Oocyte Developmental Competence Research Articles

Novel Oocyte-Secreted Factors Improve Mouse IVM Outcomes

Background: In vitro maturation (IVM) is a technology designed to obtain mature oocytes following culture of immature cumulus–oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for patients with excessive AFC or those requiring immediate fertility preservation. However, the clinical uptake of IVM has been slow, primarily due to lower embryo yield and live birth rate relative to IVF, therefore improving IVM culture is required. Aim: To assess whether supplementation of IVM culture medium with the novel in-house engineered TGFβ proteins cumulin and super-GDF9 improves subsequent embryo development. Method: Immature mouse COCs were cultured by standard IVM or bi-phasic IVM ± cumulin or super-GDF9. Following IVM, cumulus expansion was scored and COCs were fertilized, and cultured to assess embryo development. Differential staining was performed on day 6 blastocysts following bi-phasic IVM to assess cell allocation. In a separate experiment, hyperspectral imaging of autofluorescence was carried out on oocytes and cumulus cells following standard IVM ± cumulin to assess the molecular composition of these cells. Results: Both cumulin and super-GDF9 in standard IVM significantly increased cumulus expansion (P<0001; n=104-115 COCs) and blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; P=0.006; n=382-406 oocytes). Hyperspectral imaging showed that oocytes (n=115-158) and cumulus cells (n=532-600) exposed to cumulin during IVM had a distinct spectral profile that varied dramatically (P<0.005) from untreated cells, demonstrating that cumulin has a major impact on the molecular composition of these cells, likely contributing to the improved oocyte quality. In bi-phasic IVM, cumulin did not significantly alter embryo yield (n=387-424 oocytes) or blastocyst cell number or allocation (n=84-112 blastocysts). Conclusion: Cumulin did not provide an additional beneficial effect in bi-phasic IVM, however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence suggesting that their effects in human IVM should be investigated.

Optical coherence microscopy allows for quality assessment of immature mouse oocytes.

Optical coherence microscopy is a label-free and non-invasive imaging technique capable of 3D subcellular structure visualization. Here we show that this method allows for quality assessment of immature mouse oocytes based on their chromatin conformation and can be a valuable addition to the toolkit used in assisted reproduction procedures. The success of assisted reproductive technologies, and particularly in vitro maturation, is tightly linked to the quality of oocytes. Therefore, there is a need for robust, reliable, and easy-to-assess biomarkers of oocyte developmental competence. Microscopy techniques visualizing oocyte intracellular structure could provide such biomarkers. However, fluorescence imaging methods, applied frequently in biology and allowing for detailed structural and dynamic studies of single cells, require fluorescent tags to visualize cellular architecture and may cause short- and long-term photo-damage. On the other hand, traditional light microscopy, although relatively non-invasive, does not provide detailed structural information. Optical coherence microscopy (OCM) is a promising alternative, as it does not require sample pre-processing or labelling and can provide 3D images of intracellular structures. Here we applied OCM to assess the chromatin conformation of immature mouse oocytes, a feature that corresponds with their transcriptional status and developmental competence and cannot be examined by traditional light microscopy. We showed that OCM distinguished oocytes with so-called non-surrounded nucleoli (NSN) and surrounded nucleoli (SN) chromatin conformation with very high sensitivity and specificity and that OCM scanning did not decrease the quality of oocytes. Finally, we cross-referenced OCM data with the oocyte ability to undergo normal nuclear and cytoplasmic maturation and proven that indeed oocytes scored with OCM as NSN mature less effectively than oocytes scored as SN. Our results suggest that OCM may be a valuable addition to the imaging toolkit used in assisted reproduction procedures.

Characterization of sperm cell membrane charge and selection of high-quality sperm using microfluidics in stallions

Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there is no information available about approximate charges or ZP in equine sperm. Therefore, in this study we aimed to characterize equine sperm ZP and identify its associations with known measures of sperm quality. Additionally, we aimed to complete a comprehensive comparison of conventional sperm selection techniques as compared to the novel method of microfluidic sorting. Ejaculates (n = 22) were partitioned into fresh (∼23 °C, 0 h; n = 12) and cooled (∼4 °C, 24 h; n = 10) groups, and processed by swim up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and microfluidic chip (MF) sorting. Motility, progressive motility, cell viability, normal morphology, and ZP were evaluated for both unprocessed fractions and post-selected fractions. The ZP of both fresh and cooled samples was net negative and also correlated with motility and progressive motility for both fresh and cooled samples (P < 0.05). The ZP of cooled samples was also correlated with viability (P < 0.05). Among the compared methods of sperm selection, MF was highly effective in selecting high quality sperm as determined by the measured parameters. Percent motility, progressive motility, normal morphology, and viability of MF selected sperm were of higher quality than sperm selected by SU, and of similar to DG-SU and DGC without the use of potentially harmful centrifugation steps. Correlations between ZP, motility, and viability parameters may indicate a role of external charge on the motility and survival of sperm within the female reproductive tract. In conclusion, we identified an average net negative ZP on equine sperm and correlations between ZP and other measures of sperm quality, as well as having identified MF as a novel effective method of equine sperm selection for IVP.

