LAUSR

BACKGROUND Non-invasive molecular biomarkers for classifying azoospermia by origin into either obstructive (OA), or non-obstructive/secretory azoospermia (NOA/SA), as well as for inferring the spermatogenic reserve of the testis of NOA/SA patients, are of great interest for testicular sperm retrieval outcome prediction for assisted reproduction. Prior analyses of semen small non-coding RNA (sncRNA) expression in azoospermia have focused on miRNAs but there has been a lack of attention on other regulatory small RNA species. In this regard, studying more in-depth expression changes of sncRNA subtypes in small extracellular vesicles (sEVs) from semen of azoospermic individuals could be useful to select additional non-invasive biomarkers with diagnostic/prognostic purposes. MATERIAL AND METHODS A high throughput sncRNA profiling analysis to determine the expression pattern of seminal sEV miRNAs (analyzed at the isomiR level), piRNAs, and tsRNAs in normozoospermic (n = 4) and [OA-N n = 4; SA(Sp+) n = 5; SA(Sp-) n = 4] azoospermic individuals was carried out. RT-qPCR validation analysis of selected miRNAs was additionally performed in a larger number of individuals. RESULTS AND DISCUSSION Clinically relevant quantitative changes in the sncRNA levels contained in semen sEVs can be used as biomarkers for the origin of azoospermia and for predicting the presence of residual spermatogenesis. In this regard, canonical isoform miRNAs (n = 185) but also other isomiR variants (n = 238) stand out in terms of numbers and fold-change differences in expression, underlining the need to consider isomiRs when investigating miRNA-based regulation. Conversely, although tsRNAs are shown in our study to represent a high proportion of sncRNA sequences in seminal sEV samples, they are not able to discriminate the origin of azoospermia. piRNAs cluster profiles and individual piRNAs with significant differential expression were not able to discriminate either. Our study demonstrated that expression values of individual and/or combined canonical isoform miRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, miR-181b-5p; AUC>0.8) in sEVs provide considerable clinical value in identifying samples with a high likelihood of sperm retrieval, while discriminating azoospermia with different origin. Although no individual miRNA showed sufficient discriminating power on its own to identify severe spermatogenic disorders with focal spermatogenesis, multivariate miRNA models in semen sEVs have the potential of identifying those individuals with residual spermatogenesis. Availability and adoption of such non-invasive molecular biomarkers would represent a great improvement in reproductive treatment decision protocols for azoospermia in clinical practice. This article is protected by copyright. All rights reserved.