The development of an effective program that combines in vitro maturation (IVM) and cryopreservation for immature oocytes would represent a novel advance for in vitro fertilization (IVF), especially as a means to preserve the fertility of women in unique situations. The aim of this study was to analyze the ultrastructural characteristics of human oocytes, obtained after controlled ovarian stimulation, to determine whether IVM is best performed before or after vitrification. To this end, we analyzed the following features in a total of 22 MII oocytes: size, zona pellucida and perivitelline space, mitochondria number, M-SER (mitochondria-smooth endoplasmic reticulum) aggregates and M-V (mitochondria-vesicle) complexes, the number of cortical granules and microvilli, and the presence of vacuolization using transmission electron microscopy (TEM). Each oocyte presented a rounded shape, with an intact oolemma, and was surrounded by a continuous zona pellucida and perivitelline space. Statistical analysis comparing oocytes vitrified before or after IVM indicated that there were no significant differences between examined characteristics.
STUDY QUESTION Are transcriptomic profiles altered in ovarian granulosa cells (GCs) and peripheral blood mononuclear cells (PBMNCs) of women with polycystic ovary syndrome (PCOS) compared to young poor responders (YPR) and women with normal response to ovarian stimulation? SUMMARY ANSWER RNA expression profiles in ovarian GCs and PBMNCs were significantly altered in patients with PCOS compared with normoresponder controls (CONT) and YPR. WHAT IS KNOWN ALREADY PCOS is characterised by a higher number of follicles at all developmental stages. During controlled ovarian hyperstimulation, PCOS women develop a larger number of follicles as a result of an exacerbated response, with an increased risk of ovarian hyperstimulation syndrome. Despite the number of developing follicles, they are often heterogeneous in both size and maturation stage, with compromised quality and retrieval of immature oocytes. Women with PCOS appear to have a longer reproductive lifespan, with a slightly higher menopausal age than the general population, in addition to having a higher antral follicular count. As a result, the ovarian follicular dynamics appear to differ significantly from those observed in women with poor ovarian response (POR) or diminished ovarian reserve. STUDY DESIGN, SIZE, DURATION Transcriptomic profiling with RNA-sequencing and validation using quantitative reverse transcription PCR (qRT-PCR). Women with PCOS (N = 20), YPR (N = 20) and CONT (N = 20). Five patients for each group were used for sequencing and 15 samples per group were used for validation. PARTICIPANTS/MATERIALS, SETTING, METHODS PCOS was defined using the revised Rotterdam diagnostic criteria for PCOS. The YPR group included women <35 years old with <4 mature follicles (at least 15 mm) on the day of the trigger. According to internal data, this group represented the bottom 15th percentile of patients' responses in this age group. It was consistent with Patient-Oriented Strategies Encompassing Individualize D Oocyte Number (POSEIDON) criteria for POR (Group 3). The young CONT group included women <35 years without PCOS or anovulation, who developed >14 mature follicles (at least 15 mm on transvaginal ultrasound). According to internal data, a threshold of >14 mature follicles was established to represent the top 25% of patients in this age group in this clinic. Overall, n = 60 GCs and PBMNCs samples were collected and processed for total RNA extraction. To define the transcriptomic cargo of GCs and PBMNCs, RNA-seq libraries were successfully prepared from samples and analysed by RNA-seq analysis. Differential gene expression analysis was used to compare RNA-seq results between different groups of samples. Ingenuity pathway analysis was used to perform Gene Ontology and pathways analyses. MAIN RESULTS AND THE ROLE OF CHANCE In PBMNCs of PCOS, there were 65 differentially expressed genes (DEGs) compared to CONT, and 16 compared to YPR. In GCs of PCOS, 4 genes showed decreased expression compared to CONT, while 58 genes were differentially expressed compared to YPR. qRT-PCR analysis confirmed the findings of the RNA-seq. The functional enrichment analysis performed revealed that DEGs in GCs of PCOS compared to CONT and YPR were prevalently involved in protein ubiquitination, oxidative phosphorylation, mitochondrial dysfunction and sirtuin signaling pathways. LARGE SCALE DATA The data used in this study is partially available at Gene Ontology database. LIMITATIONS, REASONS FOR CAUTION The analysis in PBMNCs could be uninformative due to inter-individual variability among patients in the same study groups. Despite the fact that we considered this was the best approach for our study's novel, exploratory nature. WIDER IMPLICATIONS OF THE FINDINGS RNA expression profiles in ovarian GCs and PBMNCs were altered in patients with PCOS compared with CONT and YPR. GCs of PCOS patients showed altered expression of several genes involved in oxidative phosphorylation, mitochondrial function and sirtuin signaling pathways. This is the first study to show that the transcriptomic landscape in GCs is altered in PCOS compared to CONT and YPR. STUDY FUNDING/COMPETING INTEREST(S) This study was partially supported by grant PI18/00322 from Instituto de Salud Carlos III, and European Regional Development Fund (FEDER), ‘A way to make Europe’ awarded to S.H. M.C., S.H., S.T., L.R., M.R., I.R., A.P. and R.C. declare no conflict of interests concerning this research. E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence. TRIAL REGISTRATION NUMBER N/A.
