levels. Profiling steroid levels in follicular fluid (FF) would provide a focused evaluation of the ovarian endocrine milieu and is therefore better suited to identify key hormones potentially involved in dysfunctional follicular growth. The anti-Müllerian hormone (AMH) level in serum is a well-established clinical marker to estimate the ovarian reserve in assisted reproductive technology, while the AMH level in FF serves as a new prognostic marker for oocyte developmental potential. However, AMH-linked changes in the follicular steroid milieu have not been fully explored. We conducted an AMH-dependent analysis of the FF endocrine milieu. Hormones were quantified using LC-MS (n=42) and ELISA (n=4) in FF of 83 women undergoing natural cycle in vitro fertilisation. Patients were age-matched and classified into 4 statistically different (p&amp;lt;0.0001) groups according to serum AMH levels: group A (&amp;lt;1.1 pmol/L, n=20); group B (1 - 7.14 pmol/L, n=21); group C (7.14 - 35.5 pmol/L, n=21); and group D (&amp;gt;35.5 pmol/L, n=21). Group D was designated as our polycystic ovary syndrome (PCOS)-like group, as high serum AMH is a marker for PCOS. We characterised androgen and progesterone metabolic pathways in FF. Androstenedione (A4) and testosterone (T) levels differed significantly between the groups (p&amp;lt;0.01) and were highest in group D at 60 nmol/L and 4.7 nmol/L, respectively. The levels of 11-ketoandrostenedione (11KA4) and 11β-hydroxyandrosterone (adrenal-origin) also differed between the groups (p&amp;lt;0.05), with higher 11KA4 levels (±2.7-fold) quantified in group D. In parallel with serum AMH, FF AMH differed between the groups (p&amp;lt;0.0001), together with luteinizing hormone/FSH, A4/dehydroepiandrosterone, estradiol(E2)/T and E2/A4 ratios. In comparison to groups A, B and C combined, the PCOS-like group D showed significantly increased downstream progesterone metabolite levels –11α-hydroxyprogesterone [OHP4], 17α,20α-diOHP4, 16α-OHP4 and 5α-pregnanolone, with lower FSH levels. Our results show that the follicular milieu is significantly different when high AMH levels are measured compared to when AMH levels are low, marked by: (i) higher progesterone metabolite levels in follicles and (ii) higher A4 and T levels, concomitant with unchanged E2 levels suggesting that androgens were not fully converted to estrogens in these follicles. Our study furthermore corroborates other studies reporting an intrafollicular hyperandrogenic state in women with increased circulatory AMH levels. In summary, our study not only provides data based on a broad steroid profile that the follicular androgen endocrine milieu is affected in women with high serum AMH levels, but it also provides a model for the premature progesterone increase in PCOS women which leads to a reduction in the embryo implantation rate. Presentation: Friday, June 16, 2023

Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Aristolochic acid I exposure triggers ovarian dysfunction by activating NLRP3 inflammasome and affecting mitochondrial homeostasis.

Aristolochic acids are widely distributed in the plants of Aristolochiaceae family and Asarum species. Aristolochic acid I (AAI) is the most frequent compound of aristolochic acids, which can accumulate in the soil, and then contaminates crops and water and enters the human body. Research has shown that AAI affects the reproductive system. However, the mechanism of AAI's effects on the ovaries at the tissue level still needs to be clarified. In this research, we found AAI exposure reduced the body and ovarian growth in mice, decreased the ovarian coefficient, prevented follicular development, and increased atretic follicles. Further experiments showed that AAI upregulated nuclear factor-κB and tumor necrosis factor-α expression, activated the NOD-like receptor protein 3 inflammasome, and led to ovarian inflammation and fibrosis. AAI also affected mitochondrial complex function and the balance between mitochondrial fusion and division. Metabolomic results also showed ovarian inflammation and mitochondrial dysfunction due to AAI exposure. These disruptions reduced the oocyte developmental potential by forming abnormal microtubule organizing centers and expressing abnormal BubR1 to destroy spindle assembly. In summary, AAI exposure triggers ovarian inflammation and fibrosis, affecting the oocyte developmental potential.