Optical imaging detects metabolic signatures associated with oocyte quality†.

Oocyte developmental potential is intimately linked to metabolism. Existing approaches to measure metabolism in the cumulus oocyte complex (COC) do not provide information on the separate cumulus and oocyte compartments. Development of an assay that achieves this may lead to an accurate diagnostic for oocyte quality. Optical imaging of the autofluorescent cofactors reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides a spatially resolved indicator of metabolism via the optical redox ratio (FAD/[NAD(P)H + FAD]). This may provide an assessment of oocyte quality. Here, we determined whether the optical redox ratio is a robust methodology for measuring metabolism in the cumulus and oocyte compartments compared with oxygen consumption in the whole COC. We also determined whether optical imaging could detect metabolic differences associated with poor oocyte quality (etomoxir-treated). We used confocal microscopy to measure NAD(P)H and FAD, and extracellular flux to measure oxygen consumption. The optical redox ratio accurately reflected metabolism in the oocyte compartment when compared with oxygen consumption (whole COC). Etomoxir-treated COCs showed significantly lower levels of NAD(P)H and FAD compared to control. We further validated this approach using hyperspectral imaging, which is clinically compatible due to its low energy dose. This confirmed lower NAD(P)H and FAD in etomoxir-treated COCs. When comparing hyperspectral imaged vs non-imaged COCs, subsequent preimplantation development and post-transfer viability were comparable. Collectively, these results demonstrate that label-free optical imaging of metabolic cofactors is a safe and sensitive assay for measuring metabolism and has potential to assess oocyte developmental competence.

Biomechanical forces and signals operating in the ovary during folliculogenesis and their dysregulation: implications for fertility.

Folliculogenesis occurs in the highly dynamic environment of the ovary. Follicle cyclic recruitment, neo-angiogenesis, spatial displacement, follicle atresia and ovulation stand out as major events resulting from the interplay between mechanical forces and molecular signals. Morphological and functional changes to the growing follicle and to the surrounding tissue are required to produce oocytes capable of supporting preimplantation development to the blastocyst stage. This review will summarize the ovarian morphological and functional context that contributes to follicle recruitment, growth and ovulation, as well as to the acquisition of oocyte developmental competence. We will describe the changes occurring during folliculogenesis to the ovarian extracellular matrix (ECM) and to the vasculature, their influence on the mechanical properties of the ovarian tissue, and, in turn, their influence on the regulation of signal transduction. Also, we will outline how their dysregulation might be associated with pathologies such as polycystic ovary syndrome (PCOS), endometriosis or premature ovarian insufficiency (POI). Finally, for each of these three pathologies, we will highlight therapeutic strategies attempting to correct the altered biomechanical context in order to restore fertility. For each area discussed, a systematic bibliographical search was performed, without temporal limits, using PubMed Central, Web of Science and Scopus search engines employing the keywords extracellular matrix, mechanobiology, biomechanics, vasculature, angiogenesis or signalling pathway in combination with: ovary, oogenesis, oocyte, folliculogenesis, ovarian follicle, theca, granulosa, cumulus, follicular fluid, corpus luteum, meiosis, oocyte developmental competence, preimplantation, polycystic ovary syndrome, premature ovarian insufficiency or endometriosis. Through search engines queries, we yielded a total of 37 368 papers that were further selected based on our focus on mammals and, specifically, on rodents, bovine, equine, ovine, primates and human, and also were trimmed around each specific topic of the review. After the elimination of duplicates, this selection process resulted in 628 papers, of which 287 were cited in the manuscript. Among these, 89.2% were published in the past 22 years, while the remaining 8.0%, 2.4% or 0.3% were published during the 1990s, 1980s or before, respectively. During folliculogenesis, changes occur to the ovarian ECM composition and organization that, together with vasculature modelling around the growing follicle, are aimed to sustain its recruitment and growth, and the maturation of the enclosed oocyte. These events define the scenario in which mechanical forces are key to the regulation of cascades of molecular signals. Alterations to this context determine impaired folliculogenesis and decreased oocyte developmental potential, as observed in pathological conditions which are causes of infertility, such as PCOS, endometriosis or POI. The knowledge of these mechanisms and the rules that govern them lay a sound basis to explain how follicles recruitment and growth are modulated, and stimulate insights to develop, in clinical practice, strategies to improve follicular recruitment and oocyte competence, particularly for pathologies like PCOS, endometriosis and POI.

Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus-oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analysed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P = 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase.

Metabolic and antioxidant status during transition is associated with changes in the granulosa cell transcriptome in the preovulatory follicle in high-producing dairy cows at the time of breeding

In this study, we hypothesized that early postpartum (pp) metabolic and oxidative stress conditions in dairy cows (particularly those with severe negative energy balance, NEB) are associated with long-term changes in granulosa cell (GC) functions in the preovulatory follicle at the time of breeding. Blood samples were collected at wk 2 and wk 8 pp from 47 healthy multiparous cows. Follicular fluid (FF) and GC were collected from the preovulatory follicle after estrous synchronization at wk 8. Several metabolic and antioxidant parameters were measured in blood and FF, and their correlations were studied. Subsequently, 27 representative GC samples were selected for RNA sequencing analysis. The GC gene expression data of LH-responsive genes and the estradiol:progesterone ratio in FF were used to identify pre- and post-LH surge cohorts. We compared the transcriptomic profile of subgroups of cows within the highest and lowest quartiles (Q4 vs. Q1) of each parameter, focusing on the pre-LH surge cohort (n = 16, at least 3 in each subgroup). Differentially expressed genes (DEG: adjusted P-value < 0.05, 5% false discovery rate) were determined using DESeq2 analysis and were functionally annotated. Blood and FF β-carotene and vitamin E concentrations at wk 2, but not at wk 8, were associated with the most pronounced transcriptomic differences in the GC, with up to 341 DEG indicative for lower catabolism, increased oxidoreductase activity and signaling cascades that are known to enhance oocyte developmental competence, increased responsiveness to LH, and a higher steroidogenic activity. In contrast, elevated blood NEFA concentrations at wk 2 (and not at wk 8) were associated with a long-term carryover effect detectable in the GC transcriptome at wk 8 (64 DEG). These genes are related to response to lipids and ketones, oxidative stress, and immune responses, which suggests persistent cellular stress and oxidative damage. This effect was more pronounced in cows with antioxidant deficiencies at wk 8 (up to 148 DEG), with more genes involved in oxidative stress-dependent responses, apoptosis, autophagy and catabolic processes, and mitochondrial damage. Interestingly, within the severe NEB cows (high blood NEFA at wk 2), blood antioxidant concentrations (high vs. low) at wk 8 were associated with up to 194 DEG involved in activation of meiosis and other signaling pathways, indicating a better oocyte supportive capacity. This suggests that the cow antioxidant profile at the time of breeding might alleviate, at least in part, the effect of NEB on GC functions. In conclusion, these results provide further evidence that the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on GC functions in preovulatory follicles at the time of breeding. The interplay between the effects of antioxidants and NEFA illustrated here might be useful to develop intervention strategies to minimize the effect of severe NEB on fertility.

Development of children born after in vitro maturation with a prematuration step versus natural conception: a prospective cohort study.

IVM preceded by a prematuration step (capacitation [CAPA]-IVM) improves the acquisition of oocyte developmental competence and can enhance embryo quality. There is currently no follow-up data on babies born from CAPA-IVM. This study investigated developmental outcomes in children born after CAPA-IVM versus natural conception. This prospective cohort study was conducted at a fertility clinic in Vietnam in August/September 2019. Children born after CAPA-IVM were propensity score-matched with those born after natural conception. All parents were asked to complete the Developmental Red Flags and Ages & Stages Third Edition (ASQ-3) questionnaires. A total of 46 parents (23 in each group) of 55 babies (31 CAPA-IVM and 24 natural conception) were included in the study. Baseline characteristics, including mother's age and body mass index, gestational age at delivery, and birth weight, were comparable. The mean age of children at the end of follow-up was 15months. The overall proportion of children with any abnormal ASQ-3 score was 6.5% in the CAPA-IVM group and 20.8% in the natural conception group (p = 0.24). The proportion of children with a developmental red flag did not differ significantly between the CAPA-IVM and natural conception groups (9.7% vs. 4.2%; p = 0.80). The use of CAPA-IVM did not have any significant impact on childhood physical and mental development compared with children born as a result of natural conception.