The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration.
Study question Can the Well-of-the-Well system (WOW), applied on denuded oocytes, improve germinal vesicle breakdown (GVBD) and maturation rate? Summary answer In vitro maturation (IVM) of denuded germinal vesicle (GV) oocyte using WOW culture system increases nuclear maturation competence when compared with droplet conventional culture What is known already Further research remains necessary to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of in vitro matured oocytes. Several studies on bovine oocytes have shown that oocyte-secreted factors (an uncharacterized mix of growth factors secreted by the oocyte) enhance oocyte developmental competence during in vitro maturation. These oocyte-secreted factors may accumulate at the bottom of the micro-well, as suggested for the WOW culture system. Previous reports suggested that diffusible factors secreted by individual oocytes probably accumulated in a micro-well WOW dish, may provide a suitable microenvironment for their in vitro maturation. Study design, size, duration A total of 879 GV collected between 2017 and 2019 were included in this study. They were randomly allocated into two experimental groups: (1) single-cultured oocytes (SC) that were cultured individually in micro-droplets, and (2) group-cultured oocytes (WOW) that were cultured in a microwell culture system using the WOW dish (culture dish for time lapse incubator). The nuclear maturation was assessed after 24 hours and 48 hours of IVM Participants/materials, setting, methods GV oocytes were obtained from 609 patients undergoing controlled ovarian stimulation cycles. Oocytes from the experimental group (1) were placed individually in conventional 25μl micro-droplets in a 35 mm dish. Oocytes from the experimental group (2) were placed in 80 μl droplet individually in each of 9 microwells of WOW dish. All GV oocytes were matured in a single step embryo culture medium, supplemented with human menopausal gonadotropin and synthetic serum substitute. Main results and the role of chance Mature oocyte (MII) was considered when we observed rupture of the GV and the presence of a first polar body in the perivitelline space during the first 24 or 48 hours of culture under inverted optical microscope. GVBD noted significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [GVBD: SC group; 70% (318/455) vs. WOW group; 83% (352/424)] and 48 hours [GVBD: SC group; 77% (319/416) vs. WOW group; 94% (398/424)]. The maturation rates (MR) showed significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [MR: SC group; 51% (233/455) vs. WOW group; 80% (338/424)] and 48 hours [MR: SC group; 71% (295/416) vs. WOW group; 91% (387/424)]. Limitations, reasons for caution There is no data on cleavage and blastocyst rates. There are no previous reports comparing the maturation rates in denuded human oocytes single-cultured in individually droplet or group-cultured in WOW dish. Wider implications of the findings: Our results must be taken into account in order to improve the culture conditions for the optimization of the in vitro maturation technique in human oocytes from stimulated cycles. We now provide evidence that group-cultured oocytes in WOW dish increase GVBD and maturation rates. Trial registration number Not applicable
Study question Does the D19S884 allele 8 (A8) equally affect the pathogenesis and ovarian gene expression of PCOS and PCOM patients? Summary answer A8-allele produces metabolic, endocrine, and ovarian alterations regardless diagnose. The mechanisms involved in PCOM alterations are different from those of the ovarian phenotype of PCOS. What is known already The specific D19S884 allele 8 of the FBN3 gene may be related to polycystic ovarian syndrome (PCOS) clinical manifestations. The A8 allele participates in alternative splicing of FBN3 and produces Asprosin-3, related to glucose modulation, and Fibrillin-3. Fibrilin-3 is an extracellular matrix protein, and together with a dysregulation of the Hippo pathway, could be responsible for constraining follicular growth in the PCOS ovaries. However, it is still unknown how these pathways act in PCOS, and whether these mechanisms are also involved in pathophysiology of polycystic ovarian morphology (PCOM) in apparently normal women with regular menses. Study design, size, duration Cross-sectional and descriptive study with 139 women (24-39 years-old) undergoing an IVF cycle between 2019-2022 at Hospital La Fe (Valencia, Spain). Thirty-one patients were