P-791 Supplementation of mitochondria from endometrial mesenchymal stem cells (EnMSCs) improves oocyte quality in aged mice

Abstract Study question To improve the quality of aging oocytes by supplementing the mitochondria from endometrial mesenchymal stem cells (EnMSCs) via microinjection. Summary answer Supplementation with EnMSC-derived mitochondria can improve the quality of oocytes and promote embryo development in aging mice. What is known already Maternal aging is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age-related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aging oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. Study design, size, duration Study design: Eight-month-old ICR mice were purchased and then were further raised to ten months of age for use in the experiments. EnMSCs were stably cultivated and oocytes were extracted from ten-month-old ICR mice. Study size: About 300 GV oocytes and 134 MII oocytes were counted . 80 fertilized oocytes were transplanted into the fallopian tubes of surrogate mothers. Study duraton: About one half a year. Participants/materials, setting, methods Participants:ten-month-old ICR mice. Setting and methods: We cultivated EnMSCs from aged mice and extracted mitochondria from EnMSCs. And then GV oocytes were supplemented with mitochondria via microinjection. To further assess the effects of mitochondrial supplementation on embryo development, MII oocytes from aging mice were fertilized by ICSI combining EnMSCs mitochondria microinjection.To test whether mitochondrial supplementation could improve in vivo embryo development in aging mice, embryo transplantation was performed after mitochondrial microinjection combined with ICSI. Main results and the role of chance In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aging oocytes.Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aging mice after intracytoplasmic sperm injection (ICSI). We also found that the birth rate of mitochondria-injected aging oocytes was significantly increased after embryo transplantation. Limitations, reasons for caution Microinjection into oocytes was invasive and some oocytes died after microinjection. The ability of this method to improve oocyte quality was limited because the MII rate of oocytes after mitochondria supplementation was 35.65±1.75% while the rate of oocytes from 6–8 weeks of mice in a previous study was nearly 80%. Wider implications of the findings For clinical treatment, the EnMSCs can be obtained from the patient's own menstrual blood as the sources of mitochondrial supplementation. A noninvasive procedure for cell extraction ensures safety, and the findings of this work might provide a valuable strategy for patients with declining oocyte quality due to aging. Trial registration number Not applicable

P-406 Metal-phenolic network cell-membrane modification-mediated assembly strategy for re-establishing oocyte-granulosa cell communications and improving oocyte developmental potential

Abstract Study question Re-establish the cell-cell communication of isolated oocyte and granulosa cells (GCs) to mimic the follicle microenvironment and promote the success rate of in-vitro maturation (IVM). Summary answer The Metal-phenolic network (MPN) nanoparticles-mediated cell membrane modification system was used to achieve self-assembly of GCs on the oocyte membrane and re-establishing the Oocyte-GCs communication. What is known already How to preserve the fertility of female patients of childbearing age is an important problem faced by reproductive medicine. IVM of oocytes is an important means for female fertility preservation, but the problem of oocyte maturation rate needs to be solved urgently. The Oocyte-GCs interaction determines the key events in the oocyte development and maturation. Biological materials are gradually used for IVM, but there are still significant disadvantages. MPN has been widely used for the functionalization of bacteria and cell surface due to its unique nano-network structure and good biocompatibility. Study design, size, duration The MPN nanoparticles are respectively modified on GCs and oocytes, and the GCs can be self-assembled on the surface of the oocytes to re-establish cell communication. Then explore the biosafety and cytotoxicity of MPN nanoparticles on GCs and oocytes to rule out potential impact on the secretory function of GCs, reveal the formation of gap junction and substance exchange of Oocyte-GCs, and evaluate the effects of this technology on oocyte maturation, fertilization and embryo development. Participants/materials, setting, methods The oocyte-GCs assembly could be achieved through the MPN(EGCG-Zn2+). Then MPN@GCs and MPN@Oocytes were characterized by fluorescent protein, Zeta, SEM and TEM; the cytotoxicity was assessed by Live/Dead staining, CCK-8 and flow cytometry assays. Gap junction was observed by TEM; the expression levels changes of signaling pathways for assembled oocyte and GCs were detected by single-cell RNA-seq, RT-qPCR and WB. The oocyte and embryo developmental potential were also analyzed. Main results and the role of chance We confirmed the formation of a nano-network surrounding GCs and oocytes and achieved the assembly of GCs on the surface of oocytes. Furthermore, we illustrated that MPN was non- cytotoxicity for GCs and oocytes and have no negative effect on the secretion function of GCs, indicating high biocompatibility of MPN and shows the potential application on the re-establishment of oocyte-GCs communication. Gap junctions formation and substance exchange establishment between GCs and oocytes were also proved in the study. The single-cell RNA-seq uncovered key pathways involved in oocyte-GCs interactions after the oocyte-GCs assembly. In detail, the SUMO1 ubiquitination modification of PTEN was inhibited after re-establishing cell-cell communication and decreased the localization of PTEN at the GCs membrane, which weakened its inhibition on PI3K. Higher PI3K level further up-regulated AKT level and stimulated its activation by increasing phosphorylation modification, finally improved the developmental potential and maturation rate of oocytes. The re-establishment of cell communication significantly down-regulated the P38/MAPK pathway in oocytes, including inhibited P38/MAPK expression, thereby reducing the phosphorylation of MSK1 and the activation of ATF-1, and effectively weakened the apoptosis controlled by P38/MAPK. Finally, a significantly higher fertilization and blastocyst formation rate were observed as compared to the control group. Limitations, reasons for caution Although this study has achieved promising results, there is still gap between basic research and applications, and more fundamental research should be conducted to make it a powerful tool for clinical application in the future. Wider implications of the findings This study not only provided a promising tool for establishing oocyte-GCs communication and improving oocyte maturation rate for IVM, but also showed a versatile and powerful cell-cell assembly platform for regenerative medicine including tissue engineering and organoid studies. Trial registration number not applicable