Preovulatory follicle contributions to oocyte competence in cattle: importance of the ever-evolving intrafollicular environment leading up to the luteinizing hormone surge.

The preovulatory intrafollicular environment plays a major role in determining oocyte competence. The basis of this review is to highlight the importance of the preovulatory follicle's physiological status prior to the preovulatory luteinizing hormone (LH) surge and onset of oocyte maturation to promote an optimal follicular microenvironment and optimal oocyte developmental competence in cattle. While the underlying mechanisms remain unclear, and are likely multifactorial, the preovulatory follicle's physiological status prior to the preovulatory LH surge is highly influential on the oocyte's capacity to undergo postfertilization embryo development. Changes in the intrafollicular environment of the preovulatory follicle including steroid hormone production, metabolome profiles, and proteome profiles likely support the oocyte's developmental and metabolic competency. This review focuses on the relationship between bovine oocyte developmental competency and antral follicle progression to the preovulatory phase, the role of the preovulatory follicle in improving oocyte developmental competence in cattle, and the importance of the ever-evolving preovulatory intrafollicular environment for optimal fertility.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

P-724 Gene expression profiling of cumulus cells from women with adenomyosis disclose differential pattern to endometriosis: striking the chord for evaluation of a biomarker

Abstract Study question Can metabolic picture of cumulus cell (CC) assess assisted reproductive technology outcome/s in women with adenomyosis/endometriosis and have implications on treatment decisions? Summary answer The differentially expressed genes between uterine pathologies explain the functional gap to identify diagnostic/prognostic markers of CC and further dissect molecular contribution to oocyte maturation. What is known already A healthy intra-follicular environment supports achievement of oocyte developmental competence (ODC) largely via coordinated CC-follicular fluid (FF) crosstalk. Lately, promising studies document CC of endometriosis are involved in important pathways for ODC like oxidative phosphorylation, metabolism, mitochondrial function and acetylation to name a few. Similar presentation/s of adenomyosis to other gynecological diseases including endometriosis makes the diagnosis difficult. Given that the functional integrities of CCs are susceptible to concurrent pathological conditions, understanding the influence of CCs on oocyte quality compared to routine morphological analysis in presence of deleterious systemic or pelvic conditions may impact clinical decisions. Study design, size, duration Twenty-eight consented patients undergoing IVF-ICSI between January to December 2021at Institute of Reproductive Medicine, Kolkata were enrolled. CCs obtained and corresponding oocyte maturation was assessed. They were sub-stratified (n = 9) according to uterine pathologies: adenomyosis (Group A), and endometriosis (Group B) as diagnosed by transvaginal ultrasound (TVUS). Counterpart/s of male sub-fertility, defined by World Health Organization criteria (5th edition) was treated as control (Group C; n = 10). CC from mature MII and immature GV oocytes were compared. Participants/materials, setting, methods CCs collected following oocyte-denudation were evaluated by Trypan Blue viability test. Patient age and embryo development were recorded with respect to each cumulus complex. Real Time-quantitative polymerase-chain-reaction (qRTPCR) was performed for glycolysis (PFKP, PKM2, ALDOA, LDHA, TPI1), cholesterol-biosynthesis (CYP51, MVK, PMVK, EBP) and extracellular-matrix (PTGS2, PTX3, TNFAIP6, VCAN) related gene/s known to have important functions in cumulus expansion and metabolism. Patient characteristics and gene expression were correlated by Spearman’s correlation. P &amp;lt; 0.05 was considered statistically significant. Main results and the role of chance A significant higher (p &amp;lt; 0.001) age (A vs B: 35.77±2.33 vs 25.88± 1.76; A vs C: 35.77±2.33 vs 31.9±1.98) and increased number of oocytes retrieved (A vs B: 11.22±1.56 vs 8.55±0.88) was documented in adenomyosis cohort. Immature oocytes from Group A expressed enhanced cholesterol/steroid biosynthesis, with significant (A vs B; p &amp;lt; 0.01) up-regulation of CYP51, MVK, PMVK, EBP in spite of hyper-estrogenic environment in peripheral blood. On contrary, significant attenuation (p &amp;lt; 0.01) of cholesterol biosynthesis was documented in endometriosis cohort compared to control. Expression of PTGS2, PTX3 and VCAN was down-regulated (p &amp;lt; 0.001) (which together mediate cumulus expansion and oocyte maturation, as well as corpus luteum formation and maintenance and reduce spindle abnormalities) in both Group A and B. Expression/s of CC-PFKP and PKM2 was lower in immature compared to mature oocytes (B vs C; p &amp;lt; 0.01) in endometriosis making the duo responsible for follicular/oocyte growth, and embryonic development. This supports the role in promoting cumulus expansion and oocyte competence. Expression levels of other genes including ALDOA, LDHA, TPI1, and TNFAIP6 were not associated with oocyte maturity, and fertilization in either cohort/s. Interestingly, levels of VCAN, TNFAIP6, PTX3 changed significantly as a function of the subject’s age and embryo development. Limitations, reasons for caution Our findings are limited by the relatively small sample size and inherent biological variability of human samples. Several yet known or unknown factors are presumably involved in the implantation process of adenomyosis and/or endometriosis. Multi-centric systematic studies are needed to corroborate these findings and account for the probable confounding factors. Wider implications of the findings A differential metabolic dependence of CC play essential role in oocyte competence in adenomyosis influencing cumulus expansion and oocyte maturation. This would be useful to do further studies to know how molecular interface of implantation capacity with live-birth rate would be affected to complement embryonic morphological assessment/s. Trial registration number Not Applicable

