Transcriptomics in human cumulus cells during oocyte in vitro maturation

Advancing human oocyte in vitro maturation (IVM) requires understanding the mechanisms that govern this process. The cumulus cells (CCs) that surround the oocyte play a crucial role. Our study focused on identifying specific genes in human CCs contributing to oocyte maturation in vitro.

We used microarrays to detail the global transcriptome underlying oocyte maturation in cumulus cells and identified distinct up-and-down-regulated genes and signaling pathways during this process. Cumulus cells were removed from immatures (germinal vesicle - GV) cumulus-oocyte complexes (COCs) before IVM (Fresh GV, n = 5) and immature (GV) and mature (MII) COCs after IVM (GV-IVM, n=8; and MII-IVM, n=12). All the samples were collected ex vivo from 8 women who underwent unilateral ovariectomy and ovarian tissue cryopreservation for fertility preservation. Women did not receive prior ovarian stimulation

Methods
Total RNA was individually extracted and purified from each sample with TRIzol® reagent and 1-bromo-3-chloropropane and, subsequently, with RNeasy® Minikit 250 (Qiagen, Denmark) according to the manufacturer’s instructions. All steps were performed on ice. RNA quality and quantity were assessed by DeNovix DS-11 FX spectrophotometer and Bioanalyzer RNA 6000 Pico Kit, respectively. Only samples with RNA integrity (RIN) values ≥ 6 were included in the study. Moreover, 25 samples were selected based on their RIN values to ensure a similar distribution across all groups: Fresh GV (mean RIN 8.9, n = 5), GV (mean RIN 7.9, n = 8), and MII (mean RIN 8, n = 12) (Supplementary Table S2). The selected samples were further processed using the ClariomTM D arrays with GeneChipTM Whole Transcript (WT) PLUS Reagent Kit according to the manufacturer’s instructions. The array was washed and stained with phycoerythrin-conjugated streptavidin using the Affymetrix Fluidics Station 450 and then scanned with the Affymetrix GeneChipTM Scanner 3000 7G System to generate fluorescent images. The Expression Console Software generated cell intensity files (.CEL files). The raw CEL files were imported into the Transcriptome Analysis Console (TAC, v4.0.2.15, Applied Biosystems). Data summarization, normalization, gene summaries, and statistical analysis were performed in one analysis flow. Normalization was performed by the signal space transduction-robust multi-array average (SST-RMA) approach. The differential expression analysis among CC groups (Fresh GV, GV-IVM, and MII-IVM) was set up using ANOVA ebayes comparison and an overall false discovery rate (FDR) <0.05. For differential expression between groups, an FDR < 0.05 combined with a gene level fold change (FC) <−2 or >2 was considered significant. Furthermore, pathway analysis, principal component analysis (PCA), and hierarchical clustering were performed in TAC.