ИСТИНА

Background Within-subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood. Objectives To investigate intra-individual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men. Materials and Methods Semen parameters were assessed in samples collected 6-8 days apart from 164 18-19 year old participants of the Russian Children’s Study, a prospective cohort. Subsets of paired samples were used for label-free quantitation and targeted mass-spectrometry of proteins in seminal plasma (SP) and seminal extracellular vesicles (EV), and for small non-coding RNA (sncRNA) profiling in EVs and sperm using RNA-seq. The mean difference between two ejaculates, within-subject variation, intraclass correlation, and concordance correlation were used to assess IIV for all parameters. Low variability with high reproducibility and high reliability was considered if CVw<15% and ICC>0.90, respectively. Results Analytical variability was low for all investigated parameters in technical replicates. IIV was assessed for basic semen parameters and proteins in SPs and EVs: 319 and 777 proteins, respectively, using untargeted analysis; 9 and 10 proteins using targeted quantification. Also, the IVV for sncRNA, including miRNA, piRNA, tRNA, and tRNA-derived small RNA (tsRNA) was described in EVs (409 sncRNA and 78 tsRNA) and in sperm (265 sncRNA and 15 tsRNA). We identified 22 and 27 non-overlapping proteins in SP and EVs, respectively, and 46 and 9 sncRNA, including 5 and zero tsRNA in seminal EVs and sperm, respectively, with low variability. The fatty acid synthase (FAS) has the lowest intra-individual variability in both media in targeted protein quantification. Discussion We identified a number of proteins and sncRNA with low variability among 111 proteins, 176 sncRNA, and 12 tsRNA which were previously suggested as biomarkers of male fertility and reproductive outcomes: TRFL, CRIS3, AACT, ESPB1, GSTM3, A1AG2, SAMP, AMPN, NEP, FAS, and miR-10b-3p, miR-122-5p, miR-205-5p, miR-222-3p, miR-34c-5p, miR-509-3-5p, miR-888-5p, miR-892a, miR-363-3p, miR-941, miR-146a-5p, miR-744-5p. Conclusion These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health.