O-206 The impact of oocyte central granularity (CG) on the outcomes of ICSI cycles: live birth achievement is only compromised if CG-derived embryos are transferred

Abstract Study question Does oocyte central granularity (CG) compromise post-ICSI live birth achievement? Are morphologically normal oocytes obtained from CG cycles also affected? Summary answer Oocytes with CG, but not morphologically normal oocytes obtained from cycles providing CG oocytes, present lower competence to achieve a live birth. What is known already Ooplasm central granularity (CG) is associated with alterations in the actin cytoskeleton and meiotic spindle potentially affecting meiotic accuracy, fertilisation dynamics and embryo development. We have recently reported lower fertilisation rates and slower morphokinetics associated with CG. However, to clarify whether CG does affect oocyte developmental competence and if it is associated with endocrine alterations that may also impinge on the performance of morphologically normal oocytes obtained in the same cycle, adequately powered studies assessing the clinical outcomes of cycles transferring embryos derived from CG oocytes or from their morphologically normal companions are needed. Study design, size, duration Retrospective analysis including 866 control ICSI cycles/patients providing only morphologically normal oocytes (NO) and 211 CG cycles/patients providing NO and CG oocytes, from July 2014 to August 2021. Clinical outcomes were compared (1) between cycles providing CG oocytes (CG) or not (Control 1), and (2) among cycles achieving a fresh transfer in which only NO were obtained (Control-T), CG oocytes were obtained but NO-derived embryos were transferred (NOT), and CG-derived embryos were transferred (CGT). Participants/materials, setting, methods Patients undergoing their first ICSI cycle (n = 866; Control) or their first ICSI cycle providing CG oocytes (n = 211; Treatment-groups) were included. Cycle characteristics and outcomes were compared using the Fisher’s exact test and the Wilcoxon sum rank test. Multivariate analyses were performed to assess the potential interference of maternal age, infertility cause, number of embryos transferred and day of embryo transfer on the relationship between CG and live birth. Main results and the role of chance Patients providing CG oocytes (n = 211) presented higher maternal age (37.6± 4.5 vs. 36.5± 4.3; p= p &amp;lt; 0.001), higher basal FSH (8.5± 3.8 vs. 7.9± 4.1 IU/L; p = 0.03), lower AMH (2.2± 2.2 vs. 3.2± 3.5 ng/mL; p &amp;lt; 0.001), higher incidence of ovarian infertility (17.5% vs. 4.9%; p &amp;lt; 0.001) and higher FSH total dose (2255.3± 866.4 vs. 1825.6± 752.3 IU; p &amp;lt; 0.0001), compared to controls (n = 866). Implantation, abortion and live birth rates did not differ between cycles providing (CG) or not (Control) CG oocytes (22.7% vs. 24.9%, 17.1% vs. 27.7%, 25.8% vs. 22.9%, respectively). Nevertheless, implantation and live birth rates were lower in cycles in which CG-derived embryos were transferred (CGT; n = 87) compared to cycles in which CG oocytes were obtained but only NO-derived embryos were transferred (NOT; n = 57), or to cycles in which CG oocytes were not obtained (Control-T; n = 595). Implantation rates were 11.4%, 30.9% and 24.9%, while live birth rates were 10.5%, 30.2% and 22.9% for CGT, NOT and Control-T, respectively (p &amp;lt; 0.001 for implantation rates in CGT vs. NOT and CGT vs. Control-T; p &amp;lt; 0.04 for live birth rates in CGT vs. NOT and CGT vs. Control-T). No significant differences were observed between NOT and Control-T. Limitations, reasons for caution Our study is subjected to the limitations of a retrospective analysis and thus the data may be affected by other variables that were not controlled for. Moreover, the intrinsic difficulty in obtaining a higher number of cycles in which only CG-derived embryos are transferred, limits the power of the analysis. Wider implications of the findings Our findings shed light on a still unsolved question in ICSI practice. The present data indicate that CG is associated with markedly reduced oocyte developmental competence, while the presence of CG oocytes in a cycle does not affect the quality of the companion normal oocytes. Trial registration number not applicable

