ИСТИНА

Introduction: For the identification of small non-coding RNA (sncRNA) as prognostic markers of fertility subjects are usually recruited through infertility clinics. We examine the association of semen quality with sncRNA profile in young healthy adults. Patients and Methods: We selected 49 subjects from the prospective cohort Russian Children’s Study (n=516) enrolled at 8-9 years of age. Puberty was evaluated in annual follow-ups and semen samples were collected at 18-20 years of age. Fresh semen was evaluated for volume, sperm concentration, and motility according to ESHRE manual. Sperm RNA was extracted from 2 layers after centrifugation with 50% and 90% density gradient. Libraries of sncRNA were constructed using NEBNext Kit, n=41 (NEB) and NEXTFLEX® Kit, n=8 (Perkin-Elmer) and sequenced on NextSeq 500 (Illumina). Reads were mapped to hg38 in a sequential order: ribRNA>miRNA >piRNA>tRNA. DESeq2 R package was used for the analysis of differentially expressed sncRNA. Result: Medians (IQR) for age, semen volume, sperm concentration, total motile sperm and total progressive motile sperm (TPMS) were: 18.3 (18.2-19.1) years, 3.3 (2.6-5) ml, 38.5 (22-64.8) mill/ml, 95.1 (51.0-160.2) mill, and 83.4 (46.4-135.7) mill. Mean number of sequenced reads was 6.9 mill, with 65% alignment to sncRNA. 2225 sncRNA were identified with >=10 mean counts: 361 miRNA, 1508 piRNA and 356 tRNA. In models adjusted for library kit, sperm layer and DNAse use we identified 102 sncRNA significantly differentially expressed by TPMS: 10 miRNA, 76 piRNA and 16 tRNA, FDR<0.05. 1282 genes—targets of 5 miRNA differentially expressed by TPMS were highly enriched for developmental categories, response to growth factors and hormones Conclusion: To our knowledge, this is the first study to examine the association of sncRNA profile and semen quality in a cohort of healthy young men. We suggest some candidate markers for evaluating semen quality in young adults. Funding: RSF #18-15-00202; for parent RCS - NIEHS #R01 ES014370.