Transcription profiling of human cumulus cells that surround oocytes resulting in early or late cleaving embryos
ABSTRACT: Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of in vitro fertilization (IVF) and more particular of elective single embryo transfer (eSET). The aim of the study was to analyse genome-wide whether the embryo viability was reflected by the expression of genes in the oocyte surrounding cumulus cells. Early cleavage (EC) was chosen as a parameter for embryo viability. Experiment Overall Design: Consenting patients visiting the IVF clinic underwent an IVF or ICSI treatment. Immediately following ultrasound-guided cumulus-oocyte-complex (COC) retrieval, a proportion of the cumulus cells surrounding the oocyte were removed. Gene expression in cumulus cells from eight oocytes resulting in an early cleavage embryo (EC-CC; n=8) and from eight oocytes resulting in a non-EC embryo (NEC-CC; n=8) derived from six patients were analysed using microarrays (n=16). To exclude a differential gene expression due to differences in patient characteristics, samples were paired. From four patients both an EC-CC and a NEC-CC sample were used. From two additional patients two EC-CC as well as two NEC-CC samples were used.
Project description:Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of in vitro fertilization (IVF) and more particular of elective single embryo transfer (eSET). The aim of the study was to analyse genome-wide whether the embryo viability was reflected by the expression of genes in the oocyte surrounding cumulus cells. Early cleavage (EC) was chosen as a parameter for embryo viability. Keywords: class comparison Overall design: Consenting patients visiting the IVF clinic underwent an IVF or ICSI treatment. Immediately following ultrasound-guided cumulus-oocyte-complex (COC) retrieval, a proportion of the cumulus cells surrounding the oocyte were removed. Gene expression in cumulus cells from eight oocytes resulting in an early cleavage embryo (EC-CC; n=8) and from eight oocytes resulting in a non-EC embryo (NEC-CC; n=8) derived from six patients were analysed using microarrays (n=16). To exclude a differential gene expression due to differences in patient characteristics, samples were paired. From four patients both an EC-CC and a NEC-CC sample were used. From two additional patients two EC-CC as well as two NEC-CC samples were used.
Project description:In vitro maturation (IVM) of the oocytes is a routine method in bovine embryo production. The competence of bovine oocytes to develop into embryo after IVM and in vitro fertilization (IVF) is lower as compared to in vivo preovulatory oocytes. Cumulus cells (CC) that enclose an oocyte are involved in the acquisition of oocyte quality during maturation. Using transcriptomic approach we compared cumulus cells gene expression during IVM with that in vivo preovulatory period. Global transcriptional profiling was performed using cumulus cells collected from mature bovine oocytes (metaphase-II stage) after maturation performed either in vivo or in vitro. In vivo matured cumulus cells were collected from ovulatory follicles of Montbeliard adult cows by ovum pick-up in vivo (OPU, n=4). In vitro matured cumulus cells were recovered from the oocytes after 22h of in vitro culture of cumulus-oocyte complexes (50 COC per experiment) from 2-6 mm ovarian follicles of adult cows (MIV, n=4). Gene expression analysis was carried out between in vivo and in vitro matured cumulus representing a total of 8 slides (dye swap protocol)
Project description:Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder that is characterized by increased circulating androgen levels, anovulatory infertility, and frequently, insulin resistance and hyperinsulinemia.The abnormity of oocyte nuclear maturity is the main reason for anovulatory infertility and pregnancy loss in PCOS patients.The bidirectional exchanges between oocyte and contiguous CCs are important for oocyte competence acquisition, early embryonic development and CC expansion.Gene expression profiles of CCs has been suggested to predict embryo development and pregnancy outcome. We used microarrays to detail the global programme of gene expression of CCs isolated from oocytes at metaphase I (CCMI) and metaphase II (CCMII) stage under controlled ovarian stimulation (COS) cycle in PCOS patients. Cumulus cells were isolated from oocyte at stage metaphase 1(MI)and stage metaphase II (MII) of PCOS patients for RNA extraction and hybridization on Affymetrix microarrays. For microarray analysis, we used three chips for each CC category. That is, CCMI1,CCMI2,CCMI3,CCMII1,CCMII2 and CCMII3.