P-689 Mutated MDR-1 impairs chromosome alignment and reduces oocyte quality

Abstract Study question With this study we aim to assess the link between oocyte mitochondrial physiology and spindle formation with respect to Multidrug Resistance transporter 1 (MDR-1) function. Summary answer Our most recent data suggest that oocyte quality is compromised in young and old Mdr1 mutant mice. What is known already MDR-1 is a transmembrane ATP-dependent effluxer present in different organs that transport a variety of xenobiotics and byproducts from one side of the membrane to the other. Previous findings by Clark et al. 2019 demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Mitochondria are essential for normal oocyte physiology, quality and competence for fertilization and blastocyst development (Chappel, 2013; Dumollard et al., 2007). Therefore, any alteration of their bioenergetics and functions affects the quality of the egg, eventually compromising fertility. Study design, size, duration To carry out the experiments we used Mdr1mutant female mice, expressing an aberrant MDR-1 protein. We focused our analysis on ovaries and oocytes of young (2-3 months) and old (9-12 months) mutant mice, comparing the results with age-matched wild types. Participants/materials, setting, methods In these conditions, we performed ATP quantification and we utilized oocyte in vitro maturation (IVM), immunofluorescence analysis and Ca2+ stimulation analysis. Main results and the role of chance Clark et al. 2019 previously observed that young mutant animals ovulate more eggs compared to wild-types. However, oocytes were fragile and lysed easily and didn’t yield more pups (Clark et al 2019). Conversely, we observed that old Mdr1 mutant mice ovulated less eggs compared to age-matched controls (P &amp;lt; 0.01, n = 4), suggesting diminished ovarian reserve which is associated with poor oocyte quality. Our preliminary data concerning oocyte ATP concentration suggest that mutant germinal vesicle (GV) oocytes present lower ATP levels compared to wild types. Immunofluorescence analysis on metaphase I (MI) oocyte spindle revealed that mutant oocytes present a higher percentage of misaligned chromosomes compared to wild type oocytes (34% and 15%, respectively; P &amp;lt; 0.05, n = 3). Lastly, considering that mitochondria are fundamental for Ca2+ homeostasis, which in turn is necessary for the fertilization process, Ca2+ waves released from mutant vs wild-type metaphase II (MII) oocytes were analyzed using stimulation with strontium, ionomycin, thapsigargin and PLCZ. In all conditions, mutant MII oocytes had altered Ca2+ response (n=∼10 cells per genotype). Limitations, reasons for caution Although these findings demonstrate the importance of MDR-1 for proper maturation and subsequent oocyte development, further studies are necessary to elucidate the specific role and localization of this transporter in the oocyte’s mitochondrial membranes. Wider implications of the findings Our mouse model indicates the importance of mitochondrial homeostasis in infertility. Moreover, our study will provide enhanced understanding of the molecular mechanisms defining oocyte developmental competence, providing insights into human fertility studies. Trial registration number not applicable