O-208 Maternal spindle transfer restores the developmental competence of in vitro aged oocytes with diminished metabolic activity identified by hyperspectral imaging

Abstract Study question Can the developmental competence of poor-quality oocytes identified by hyperspectral imaging be restored by maternal spindle transfer? Summary answer Maternal spindle transfer can be efficiently assisted by hyperspectral imaging to identify and subsequently repair the developmental competence of poor-quality oocytes. What is known already Oocyte cytoplasmic dysfunctions (including, but not limited to the mitochondria) are a major cause for poor embryonic development. Given that maternal spindle transfer (MST) allows the replacement of the entire cytoplasm of an oocyte, it is a promising method for the enhancement of oocyte developmental competence. However, accurate diagnostic tools for oocyte quality assessment are lacking. Recently, a novel hyperspectral imaging approach has been shown to classify oocytes non-invasively based on their metabolic profile, which could assist in the evaluation of oocyte developmental potential prior to MST. Study design, size, duration Oocytes were obtained from young female mice and analyzed blindly by hyperspectral imaging either immediately after collection (fresh group, n = 122), after overnight culture (in-vitro aged group, n = 58) or on a group of MST oocytes (n = 36), where the meiotic spindle from in-vitro aged oocytes was transferred into enucleated fresh oocytes. Oocytes were inseminated by ICSI and cultured in-vitro individually. Correlations between the oocyte’s metabolic profiles and rate of development to the blastocyst stage were performed. Participants/materials, setting, methods Oocytes were collected from 6-10 weeks-old superovulated B6CBAF1 females. MST was performed in manipulation medium supplemented with cytochalasin B. After enucleation, karyoplasts were exposed to an inactivated Sendai virus solution to promote membrane fusion. ICSI was performed using a Piezo-Drill actuator. Hyperspectral imaging was done in the near-infra-red regime to avoid photoxicity. Image processing was done using dimensionality reduction, which was then feed to an Akaike Information Criterion algorithm to classify the different metabolic subpopulations. Main results and the role of chance Fresh oocytes showed metabolic profiles that significantly differed from the profiles of in-vitro aged oocytes, which were characterized by a pronounced diminished metabolic activity. While in the fresh group, 67% of the oocytes developed into expanded blastocysts, none of the in-vitro aged oocytes with low metabolism produced a blastocyst (p &amp;lt; 1x10-05). Interestingly, MST oocytes that were imaged 1h after nuclear reconstruction, showed a restored metabolic spectrum that resembled the profile from fresh non-manipulated controls. This restoration of the metabolic activity was concomitantly associated with an enhancement of developmental competence, with expanded blastocyst rates (63%) comparable to those of fresh non-manipulated controls (p = 0.668). Blastocyst developmental rates of fresh oocytes after hyperspectral imaging were comparable to oocytes from the same cohort that were not imaged and used as controls. Limitations, reasons for caution The metabolic profiles obtained from these experimental groups need to be correlated with implantation rates in mouse. The impaired embryo developmental pattern characteristic of poor-quality oocytes may not be exclusively attributed to metabolic deficiencies, so caution must be exerted when generalizing these results to oocytes with other cytoplasmic dysfunctions. Wider implications of the findings MST experiments confirm that the developmental decline of these oocytes is primarily attributed to abnormal function of cytoplasmic factors involved in metabolism, while the nuclear genome remains developmentally competent. MST can benefit from hyperspectral imaging to identify oocytes with impaired developmental potential within a cohort. Trial registration number not applicable