considered PCOS, twenty-eight were classified as PCOM while the remaining eighty were controls. After recruitment women were screened for A8 allele, metabolic status, hormone profile, follicular fluid protein determination and expression of Hippo pathway and extracellular matrix genes. The IVF cycle parameters were recorded to determine their relationship with study variables. Participants/materials, setting, methods Women with two or more Rotterdam-criteria were considered as PCOS. PCOM patients were defined by polycystic ovarian morphology on ultrasound with regular ovulatory cycles. Genomic DNA was isolated from blood samples to assess the presence of A8 allele by capillary electrophoresis and FBN3 concentration was measured on follicular fluid by ELISA. TaqMan qPCR assay was used to analyze the expression of Hippo pathway (BIRC1 and CCN2) and extracellular matrix (ECM) genes in cumulus cells. Main results and the role of chance PCOS patients showed statistically significant metabolic and endocrine alterations with higher BMI, free androgen index (FAI), and glucose levels compared to controls. Despite having increased AMH levels (PCOS:45.0±21.7; PCOM:33.8±21.5; Control:17.6±7.19pmol/L, p < 0.05), and AMH/AFC ratio (PCOS:1.9±0.9; Control:1.2±0.5, p = 0.008), PCOS showed lower fertilization rates (PCOS:64±26; Control:80±18%, p = 0.008), reduced good quality (PCOS:0.6±1.0; Control:1.3±1, p = 0.003) and transferred embryos (PCOS:1.3±0.9; Control:1.9±1.0, p = 0.018). In PCOM, metabolic and androgen profiles were not different from controls. Although AMH, AFC (PCOM:24.4±13.7; Control:15.5±7.1, p = 0.007) and aspirated follicles (PCOM:17.3±5.7; Control:14.3±7.0, p = 0.026) were increased, fewer embryos were transferred (PCOM:1.2±1.1; Control:1.9±1.0, p = 0.027). PCOM women also showed ovarian downregulation of the Hippo-pathway (CCN2-PCOM:-4.8±10.8; Control:0.1±3.0, p = 0.010) and increased FBN3 concentration (PCOM:19.6±8.8; Control:14.7±5.6ng/ml, p=N.S). The A8 allele was detected in 17% of our patients: 4% PCOS, 50% PCOM and 46% controls. Overall, A8 presence associated a Hippo-pathway downregulation (BIRC1-A8+:-2.7±4.5; A8-:0.3±2.2, p = 0.034) and increased ECM expression (EMILIN1-A8+:2.1±1.0; A8-:1.2±1.5, p = 0.04). Interestingly, in controls, higher glucose (A8+:95.1±6.7; A8-:83.5±10.1mg/dl, p = 0.002), cholesterol (A8+:182.5±11.1; A8-:165.4±27.3mg/dl, p = 0.029), LDL (A8+:117.8±10.4; A8-:83.9±29.4mg/dl, p = 0.002), and DHEAS levels (A8+:2633.0±670.7; A8-:1585.8±670.0ng/ml, p = 0.026) were found, consistent with a reduction in HDL (A8+:51.8±8.8; A8-:67.4±15.43mg/dl, p = 0.026) and a downregulation of the Hippo-pathway (BIRC1-A8+:-4.8±4.6; A8-:0.2±4.6, p = 0.013). In PCOM, A8+ promoted higher FAI (A8+:1.6±1.5; A8-:0.8±0.7, p=N.S), and less embryos obtained (A8+:5.9±3.8; A8-:10.7±3.8, p = 0.04). Limitations, reasons for caution Due to the low prevalence of A8 in our PCOS population, its effects cannot be evaluated in this group. Further validation of gene expression results by RNA-seq will be required to assess the broad spectrum of ovarian transcriptomic changes induced by both, the PCOM phenotype and the A8 allele. Wider implications of the findings Polycystic ovarian morphology is not unique to clinical disorder of PCOS and the triggering mechanisms appear to be distinct from those observed in women with regular cycles. Our findings suggest that the presence of A8 allele impacts on metabolic profile, androgen levels, and ovarian pathway dysregulations, despite the clinical diagnose. Trial registration number Not applicable
How does the in vitro maturation (IVM) medium and the vitrification procedure affect the survival of germinal vesicle (GV) oocytes obtained from stimulated cycles and their development to the blastocyst stage? In total, 1085 GV human oocytes were obtained after women underwent a cycle of controlled ovarian stimulation, and these oocytes were subjected to IVM before or after their vitrification. IVM was carried out in two commercial culture media not specifically designed for maturation. MII oocytes were then activated and embryo development until day 6 was evaluated. According to the results, a higher percentage of oocytes reach the MII stage if they are vitrified before they undergo IVM. Nevertheless, the medium used and the sample size determine whether these differences become significant or not. Similar survival rates and development to blastocysts were observed in all the conditions studied.
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