P-144 Amphiregulin - a new indicator for zygote vitrification

Abstract Study question Does the concentration of amphiregulin in follicular fluid affect survival rate and embryo quality after the vitrification process? Summary answer The concentration of amphiregulin affects development potential and embryo quality after zygote vitrification and can be used as a predictive biomarker. What is known already Epidermal growth factor receptors and its ligands, such as amphiregulin(AR), which are expressed in female reproductive tissues, have been shown to regulate various important reproductive functions. Amphiregulin concentration in follicular fluid is correlated with human oocyte developmental competence and IVF outcomes. Unlike other countries, Germany has a specific Embryo Protection Act (EPA), where the choice of embryos that have the highest potential for successful implantation must be made at the pronuclear stage(PN); in addition, all supernumerary fertilized oocytes are cryopreserved on the zygote stage. Due to EPA, the determination of potential biomarkers is key for successful IVF outcomes in Germany. Study design, size, duration This study consisted a total of 43 vitrified 2PN stage zygotes from 19 patients who underwent ICSI at UKS Homburg, (Germany) between April 2020 and November 2020. For the study purpose, follicles were aspirated separately, and the follicular fluid (FF) was centrifuged for 5 min at 1,500×g, after that supernatant was removed and stored at -20. Zygote vitrification was performed according to company guidelines (RapidVit Omni, Vitrolife). Participants/materials, setting, methods Concentrations of AR in FF were determined by using a commercially available sandwich ELISA kit (DuoSet Kit; R&amp;D Systems Inc., Minneapolis) in accordance with the manufacturer’s instructions. Good embryo quality on day 3 was defined as a 7-9 cell stage embryo with less than 25% of fragmentation and with equally sized blastomeres. Between-group comparisons were tested using a t-test. Logistic regression analysis was used to determine the influence of amphiregulin on vitrification outcomes. Main results and the role of chance The mean age of all patients was 31.6 (±3.5) years. Patients included in the study were stimulated according to a gonadotropin-releasing hormone (GnRH) antagonist protocol. All zygotes were frozen 20h after injection. Out of the 43 frozen zygotes, 38 (88.3%) survived the thawing process and continued development. Obtained results showed that concentrations of amphiregulin were significantly higher in FF where zygote continued the development [(95.88±37.02 ng/mL vs 79.19±9.92 ng/mL) respectively; p &amp;lt; 0.05]. In addition, 26 (68.4%) vitrified zygotes developed into good-quality embryos, while 17 (31.6%) embryos had poor quality. Data comparisons between good and poor-quality embryos indicated that good-quality embryos came from follicles where amphiregulin concentration was significantly higher [(113.52 ±30.36 ng/mL. vs 63.98 ±1.93 ng/mL) respectively; p &amp;lt; 0.0001]. What is more, logistic regression analysis confirmed the positive effect of amphiregulin concentration on embryo quality (p &amp;lt; 0.01). Limitations, reasons for caution The limitation of the presented study was a relatively small patient cohort. Although the pregnancy rate in the presented study was 36.8 % single embryo transfer was not performed, therefore further research should link amphiregulin concentrations with blastocyst quality and pregnancy rate as well. Wider implications of the findings The literature already confirmed a lower development potential of vitrified zygotes compared to cleaved embryos or blastocyst. Therefore, obtained findings might be helpful for the success of the vitrification process in Germany. Additionally, our findings can be a basis for the future study of amphiregulin as a biomarker. Trial registration number not applicable

P-156 cellular uptake of polystyrene nanoplastics by oocytes and their impact on subsequent embryo development: a preliminary insight using a bovine in vitro model

