Seminal extracellular vesicle sncRNA profiles reveal miRNAs/isomiRs as predictive biomarkers of testicular sperm retrieval outcome in azoospermic patients

Non-invasive molecular biomarkers for differential diagnosis of the origin of azoospermia into obstructive azoospermia (OA) or secretory azoospermia (SA), as well as for prediction of the presence of residual spermatogenesis in testicular tissue of patients presenting with SA, are of great interest for TESE outcome prediction in assisted reproduction. Our previous study of several hundreds of miRNAs suggested the use of some sEV-miRNAs from seminal plasma as biomarkers for the origin of azoospermia. In this regard, studying more in depth sncRNAs in EVs from semen of azoospermic individuals will be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes which could help in the identification of those individuals with real chances of positive sperm recovery on the testicular biopsy prior to assisted reproduction technology (ART) treatment. A high-throughput sncRNA profiling analysis using paired-end small RNA-seq was performed in seminal sEVs from azoospermia and normozoospermic individuals. Overall design: A case and control prospective study was performed. In this pilot study we aimed at extensively characterizing seminal sEV sncRNA profiles of azoospermic individuals with different testicular sperm recovery outcome. For that, we have profiled and quantified the expression of annotated sncRNAs by paired-end small RNA-seq and determined the expression pattern of not only miRNAs (analyzed at the isomiR level), but also piRNAs, and tsRNAs within the human seminal sEVs in a small number of normozoospermic (n=4) and azoospermic [OA-N n=4; SA(Sp+) n=5; SA(Sp-) n=4] individuals. RT-qPCR validation analysis of selected miRNAs was additionally performed in a larger number of individuals.