Project description:The objective of this study was to provide valuable insights into the determination of clinical pregnancy and live birth outcomes in the context of differential gene expression analysis of cumulus cells as predictors of clinical pregnancy and/or live birth after single embryo transfers. This study was performed with the consideration that each oocyte and each group of cumulus cells surrounding different oocytes might have different genetic expression patterns that might affect human reproduction. Differential gene expression analysis of cumulus cells was performed in 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes of 10 patients. Same procedures related to the oocyte maturation, microinjection, and microarray analyses were applied to the group of cumulus cells, individually. The results of the microarray analyses were enriched according to the clinical pregnancy and live birth outcomes of the patients. In conclusion, our data indicated significant differences in gene expression related with a variety of signaling pathways; the RAS signaling, the mitogen-activated protein kinases signalling, the ERBB signaling, the RAP1 signaling, the transcription factor nuclear factor-kb signaling, the transforming growth factor-b signaling pathways and neurological signaling pathways such as glutamatergic synapse, cholinergic synapse, dopaminergic synapse, GABAergic synapse, long-term potentiation, the neuroactive ligand-receptor interaction pathways. Circadian entrainment pathway was also affected in both clinical pregnancy and live birth at different significance level. These pathways can be exploited to identify biomarkers related to clinical pregnancy and live birth outcomes. Overall design: This prospective case study included 10 women (aged 23-35 years) who were referred to a 3rd level reference center university hospital for idiopathic infertility and implemented Assisted Reproductive Technologies. The total sample size was 10 oocyte-cumulus complexes obtained over a period of 14 months between October 2011 and December 2012. Cumulus cells gene expressions in 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes of 10 patients were individually evaluated by microarrays using a NimbleGen Human Gene Expression 12x135K Microarray Kit (NimbleGen, Roche, Germany). Although the number of cumulus cells belonging to the same oocyte was very few, the oocytes and the cumulus cells sourrounding the same oocyte were analysed individually. The cumulus cells from each oocyte that developed an embryo that were transferred, applied with microarray analysis. Cumulus cells gene expressions were individually evaluated by microarrays in10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes of 10 different paitents using a NimbleGen Human Gene Expression 12x135K Microarray Kit (NimbleGen, Roche) according to the manufacturer’s instructions. Twelve-sample hybridization was performed on a single microarray slide, with three probes per gene. Total RNA was isolated using an RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA synthesis from total RNA was carried out using a Superscript Double Chain cDNA Synthesis Kit (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was marked using a Single Color DNA Labeling Kit (NimbleGen, Roche, Germany). cDNA was hybridized using the NimbleGen Hybridization System. Washing and drying of the array were carried out with a NimbleGen MS 2000 Microarray Scanner, and raw probe expression data was collected from microarray images using DEVA software.
Project description:Previous studies have shown that GOLPH3 mediates cell growth, proliferation and differentiation and inhibits cell apoptosis; however, the role of GOLPH3 in cumulus granulosa cells and the value of GOLPH3 in predicting ICSI pregnancy outcomes remain unknown until now. Our findings showed higher positive expression rate, score of staining intensity, and immunohistochemical score of GOLPH3 in the cumulus granulosa cells of the pregnant women relative to non-pregnant women, and a higher apoptotic rate of cumulus granulosa cells was detected in non-pregnant women than in pregnant women. Pearson correlation analyses revealed that pregnancy correlated negatively with GOLPH3 expression and apoptosis of cumulus granulosa cells, and positively with the number of follicles punctured, number of grade III oocytes, number of eggs retrieved for ICSI, number of zygotes, number of cleavage-stage embryos, number of top-quality embryos, number of blastocysts, number of top-quality blastocysts, and number of frozen embryos. GOLPH3 may be involved in the apoptosis of cumulus granulosa cells, which may correlate with oocyte maturation and egg development. GOLPH3 expression in cumulus granulosa cells may facilitate the selection of top-quality eggs and embryos, the prediction of the clinical pregnancy outcomes of ICSI, and the increase of the pregnancy rate.
Project description:PURPOSE:Clinical pregnancy rate after IVF with eSET stagnates between 30 and 40%. In order to increase pregnancy and live birth rates, multiple embryo transfer is still common practice. Providing additional non-invasive tools to choose the competent embryo for transfer could avoid multiple pregnancy and improve time to pregnancy. Cumulus mRNA analysis with quantitative PCR (QPCR) is a non-invasive approach. However, so far, no gene sets have been validated in prospective interventional studies. METHODS:A prospective interventional single-center pilot study with two matched controls (day-3 and day-5 eSET) was performed in 96 patients consenting to the analysis of the cumulus-corona of their oocytes. All patients were super-ovulated for ICSI and eSET at day 3. All oocytes were denuded individually and cumulus was analyzed by quantitative PCR using three predictive genes (EFNB2, SASH1, CAMK1D) and two housekeeping genes (UBC and ?2M). Patients (n?=?62) with 2 or more day-3 embryos (good or excellent morphology) had their embryo chosen following the normalized expression of the genes. RESULTS:Corona testing significantly increased the clinical pregnancy and live births rates (63% and 55%) compared to single embryo transfer (eSET) on day 3 (27% and 23%: p?<?0.001) and day 5 (43% and 39%: p?=?0.022 and p?=?0.050) fresh transfer cycle controls with morphology-only selection. Time-to-pregnancy was significantly reduced, regardless of the number of good-quality embryos available on day 3. CONCLUSION:Combining standard morphology scoring and cumulus/corona gene expression analysis increases day-3 eSET results and significantly reduces the time to pregnancy. TRIAL REGISTRATION NUMBER:This is not an RCT study and was only registered by the ethical committee of the University Hospital UZBRUSSEL of the Vrije Universiteit Brussel VUB (BUN: 143201318000).
