Differential Gene Expression Analysis of Human Cumulus Cells as a Predictor of Pregnancy

The objective of this study was to provide valuable insights into the determination of clinical pregnancy and live birth outcomes in the context of differential gene expression analysis of cumulus cells as predictors of clinical pregnancy and/or live birth after single embryo transfers. This study was performed with the consideration that each oocyte and each group of cumulus cells surrounding different oocytes might have different genetic expression patterns that might affect human reproduction. Differential gene expression analysis of cumulus cells was performed in 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes of 10 patients. Same procedures related to the oocyte maturation, microinjection, and microarray analyses were applied to the group of cumulus cells, individually. The results of the microarray analyses were enriched according to the clinical pregnancy and live birth outcomes of the patients. In conclusion, our data indicated significant differences in gene expression related with a variety of signaling pathways; the RAS signaling, the mitogen-activated protein kinases signalling, the ERBB signaling, the RAP1 signaling, the transcription factor nuclear factor-kb signaling, the transforming growth factor-b signaling pathways and neurological signaling pathways such as glutamatergic synapse, cholinergic synapse, dopaminergic synapse, GABAergic synapse, long-term potentiation, the neuroactive ligand-receptor interaction pathways. Circadian entrainment pathway was also affected in both clinical pregnancy and live birth at different significance level. These pathways can be exploited to identify biomarkers related to clinical pregnancy and live birth outcomes. This prospective case study included 10 women (aged 23-35 years) who were referred to a 3rd level reference center university hospital for idiopathic infertility and implemented Assisted Reproductive Technologies. The total sample size was 10 oocyte-cumulus complexes obtained over a period of 14 months between October 2011 and December 2012. Cumulus cells gene expressions in 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes of 10 patients were individually evaluated by microarrays using a NimbleGen Human Gene Expression 12x135K Microarray Kit (NimbleGen, Roche, Germany). Although the number of cumulus cells belonging to the same oocyte was very few, the oocytes and the cumulus cells sourrounding the same oocyte were analysed individually. The cumulus cells from each oocyte that developed an embryo that were transferred, applied with microarray analysis. Cumulus cells gene expressions were individually evaluated by microarrays in10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes of 10 different paitents using a NimbleGen Human Gene Expression 12x135K Microarray Kit (NimbleGen, Roche) according to the manufacturer’s instructions. Twelve-sample hybridization was performed on a single microarray slide, with three probes per gene. Total RNA was isolated using an RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA synthesis from total RNA was carried out using a Superscript Double Chain cDNA Synthesis Kit (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was marked using a Single Color DNA Labeling Kit (NimbleGen, Roche, Germany). cDNA was hybridized using the NimbleGen Hybridization System. Washing and drying of the array were carried out with a NimbleGen MS 2000 Microarray Scanner, and raw probe expression data was collected from microarray images using DEVA software.