In vitro derived embryos produced from mares treated with intravenous clomiphene citrate

Optimizing the number of follicles available for ovum pick-up and in vitro embryo production (OPU-IVP) would increase the efficiency of OPU-IVP sessions. In other species, clomiphene citrate (CC) has been credited with enhancing follicular recruitment through augmented GnRH pulsatility. Our goal was to describe the number of follicles, reproductive hormone secretion patterns, and oocyte developmental potential in mares treated with CC and subjected to OPU-IVP. Six light-breed mares underwent follicle ablation (Day 0) to induce a new follicular wave, followed by administration of 250mcg of cloprostenol (PGF2-α) on Day 6. On Days 10-14, a five-day protocol of 0.5% intravenous CC (Annaji.et.al. AAPS PharmSciTech. 2023; 24:48) was administered at a rate of 2grams/mare/once daily; all mares subsequently underwent OPU on Day 17. Collected oocytes were shipped counter-to-counter to a laboratory for fertilization via intracytoplasmic sperm injection (ICSI). Ovarian follicle counts were performed via transrectal ultrasonography immediately prior to each daily administration of CC (Days 10-14) and on the day of OPU (Day 17); the total number of follicles, in addition to the diameter of the largest follicle (mm) were recorded. Serum samples were collected to quantify and characterize systemic reproductive hormone patterns (i.e., LH, FSH, AMH, and P4); sample collection started on the day of ablation (Day 0) and concluded one week following OPU (Day 24).CORR and PRINQUAL procedures of SAS version 9.4 (SAS Institute, Cary, North Carolina) were utilized to evaluate correlation amongst hormones. The development rate of embryos originating from CC treated mares were compared to development rates of commercial embryos produced during the same time period at the ICSI laboratory. Parametric (T-test) and nonparametric (NPAR1WAY) procedures (SAS) were utilized to compare embryo development rates. For all statistical analyses, significance level was set at p≤0.05. Upon completion of CC treatment, the mean (+/- SEM) diameter of the largest follicle and the mean (+/- SEM) total number of follicles was 40 mm (+/- 5.1 mm) and 11.3 (+/- 1.2), respectively. Significant negative correlations were found between LH and FSH (p=0.007), FSH and the largest follicle (p<0.001), and FSH and total follicle number (p=0.01). Conversely, significant positive correlations were found between LH and the largest follicle (p=0.003), AMH and total follicle number (p=0.01), and AMH and P4 (p=0.008). The overall embryo development rate was 50% for CC treated mares (15/30 injected oocytes) and 36.8% (14/38 injected oocytes) for commercial embryos (p=0.56). Although experimental control mares were not utilized in this pilot study, our results warrant continued investigation to identify the impact(s) of intravenous CC administration on follicular dynamics, OPU-IVP and foal production.

The expression levels of NOS2, HMOX1, and VEGFC in cumulus cells are markers of oocyte maturation and fertilization rate.

