Unravelling the genetic basis of Azoospermia: RNA-Sequence and transcriptome profiling analyses in a Greek Population.

Abstract

ObjectiveTo investigate whether idiopathic non-obstructive azoospermia (iNOA) has its own transcriptomic signature. DesignTesticular tissue biopsies, were retrieved, processed and prepared for RNA extraction from 26 consented patients diagnosed with iNOA. Samples were grouped into four pools based on the presence of testicular spermatozoa: two replicate pools for “No presence” (Null-spz-1 & Null-spz-2 pools), one for “High presence” (High-spz pool) and one for “Rare presence” (Rare-spz pool). A second set of replicate pools (CF-1 & CF-2) was used from patients with obstructive azoospermia (OA), served as controls. RNA sequencing (RNA-seq) and comparative Transcriptomics Analysis (CTA) were performed, followed by differential gene expression analysis (DGE) focused on protein-coding genes only. Differentially expressed genes (DEGs) exclusively upregulated or downregulated were further analyzed using the Gene Ontology (GO), STRING, and Kyoto Encyclopedia of Genes and Genome (KEGG) bioinformatic platforms. SubjectsMales diagnosed with iNOA. ExposureTesticular biopsies from men diagnosed with iNOA. Main Outcome MeasuresProtein-coding differentially expressed genes (DEGs). ResultsA significantly altered transcriptomic profile of protein-coding genes was identified in the testicular tissues from men with iNOA. A total of 3858 genes, exhibited dysregulated expression, with 1994 genes being exclusively downregulated and 1734 upregulated. Biological processes (BPs) such as, male gamete generation (GO:0048232) and meiotic cycle (GO:0051321) were significantly enriched by the downregulated DEGs while the upregulated DEGs enriched BPs such as, regulation of cell death (GO:0010941), regulation of cell adhesion (GO:0030155) and defense response (GO:0006952). Interactome analysis identified hub genes among the downregulated DEGs, including PCNA, PLK1, MCM4, CDK1, CCNB1, AURKA, CCNA2, and CDC6 and among the upregulated DEGs, including EGFR, RELA, CTNNB1, MYC, JUN, SMAD3, STAT3 NFKB1, TGFB1 and ACTB. Additionally, KEGG analysis demonstrated that pathways such as cell cycle (hsa04110) and Oocyte meiosis (hsa04114) are the primarily affected by the downregulated genes, while the upregulated genes mainly affected pathways such as, the Focal adhesion (hsa04510) and the PI3-Akt signaling pathway (hsa04151). ConclusionA distinct mRNA expression profile and altered transcriptomic activity were identified in the testicular tissues of men with iNOA.