Altered Non-coding RNA Profiles of Seminal Plasma Extracellular Vesicles of Men with Poor Semen Quality Undergoing in Vitro Fertilization Treatment

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

Method: We assessed sperm-free seminal plasma extracellular vesicle non-coding RNA 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 sequencing reads were mapped to standard biotype-specific transcriptomes in the order micro RNA > transfer RNA > piwi-interacting RNA > ribosomal RNA  > ribosomal RNA > circular RNA > long non-coding RNA using STAR. Differential expression of normalized non-coding RNA read counts between the two groups was conducted by EdgeR (Fold change ≥1.5 and (false discovery rate [FDR] < 0.05).

Result: Small RNA sequencing identified a wide variety of seminal plasma extracellular vesicle non-coding RNA biotypes including micro RNA, ribosomal RNAs, piwi-interacting RNAs, transfer RNA, long non-coding RNAs as well as circular RNAs, and fragments associated with pseudogenes, and nonsense-mediated decay. The expression levels of 57 seminal plasma extracellular vesicle non-coding RNAs (micro RNA: 6, piwi-interacting RNA: 4, ribosomal RNA: 6, circular RNA: 34, and long non-coding RNA: 7) were altered in men with poor semen quality relative to normal semen parameters, many (60%) of which were circular RNA species. Ontology analysis of differentially expressed micro RNAs and circular RNAs showed enrichment in functional terms related to cellular communication and early development.

Conclusion: This is the first study to generate comprehensive seminal plasma extracellular vesicle non-coding RNA profiles in a clinical setting and to determine the differences between men with normal and abnormal semen parameters. Thus, our study suggests that seminal plasma extracellular vesicle non-coding RNAs may represent novel biomarkers of male reproductive phenotypes.