Project description:Cumulus cells surrounding mature oocytes that developed to moruale/blastocyst stage on day 5 of IVF cycle were collected and used for gene expression profiling using Affymetrix Human Gene 1.0 ST Arrays in order to determine differences in gene expression between the modified natural and stimulated in vitro fertilization (IVF) procedures. Microarray analysis was made on 8 individual CC samples, derived from 5 patients that first underwent MNIVF, followed by a controlled ovarian hyperstimulation (COH) cycle. As the quality of the isolated RNA derived from 2 of the MNIVF CC samples was not good, these samples were not included in the analysis. Altogether, 3 CC samples from MNIVF and 5 CC samples from COH cycles were included in microarray analysis.
Project description:This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus–oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis and qRT-PCR.
Project description:In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).
Project description:RNA sequencing profiling of cumulus cells revealed that the follicles of older women have a compromised environment with hypoxic stress being the main contributor to age-related oocyte quality decay. Twenty cumulus cells (CC) samples (from a total of 15 patients) were obtained from oocytes of either male factor or egg donor patients. RNA sequencing and bioinformatic tools were used to identify differentially expressed genes (DEGs) between CCs from seven aged and eight young patients (<35 (years old) y.o. vs >40 y.o.). Quantitative-PCR and immunoflourescent staining were used for validation.
Project description:The elaboration of a quality oocyte is integrally linked to the correct developmental progression of cumulus cell phenotype. In humans and non-human primates, oocyte quality is diminished with in vitro maturation. To determine the changes in gene expression in rhesus monkey cumulus cells (CC) that occur during the final day prior to oocyte maturation and how these changes differ between in vitro and in vivo maturation (IVM and VVM), we completed a detailed comparison of transcriptomes using the Affymetrix gene array. We observe a large number of genes differing in expression when comparing IVM-CC and VVM-CC directly, but a much larger number of differences comparing the transitions from the pre-oocyte maturation to post- IVM and post-VVM state. We observe a truncation or delay in the normal pattern of gene regulation, but also remarkable compensatory changes in gene expression during IVM. Among the genes affected in cumulus cells by IVM are those that contribute to productive cell-cell interactions between cumulus cell and oocyte and between cumulus cells. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not suppressed during IVM. We identify a panel of 24 marker genes, the expression of which should provide the foundation for understanding how IVM can be improved, for monitoring IVM conditions, and for diagnosing oocyte quality. We compared transcriptomes of cumulus cells isolated from in vitro matured cumulus-oocyte complexes COCs (IVM-CC) and from in vivo matured COCs (VVM-CC) to identify potential cumulus cell markers of oocyte quality. The global gene expression profile of pre-maturation cumulus cells (PM-CC) was used to developmental transitions between IVM and VVM.
Project description:Affymetrix gene expression profiling in cumulus cells (CC) retrieved from patients undergoing GnRH agonists and GnRH antagonists IVF treatment. Oocytes from three different maturity stages were considered: metaphase I oocytes (MI), nonfertilized metaphase II (MII) oocytes (MII-NF) and MII oocytes developed to blactocyst stage embryo (MII-BL). From 4 GnRH agonist treated patients, CC MI, CC MII-NF and CC MII-BL samples were collected; from 5 GnRH agonist and 6 GnRH antagonist treated patients, CC MII-NF and CC MII-BL samples were collected; and from 2 GnRH agonist and 4 GnRH antagonist treated patients, CC MI and CC MII-BL were collected. Altogether, 10 CC MI, 15 CC MII-NF and 21 CC MII-BL were collected and considered for transcriptome analysis.
Project description:Cumulus-oocyte complexes were isolated at seperate time-points to generate temporal complexes. Targets from two biological replicates at each time point (0h, 8h, 16h post-hCG treatment) were generated and the expression profiles were determined using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Comparisons between the sample groups allow the identification of genes with temporal expression patterns. Experiment Overall Design: 2 eCG-primed (48h) pooled cumulus-oocyte complexes, 2 eCG-primed (48h) hCG-treated (8h) pooled cumulus-oocyte complexes, and 2 eCG-primed (48h) hCG-treated (16h) pooled cumulus-oocyte complexes replicates were analyzed