A comprehensive profile of cell-free RNAs in seminal plasma reveals their noninvasive potential in male infertility

Abstract Study question Could the cell-free RNAs (cfRNAs) in seminal plasma be associated with male infertility and used as clinical biomarkers? Summary answer CfRNAs in seminal plasma show different patterns in men with infertility and could serve as diagnostic and predictive factors in male infertility. What is known already Seminal plasma is a biological fluid containing large amounts of tissue-specific components secreted by organs of the male reproductive system. Thus, seminal plasma has great potential as a clinical sample for biomarker discovery and noninvasive diagnostics of male reproductive disorders. Several metabolites, metal ions, proteins and nucleotides are associated with semen quality and could be predictive factors of azoospermia forms and subtypes. However, the comprehensive transcriptome of cfRNAs in seminal plasma and their clinical value is not clear. Study design, size, duration We applied a previously developed library method of polyadenylation ligation-mediated sequencing (PALM-Seq) to capture and sequence the cfRNAs of seminal plasma from adult men, including 129 patients with asthenozoospermia, 101 patients with oligozoospermia, 102 patients with azoospermia (obstructive and nonobstructive azoospermia) and 107 healthy individuals. Participants/materials, setting, methods We performed PALM-Seq on the seminal plasma of recruited individuals and comprehensively profiled cfRNAs including miRNA, tRNA, piRNA, lncRNA and mRNA. In addition, we followed up the clinical outcome of assisted reproductive technology (ART) during reproductive therapy. Main results and the role of chance Hundreds of miRNA and tRNA could be detected in all samples. Unlike other body biofluids, we defined a large amount of piRNA in seminal plasma since piRNA is highly expressed in germ cells. Besides, the piRNA showed a group-specific pattern in men with infertility, especially in patients with azoospermia. Differentially abundant mRNA in patients with asthenozoospermia, oligozoospermia and azoospermia were mainly involved in regulating mononuclear cell migration, thermogenesis and germ cell development, respectively. The abundance of seminal plasma cfRNAs was associated with parameters of semen quality, as well as sperm DNA fragmentation. At last, using the collected omics data and machine learning models, we could accurately classify the subtypes of azoospermia and successfully predict the ART outcome before treatment. Limitations, reasons for caution We only recruited a limited number of individuals in this research and more studies focused on the ART outcome are needed. Wider implications of the findings We comprehensively characterized kinds of cfRNAs in seminal plasma for the first time and revealed changes in men with infertility. Our results contribute a new insight into understanding the mechanism of male infertility. Our findings could provide noninvasive diagnostics before invasive biopsies and help to improve the treatment of infertility. Trial registration number Not applocable