Dataset Information

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.

ABSTRACT:

Background

Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence.

Methodology/principal findings

197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy.

Conclusion/significance

RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.

SUBMITTER: Feuerstein P

PROVIDER: S-EPMC3407221 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.

Feuerstein Prisca P Puard Vincent V Chevalier Catherine C Teusan Raluca R Cadoret Veronique V Guerif Fabrice F Houlgatte Remi R Royere Dominique D

PloS one 20120727 7

<h4>Background</h4>Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its development ...[more]

PMID: 22848380

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Project description:BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied. METHODS: The mRNA expression levels of cumulus cells were detected by qRT- PCR. RESULTS: Consistent with our previous cDNA microarray data, with the exception of GPX3 and PTX3, the selected 7 genes were related to oocyte nuclear maturation in PCOS patients. Noticeably, the expression level of RUNX2 was lower in cumulus cells derived from oocytes that could develop into blastocysts than the level of expression from oocytes that could not. The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization. GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence. CONCLUSIONS: Several cumulus cell genes were associated with oocyte maturation, fertilization and embryo quality in PCOS patients. RUNX2 and GPX3 are candidate genetic markers in the monitoring of embryo quality for PCOS patients, whereas PTX3 mainly played a role in fertilization process. Together with morphological evaluation, cumulus cells genes may serve as biomarkers of oocyte and embryo selection during the IVF process for PCOS patients and may advance our understanding of PCOS.

Project description:Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter (p < 0.001), oocyte diameter (p < 0.05), and ZP thickness (p < 0.001). Furthermore, the relative gene expression of BAX, BCL2, GDF9, and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX, STAR, and PTGS2, while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.

Project description:Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.

Dataset Information

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.

Background

Methodology/principal findings

Conclusion/significance

Publications

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.

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