P-694: Gene expression profile of human immature and mature oocytes: A microarray approach

Abstract

The understanding of the mechanisms regulating human oocyte maturation are still rudimentary. The aim of this study is to determine gene expression profiles of human nuclear mature and immature oocytes (germinal vesicle and metaphase I). We have identified transcripts differentially expressed between imature and mature oocytes and cumulus cells. The patients referred for cIVF or ICSI were stimulated with a combination of gonadotropin-releasing hormone agonist (GnRH-a) and recombinants FSH or Menopur. Germinal vesicle (GV, n=20), metaphase I (MI, n=20) and unfertilized MII (n=16) oocytes were collected 21 hours or 44 hours post insemination and frozen at -80°C in RLT buffer from the RNeasy kit (Qiagen) before RNA extraction. Pools of oocytes were analyzed by DNA microarrays on Affymetrix® HG-U133 plus 2.0 GeneChip oligonucleotide array chips made of 54675 probesets and representing roughly 35000 unique human known or predicted genes. The Affymetrix® GeneChip Operating Software 1.2 (GCOS) was used to evaluate the signal intensity for each probeset (detection call “present” or “absent”) and also to perform pair wise comparison between single gene expression raw values from oocytes of different stages. We determined genes exclusively expressed during one stage and a list of genes which expression progressively increased during oocyte maturation by selecting the probesets with the following ratios constraints: MI/GV > 1.73, MII/MI > 1.73 and MII/GV > 3, with a significant change p-value (P < 0.01) and where GV, MI and MII stands for signals values in these samples. Immature and mature oocytes express in average 8728 genes, but there is a clear decline from the GV and MI stages (n = 10 892 and 9682 respectively) to MII oocytes (n = 5 633). Nevertheless, within this contingent expression varies little between the GV, MI and MII samples (tight scatter plots = 0.63 and high correlation coefficients = 0.92). The most specific genes only found in one category were HKR3, ZNF165 and MAK for the GV stage, PTPN12 for the MI stage. Despite showing the lowest diversity of gene expression, MII oocytes displayed the most specific pattern among all oocytes by up-regulation of 444 genes. Some are specific to MII oocytes (SOX2OT, DMBX1/OTX3, TGFB3, BCL2L11 and CAS2) whereas others were gradually induced during maturation (HIC2, CDC25A, CCNJ, CCNG2, SOCS7 and CUL5). Very few genes were specifically over- or under-expressed in GV and MI cells. Interestingly, a marker like Epithin (ST14) is very specific of both GV and MI oocytes whereas it is absent from mature MII oocytes. Such gene represents a potential marker of human immature oocytes. We identified human genes that are specific to three oocyte developmental stages as well as genes which expression increase gradually with the oocyte maturation process. Understanding these factors’ function will greatly improve our command of in vitro fertilization procedures. These genes already give insights about vital regulation pathways and will provide candidate cytoplasmic and nuclear markers of human oocyte maturation.