Throughout the reproductive life of women, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment, and act as a gatekeeper for oocyte developmental potential. One such mechanism relies on the hypoxia-tolerance response, which, with age, decreases systematically, including in the ovary. We aimed to evaluate the association between gene expression related to hypoxia and aging in CC and reproductive results in in vitro fertilization cycles. We recruited 94 women undergoing controlled ovarian stimulation. Total RNA was extracted from pooled CCs collected after oocyte pick-up (OPU) and reverse-transcribed to complementary DNA using random hexamers to test 14 genes related to hypoxia response via HIF1α activation, oxidative stress, and angiogenic responses. The expression of CLU, NOS2, and TXNIP had a positive correlation with age (rs = 0.25, rs = 0.24, and rs = 0.35, respectively). Additionally, NOS2 and HMOX1 expression correlated positively with the retrieval of immature oocytes (rs = 0.22 and rs = 0.40, respectively). Moreover, VEGFC levels decreased overall with increasing fertilization rate, independently of age (rs = -0.29). We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia with age, pointing at NOS2, HMOX1, and VEGFC expression as markers for oocyte maturation and fertilization success.

General anesthesia with propofol during oocyte retrieval and in vitro fertilization outcomes: retrospective cohort study

General anesthesia is frequently administered during oocyte retrieval. Its effects on the outcomes of IVF cycles are uncertain. This study investigated whether administration of general anesthesia (specifically propofol) during oocyte retrieval affects IVF outcomes. A total of 245 women undergoing IVF cycles were included in this retrospective cohort study. IVF outcomes of 129 women who underwent oocyte retrieval under propofol anesthesia and 116 without anesthesia were compared. Data were adjusted for age, BMI, estradiol on triggering day and total gonadotropin dose. The primary outcomes were fertilization, pregnancy and live birth rates. A secondary outcome was the efficiency of follicle retrieval associated with the use of anesthesia. Fertilization rate was lower in retrievals under anesthesia compared to without (53.4% ± 34.8 vs. 63.7% ± 33.6, respectively; p = 0.02). There was no significant difference in the ratio of expected to retrieved oocytes between retrievals with and without anesthesia (0.8 ± 0.4 vs. 0.8 ± 0.8, respectively, p = 0.96). The differences in pregnancy and live birth rates between the groups were not statistically significant. General anesthesia administered during oocyte retrieval may have adverse effects on the fertilization potential of oocytes. This impact on the developmental potential of oocytes may lead to negative IVF outcomes and should be investigated further.

040 Excessive FSH doses during superovulation of small ovarian reserve heifers induce premature cumulus expansion and diminish oocyte developmental potential

040 Excessive FSH doses during superovulation of small ovarian reserve heifers induce premature cumulus expansion and diminish oocyte developmental potential

Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation.

In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), and (3) oocytes vitrified immature, warmed, and in vitro matured (VIM). In comparison with fresh oocytes, VIM resulted in 46 differentially expressed (DE) genes (14 upregulated and 32 downregulated), while VMAT showed 36 DE genes (18 in each category). A comparison of VIM vs. VMAT resulted in 44 DE genes (20 upregulated and 24 downregulated). Pathway analyses highlighted cytoskeleton, spindle formation, and calcium and cation ion transport and homeostasis as the main affected pathways in vitrified oocytes. The vitrification of in vitro matured oocytes presented subtle advantages in terms of the mRNA profile over the vitrification of immature oocytes. Therefore, this study provides a new perspective for understanding the impact of vitrification on equine oocytes and can be the basis for further improvements in the efficiency of equine oocyte vitrification.

Cobalt chloride exposure disturbs spindle assembly and decreases mouse oocyte development potential

Cobalt is a kind of heavy metal which is widely used in petrochemical and biomedical industries. Animal studies have reported that cobalt would exert systemic toxicity, but its effects on the ovarian function in mammals, especially for oocyte quality remains unknown. In the present study, we report that cobalt chloride treatment affects ovary coefficient and follicular growth. Oocytes in cobalt chloride exposed mice exhibited a decreased development potential, with the evidence of decreased occurrence rate of germ vesicle breakdown and polar body extrusion. Besides, cobalt chloride disorganized meiotic spindle formation and movement, mechanically associated with affecting TACC3 and Ac-a-tubulin levels, and disturbing actin reorganization. In addition, cobalt chloride exposure result in mitochondrial cristae structures disappear, cluster distribution and potential depolarization. Altogether, these findings suggest that cobalt chloride impairs the ovarian follicle growth and affects oocyte development by disrupted spindle assembly and mitochondrial function.