Altered non-coding RNA profiles of seminal plasma extracellular vesicles of men with poor semen quality undergoing In Vitro fertilization treatment

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Background: Currently, the precise mechanisms that underline male infertility are still unclear. Accumulating data implicate non-coding RNA (ncRNA) cargo of seminal plasma extracellular vesicles (spEVs) due to their association with poor semen quality and higher expression level relative to vesicle-free seminal plasma.

Method: We assessed sperm-free spEV ncRNA profiles from 91 semen samples collected from male participants of couples seeking infertility treatment. Men were classified into two groups (poor, n = 32; normal, n = 59) based on World Health Organization semen cutoffs. Small RNA-seq reads were mapped to standard biotype-specific transcriptomes in the order miRNA > tRNA > piRNA > rRNA > "other" RNA > circRNA > lncRNA using STAR. Differential expression of normalized ncRNA read counts between the two groups was conducted by EdgeR (Fold change ≥1.5 & FDR < 0.05).

Result: Small RNA-seq identified a wide variety of spEV ncRNA biotypes including miRNA, rRNAs, piRNAs, tRNA, lncRNAs as well as circRNAs and fragments associated with pseudogenes, and nonsense mediated decay. The expression levels of 57 spEV ncRNAs (miRNA:6, piRNA:4, rRNA:6, circRNA:34, lncRNA:7) were altered in men with poor semen quality relative to normal semen parameters, many (60%) of which were circRNA species. Ontology analysis of differentially expressed miRNAs and circRNAs showed enrichment in functional terms related to cellular communication and early development.

Conclusion: This is the first study to generate comprehensive spEV ncRNA profiles in a clinical setting and to determine their differences between men with normal and abnormal semen parameters. Thus, our study suggests that spEV ncRNAs may represent novel biomarkers of male reproductive phenotypes. This article is protected by copyright. All rights reserved.

Keywords: IVF; Seminal plasma; extracellular vesicles; infertility; small non-coding RNA.