Abstract Study question Can polystyrene nanoplastics enter cumulus-enclosed oocytes (CEOs) and consequently alter oocyte quality and embryo development? Is this effect particle size-dependent? Summary answer After 24h in vitro exposure, polystyrene-nanoplastics entered the extracellular matrix and cytoplasm of peripheral cumulus cells, but not oocytes. Preimplantation embryo development was not altered. What is known already The ubiquitous use of plastic resulted in extreme global environmental plastic pollution. Chemical and physical processes contribute to the fragmentation of these macroplastic pollutants into heterogenous nanoplastics (NPs). Plastic intake is inevitable for humans by inhalation and ingestion of contaminated air, food (e.g. plastic ingested fish) and drinks (e.g. leakage of plastic bottles). Once imbibed, particles smaller than 150 µm can spread across the entire human body and reach the blood and lymphatic tissue. Due to the neovascularisation associated with an increased vascular permeability during follicular development, nanoparticles might reach the ovarian follicles, potentially affecting oocyte quality and embryo development. Study design, size, duration In a bottom-up approach, using a bovine in vitro oocyte maturation model, CEOs were exposed for 24h to 100µg/mL plain Nile Red-labelled polystyrene-nanoplastics (PS-NP) with average diameter of 50nm (PS-NP-50) or plain Flash Red-labelled PS-NP with average diameter of 200nm (PS-NP-200) (4 replicates using 714 CEOs). CEOs in NP-free control media (CONT) and in-vehicle control (SOLV) were included. Oocytes were in vitro fertilized (for 20h) and presumptive zygotes were cultured until 8 days post-insemination (p.i.). Participants/materials, setting, methods CEOs were fixed after IVM and examined for cumulus cell (CCs) expansion. The presence of NPs was assessed with laser-scanning Leica SP8 confocal microscope. Cleavage (48h p.i.) and blastocyst rates (8 days p.i.) were recorded together with differential staining of trophectoderm (TE, CDX2) and inner cell mass (ICM, Hoechst). The apoptotic-cell-index of the CCs and blastocysts was examined with Caspase-3 immunostaining. Images were quantified using ImageJ. Data were analysed using binary logistic regression and ANOVA. Main results and the role of chance At 24h of IVM, both PS-NP-50 and PS-NP-200 could be detected in the extracellular matrix surrounding the CCs and even within the cytoplasm of a few CCs in the outer layers of the CEOs. No NPs could be detected within the oocytes. Irrespective of the size of the NP’s, no significant differences in cumulus expansion rate or embryo cleavage rate were observed and no difference in apoptotic-cell-index of the CCs could be demonstrated due to a plastic effect (P &amp;gt; 0.1). However, the blastocyst/cleaved rate of PS-NP-50 (56%) was significantly higher than SOLV (36%, p &amp;lt; 0.05). On top, PS-NP-50 showed a lower apoptotic-cell-index (4.11±0.90) of the TE compared to CONT (7.08±0.99, p &amp;lt; 0.1), SOLV (9.09±1.49, p &amp;lt; 0.05) and PS-NP-200 (7.65±1.09, p &amp;lt; 0.05). There is a complete lack of univocal and sound scientific evidence of the actual amount of human plastic intake, making it difficult to determine a physiologically relevant concentration for testing possible effects. However, despite the high plastic concentration used in our model, no deteriorating effects on oocyte quality and embryo development were proven. Limitations, reasons for caution Exposure for only 24h during IVM resulted in a limited timeframe for uptake, consequently exerting its effects. However, long-term in vitro follicle culture models are not as straightforward. Limited reports give insight into the final destination of NPs inside the body, addressing the necessity for in vivo trials as confirmation. Wider implications of the findings While this study indicate that PS-NPs are not toxic for oocyte developmental competence upon direct exposure, this does not rule out the potential impact on e.g. embryo gene expression patterns and epigenetic programming. Future studies should also focus on indirect effects on the oocyte as PS-NP may alter follicular physiology. Trial registration number not applicable

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.

Effect of heat exposure on the growth and developmental competence of bovine oocytes derived from early antral follicles

In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte–cumulus–granulosa complexes (OCGCs) derived from early antral follicles (0.5–1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.

MtDNA content in cumulus cells does not predict development to blastocyst or implantation.

STUDY QUESTIONIs relative mitochondrial DNA (mtDNA) content in cumulus cells (CCs) related to embryo developmental competence in humans and/or the bovine model?SUMMARY ANSWERmtDNA content in CCs provides a poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer.WHAT IS KNOWN ALREADYCCs are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in CCs could be used as a proxy to infer oocyte developmental potentialSTUDY DESIGN, SIZE, DURATIONmtDNA content was analyzed in CCs obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage or oocytes developing to blastocysts.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman CCs samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis.MAIN RESULTS AND THE ROLE OF CHANCEmtDNA content in human and bovine CCs did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding CCs.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThe lack of correlation found between mtDNA content in human CCs and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, mono-ovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable.WIDER IMPLICATIONS OF THE FINDINGSThe use of molecular markers for oocyte developmental potential in CCs could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in CCs is not a good proxy for oocyte quality.STUDY FUNDING/COMPETING INTEREST(S)This research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by the Madrid Region Government. The authors declare no competing interests.

Effects of time-restricted feeding and type of food on fertility competence in female mice

We assessed the effects of feeding regimen (ad libitum vs. time-restricted food access) and type of food (normal chow (NC: 12% fat) vs. moderately high calorie diet (mHCD: 31% fat)) on fertility competence of female mice. Mice fed mHCD had higher number of oocytes than mice fed NC. On the other hand, when mice were fed NC under time-restricted access to food (NT), the developmental rate to the blastocyst per number of normally fertilized ova was significantly decreased compared to others. The reactive oxygen species (ROS) level in oocytes increased in time-restricted food access and NC group. Transcriptome analysis of whole ovarian tissues from these mice showed a change in the cholesterol metabolism among the four groups. Time-restricted food access decreased serum LDL cholesterol level in both NC and mHCD groups. Moreover, the number of atretic follicles increased in NT mice compared to ad libitum food access mice. The present study shows that mHCD feeding increases the number of ovulated oocytes and that time-restricted feeding of NC impairs the developmental competence of oocytes after fertilization, probably due to the changes in serum cholesterol levels and an increase in the ROS content in oocytes.