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.

Figure 1. Distribution of patients included in study.

Patients were separated into two main groups: microarray and qPCR. The variability group was composed of patients who had one CC_B+ and at least one CC_B-. The pregnancy group was composed of CC_B+ transferred from patients included in the variability group. CC_B+, cumulus cells from a mature oocyte yielding a blastocyst at day 5/6 of in vitro culture once fertilised; CC_B-, cumulus cells from mature oocyte which stopped developing at the embryo stage at day 5/6 of in vitro culture once fertilised; CC_GV, cumulus cells from immature oocyte at germinal vesicle stage; P+, pregnancy; P-, no pregnancy.

Figure 2. Heat map and cluster dendograms of gene clusters differentially expressed according to oocyte nuclear maturity.

Hierarchical clustering of cumulus cell samples (columns) and the 132 most significant probes (rows). Upregulated genes are marked in red, downregulated genes are marked in green. CC_GV (blue), cumulus cells from immature oocyte at germinal vesicle stage; CC_MII (pink), cumulus cells from mature oocyte.

Figure 3. Heat map and cluster dendograms of gene clusters differentially expressed according to oocyte developmental competence.

Hierarchical clustering of cumulus cell samples (columns) and the 354 most significant probes (rows). Upregulated genes are marked in red, downregulated genes are marked in green. CC_B- (blue), cumulus cells from mature oocyte which arrested at the embryo stage at day 5/6 of in vitro culture once fertilised; CC_B+ (red), cumulus cells from mature oocyte yielding a blastocyst at day 5/6 of in vitro culture once fertilised.

Figure 4. Hierarchical clustering of microarray datasets.

Measurements of the 308 genes discriminating CC_B- and CC_B+ were extracted from different datasets: our study (A, B), GSE4260 (C), GSE18559 (D), GSE21005 (E) and GSE9526 (F). They were subjected to hierarchical clustering after log transformation and median centering of genes. The different types of sample are shown as coloured squares. The quality of the separation was measured by Fisher’s exact test on the main branch. CC_GV, cumulus cells from immature oocyte at germinal vesicle stage; CC_MII, cumulus cells from mature oocyte; CC_B+, cumulus cells from mature oocyte yielding a blastocyst at day 5/6 of in vitro culture once fertilised; CC_B-, cumulus cells from mature oocyte which stopped developing at the embryo stage at day 5/6 of in vitro culture once fertilised.

Figure 5. Relative expression level obtained by qPCR of 9 genes differentially expressed according to oocyte developmental competence.

Results were expressed as means ± SEM of relative expression to the reference gene RPL19. CC_B+, cumulus cells from mature oocyte yielding a blastocyst at day 5/6 of in vitro culture once fertilised; CC_B-, cumulus cells from mature oocyte which stopped developing at the embryo stage at day 5/6 of in vitro culture once fertilised; *, significant difference (p<0.05).