Transcriptomic evaluation of bovine embryo quality on blastocysts obtained from peri-pubertal oocyte donors [8 mo vs 14 mo]
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ABSTRACT: Assisted reproduction technologies (ART) and high selection pressure observed in the dairy industry are leading towards the use of younger females for reproduction purpose, reducing the interval between generations. This situation might have an impact on embryo quality, which can affect the success rate of the procedures. This study aims to document the effect of donor age on embryo quality, on transcriptomic level, in order to caracterize the effect of using young animals for reproduction purpose. 10 young Holstein cows were used 3 times each at different ages for ovarian stimulation protocols and oocyte collections (at 8, 11 and 14 months). These oocytes were then fertilized in vitro with adult bull semen, generating 3 lots of embryos per animal. Semen from the same bull was used for all females, at all times. Each animal was used as its own control for age-effect evaluation. EmbryoGENE plateform allowed whole genome assessment of gene expression patterns at the blastocyst stage. Embryos from animals at 8 vs 14 months and at 11 vs 14 months were used for microarray hybridization. Validation was done performing RT-qPCR tests on 7 candidate genes.
Project description:Assisted reproduction technologies (ART) and high selection pressure observed in the dairy industry are leading towards the use of younger females for reproduction purpose, reducing the interval between generations. This situation might have an impact on 10 young Holstein cows were used 3 times each at different ages for ovarian stimulation protocols and oocyte collections (at 8, 11 and 14 months). These oocytes were then fertilized in vitro with adult bull semen, generating 3 lots of embryos per animal.
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 cumulusâoocyte 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:Polycystic ovary Syndrome (PCOS) is a heterogeneous endocrine disorder that shows evidence of genetic predidposition among affected individuals. We have utilized the Microarray data from granulosa cells of normal and PCOS women for network construction. Human granulosa cells were isolated from ovarian aspirates from normal and PCOS women undergoing IVF and for each sample, RNA was extracted and hybridized to an Affymetrix GeneChip.
Project description:Four groups of human oocytes from in vitro fertilization programme were analysed using microarrays: 1.) oocytes matured in vivo (retrieved as mature by ultrasound follicular aspiration but not fertilized after in vitro fertilization), 2.) oocytes matured in vitro in a conventional way (in a maturation medium with added gonadotropins FSH and HCG), 3.) oocytes matured in vitro by a new approach (in a maturation medium with added gonadotropins FSH and HCG, and in a co-culture with cumulus cells from mature oocytes of the same patient), and 4.) not matured oocytes which did not mature in spite of in vitro maturation procedure. Each group of oocytes consisted of three biological replicates with 10 oocytes per replicate therefore 12 oocyte samples and altogether 120 human oocytes were analyzed by microarrays.
Project description:Our aim with this work is to identify and quantify the miRNA profile of the human follicular fluid using a miRNA microarray approach in young and advanced-aged women and in follicles with different oocyte quality. Data collected in this study would be utilized in a future as a marker to predict the oocyte quality. This observational prospective study includes women enrolled in the assisted reproduction program of Hospital Universitari i Politècnic la Fe (Valencia, Spain). The study was accepted by IRB of this Hospital and all couples received informed consent of our study and assisted technique according to service protocols. The study was divided in two stages: Stage 1: Patients (n=21) who had follicles yielding oocytes with different degree of maturation were included (n=21). The follicular fluid samples from 21 patients were pooled in different groups. Expression of miRNA in the follicular fluids was compared within each patient according the degree of oocyte maturation. Stage 2: Patients were allocated into two groups according to the patient's age: advanced age (AA; >36 years old) and young age (YA; <36 years old). Follicular fluids from follicles containing MII oocytes were pooled and miRNA expression was subsequently compared. Please note that the MII samples with underscore (such as MII1 vs MII_1) indicate different pools so they are not the same sample, but we maintained the number because they correspond to paired samples pools for GV and MI.
Project description:Conventional Superstimulation (group 1) vs. Long Superstimulation (group 3) A genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells collected from ovarian follicles after differing durations of the growing phase induced by exogenous FSH treatment. Cows were given a conventional (4-day) or long (7-day) superstimulatory treatment (25mg FSH im at 12-h intervals; n=6 per group), followed by prostaglandin treatment with last FSH and LH treatment 24 hr later. Granulosa cells were harvested 24 h after LH treatment. The expression of 416 genes was down-regulated and 615 genes was up-regulated in the long FSH group compared to the conventional FSH group. Quantification by RT-PCR of 7 genes (NTS, PTGS2, PTX3, RGS2, INHBA, CCND2 and LRP8) followed the same trend as in microarrays. Compared to the conventional group, the long FSH group responded to exogenous LH with down-regulation of mRNA for cyclinD2, lipoprotein receptorP8, CYP51A1, CYP19A1, CJA1, INHBA, SRPINE1, and up-regulation of Vannin, POSTN, GTPAse, Cysteine. Markers of fertility and follicle maturity were up-regulated in the long FSH group. We conclude that a prolonged FSH-induced growing phase is associated with transcriptomic characteristics of greater follicular maturity and may therefore be more appropriate for optimizing the superovulatory response and developmental competence of oocytes in cattle. straight comparison of Short Superstimulation group (the reference; group 1) versus a Long Superstimulation protocol ( the treatment; group 3) using 3 different animals (biological replicates) in each group and performed dye swap. For example on array 1: group 1 cow 1 versus group 3 cow 1 (cy3 vs cy5) and on array 4 is the dye swap group 3 cow 1 versus group 1 cow 1 (cy3 vs cy5).
Project description:This group consist of human embryologists from the reproductive medical center for of the 1st affiliated hospital of Anhui Medical University. Our research is specifically focused on women ovarian reserve and the relevant female infertility. By deep sequencing, the current experiment determined the small non-coding RNA profile of cumulus cells from patients with or without diminished ovarian reserve undergoing controlled ovarian stimulation and in vitro fertilization treatment. Ovarian follicles, which are a densely-packed shell of granulosa cells that contains an immature or mature oocyte, are above all responsible for the development, maturation, and release of mature egg for fertilization. They are also responsible for synthesizing and secreting hormones that are essential for follicular development, menstrual and estrous cycle, maintenance of the reproductive tracts and their functions, development of female secondary sex characteristics, and metabolism. During folliculogenesis, ovarian granulosa cells surrounding the oocyte differentiate into mural granulosa cells, involved in gonadal steroidogenesis, and into cumulus cells, which are ovulated with the oocyte at ovulation. In the present study, we described the small non-coding RNA expression profile to characterize the ensemble of both known and novel ncRNAs expressed in cumulus cells from patients with or without Diminished ovarian reserve, by using high-throughput Solexa technology.