Project description:To identify reliable genomic biomarkers expressed in cumulus cells that accurately and non-invasively predict the oocyte developmental competence and reinforce the already used morphological criteria.Eight consenting patients were selected for ovarian stimulation and ICSI procedures. Cumulus-oocyte complexes were transvaginally punctured and individually selected based on both good morphological criteria and high zona pellucida birefringence. Following ICSI, two 3-day embryos per patient were transferred. Pregnancy outcome was recorded and proven implantation was thereafter confirmed. Differential gene expression was assessed using two microarray platforms. Further real-time PCR validation, Ingenuity pathways analysis and intra-patient analysis were performed on 17 selected candidates.Seven genes were differentially (p???0.05) associated to successful pregnancy and implantation. These biomarkers could be used to predict the oocyte developmental competence.These genomic markers are a powerful reinforcement of morphological approaches of oocyte selection. Their large-scale validation could increase pregnancy outcome and single embryo transfer efficiency.
Project description:GJA1 and PTX3 were proposed as gene markers for oocyte and embryo developmental competence, while SERPINE2 was reported to be associated with pregnancy outcome. PRSS35, which is exclusively expressed in the ovary, may be correlated with oocyte competence. This study was conducted to evaluate the correlation of cumulus GJA1, PRSS35, PTX3, and SERPINE2 gene expression levels with oocyte maturation, fertilization, and early embryo development.In total, 308 cumulus cell samples separated from individual cumulus-oocyte complex were obtained from 40 patients undergoing the intracytoplasmic sperm injection treatment procedure. Gene expression levels (mRNA levels) in cumulus cells were assessed using quantitative real-time polymerase chain reaction.Gene expression levels of GJA1 and SERPINE2 in cumulus cells surrounding mature oocytes were significantly lower than those in cumulus cells enclosing immature oocytes. PRSS35 mRNA levels in cumulus cells of fertilized oocytes were significantly higher than those in cumulus cells of unfertilized oocytes. GJA1 and SERPINE2 seemed to express higher mRNA levels, while PRSS35 showed lower expression in cumulus cells of oocytes that developed into embryos with good morphology; however, the expression levels of all three genes and PTX3 showed no significant differences between embryos with good or poor morphology.GJA1 and SERPINE2 represent potential gene markers associated with oocyte maturation. PRSS35 may be correlated with oocyte fertilization potential. However, GJA1, PRSS35, PTX3, and SERPINE2 may not be considered as marker genes for predicting embryo morphology.
Project description:Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females.
Project description:Study the effect of recombinant human FSH supplementation in the in vitro maturation medium for an incubation of 6 hrs on the transcriptomics of bovine cumulus cells. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) supplemented with FSH (CC-FSH) vs. cumulus cells without FSH (Ct-6H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:BACKGROUND:Mural Granulosa Cells (MGCs) and Cumulus Cells (CCs) are two specialized cell types that differentiate from a common progenitor during folliculogenesis. Although these two cell types have specialized functions and gene expression profiles, little is known about their microRNA (miRNA) expression patterns. OBJECTIVE:To describe the miRNA profile of mural and cumulus granulosa cells from human preovulatory follicles. METHODS:Using small RNA sequencing, we defined the miRNA expression profiles of human primary MGCs and CCs, isolated from healthy women undergoing ovum pick-up for in vitro Fertilization (IVF). RESULTS:Small RNA sequencing revealed the expression of several hundreds of miRNAs in MGCs and CCs with 53 miRNAs being significantly differentially expressed between MGCs and CCs. We validated the differential expression of miR-146a-5p, miR-149-5p, miR-509-3p and miR-182-5p by RT-qPCR. Analysis of proven targets revealed 37 targets for miR-146a-5p, 43 for miR-182-5p, 2 for miR-509-3p and 9 for miR-149-5p. Gene Ontology (GO) analysis for these 4 target gene sets revealed enrichment of 12 GO terms for miR-146a-5p and 10 for miR-182-5p. The GO term ubiquitin-like protein conjugation was enriched within both miRNA target gene sets. CONCLUSION:We generated miRNA expression profiles for MGCs and CCs and identified several differentially expressed miRNAs.
Project description:Study the effect of forskolin, dipyridamol and IBMX (here called FID) supplementation in the in vitro maturation medium for an incubation of 6 hrs on the transcriptomics of bovine cumulus cells. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) supplemented with FID (CC-FID) vs. cumulus cells without FID (Ct-6H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.