Genomic Assessment of Human Cumulus Cell Marker Genes as Predictors of Oocyte Developmental Competence: Impact of Various Experimental Factors

INRA, UMR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France.
PLoS ONE (Impact Factor: 3.23). 07/2012; 7(7):e40449. DOI: 10.1371/journal.pone.0040449
Source: PubMed


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.
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.
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.

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Available from: Vincent Puard, Aug 13, 2014
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    • "Its statistical relevance was evaluated by the Fisher's exact test. Besides, among the results, the ability of the targeted output cluster to statistically discriminate between experimental conditions (stress vs. control) was evaluated by the Fisher's exact test as previously described (Feuerstein et al. 2012). Currently, the database contains the datasets from 355 microarray experiments from public and private repositories (listed in Supplemental Table A1) up to April 2013, which were conducted on zebrafish (Danio rerio, H.) (188 datasets), European sea bass (Dicentrarchus labrax, L.) (four datasets), Atlantic cod (Gadus morhua, L.) (five datasets), longjaw mudsucker (Gillichthys mirabilis, C.) (four datasets), rainbow trout (Oncorhynchus mykiss, W.) (73 datasets), Atlantic salmon (Salmo salar, L.) (60 datasets), and gilthead sea bream (21 datasets). "
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    • "The oocyte-cumulus interaction through the expression of specific genes helps the oocyte to acquire its developmental competence. Using microarrays Feuerstein et al. [17] attempted to determine genes related to oocyte developmental competence. Gene expression of oocytecumulus was studied according to the nuclear maturity of the oocyte (immature versus mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). "
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    ABSTRACT: The quality of follicular oocytes depends on interactions with surrounding granulosa cells. Development of molecular techniques and methods enables better understanding of processes underlying mammalian reproduction on cellular level. The success in reproductive biology and medicine in different species depends on reliable assessment of oocyte and embryo viability which presently mainly bases on embryo morphology. Although successful pregnancies have been achieved using this approach, its precision still should be improved and completed with other, more objective, and accurate assessment strategies. Global profiling of gene expression in follicular cumulus cells using microarrays is continuously leading to the establishment of new biomarkers which can be used to select oocytes with highest developmental potential. Even more potential applications and greater precision could be achieved using next generation sequencing (NGS) of granulosa and cumulus cell RNA (RNA-seq). However, due to the high cost, this method is not used as frequently as microarrays at the moment. In any case, high-throughput technologies offer the possibilities and advantages in ovarian somatic cell analysis on scale that has not been noted so far. The aim of this work is to present current directions and examples of global molecular profiling of granulosa cells and underline its impact on reproductive biology and medicine.
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    • "Some researchers have considered that cumulus cell function may be a more major determinant than the oocyte itself of the quality of the mitochondria in the activated oocytes. In elegant studies undertaken to determine which cumulus genes were both induced by the LH peak and critical to oocyte competence, mRNA expression in cumulus cells was prospectively studied in human oocytes that would be used for intracytoplasmic sperm injection [14]. Gene functions that were up-regulated in cumulus cells were identified in mature oocytes (meiosis II compared to Germinal vesicle (GV) stage cells) and those that were up-regulated in cumulus cells from mature oocytes that yielded a blastocyst at day 5/6 of in vitro culture. "
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