SAT-170 Reproductive hormone levels in seminal plasma, molecular biomarkers in ejaculate components and semen quality among healthy young adults

Abstract Disclosure: O. Sergeyev: None. V. Bezuglov: None. N. Soloveva: None. V. Shtratnikova: None. N. Vavilov: None. A. Zubritskiy: None. M.M. Lee: None. V. Ashapkin: None. V. Zgoda: None. J. Pilsner: None. A. Suvorov: None. Objectives. Recently, we reported the intra-individual variability of molecular markers in repeated ejaculates among healthy young men (Sergeyev et al, 2024). We now comprehensively investigate the association of reproductive hormones in seminal plasma (SP) with semen quality, proteome and small non-coding RNA (sncRNA) profiles in the different components of the same set of ejaculates. Methods. Subsets of 85 ejaculate samples were randomly selected from 443 collected ejaculates by 18-19-year-old participants of the Russian Children’s Study (RCS) prospective cohort for basic semen analysis and reproductive hormone measurements (testosterone (T), estradiol (E2), LH, FSH, DHEAS, prolactin (P)) in SP (Architect i1000SR and chemiluminescent microparticle immunoassay). Intra-assay coefficient of variation for all 6 hormones ranged 0.75-4.9%. In a subset of 10 samples, label-free quantitation (LFQ) and targeted isotope labeling-based quantitation (TILBQ) of 10 proteins in SP and seminal extracellular vesicles (EV), and RNA-seq of sncRNA in EVs and sperm were performed. Data was analyzed by univariate and multivariate linear regression. Results. Median (25-75%) values for total motile sperm were 80.5 (36.9-614) 106/ejaculate and for hormones were T 0.95 (0.68-1.29) nmol/L; E2 118 (97.5-136) pg/mL; LH 0.06 (0.05-0.09) iu/L; FSH 0.25 (0.16-0.51) iu/L; DHEAS 100.7 (52.5-147.2) ug/dL; and P 7.3 (5.5-9.0) ng/mL. Using strict quantification criteria, we identified 420 and 979 proteins by LFQ in SP and EV, respectively; 256 microRNA (miRNA), 62 piwi-interacting RNA (piRNA), 159 mature tRNA, and 77 tRNA-derived small RNA (tsRNA) by RNA-seq in EV and 104 miRNA, 230 piRNA, 136 mature tRNA, and 13 tsRNA in sperm samples. The following significant (p<0.05) positive associations were identified using TILBQ data: E2 with LAMP2, LAMA5, ALDOA, KLK3, RL7 and FAS, and DHEAS with LAMP2 in EV; P and DHEAS with PPAP, P with LAMA5, and DHEAS with LAMP2 in SP. Based on LFQ in SP, T was positively and E2 was negatively associated with 47 and 7 proteins, respectively (p<0.001). Based on LFQ in EV, T, E2 and DHEAS were associated with 15, 9 and 12 proteins, respectively (p<0.001). P was also positively associated with 25 sncRNA in EV (p<0.001). In multivariate models adjusted for BMI and abstinence time, higher P, T and DHEAS in SP were associated with higher total motile sperm, b-estimate (p-value) 10.5 (0.0008), 28.2 (0.04) and 0.34 (0.049), respectively. Conclusions. Our study establishes associations with seminal plasma reproductive hormones, semen quality and molecular biomarkers that might have diagnostic utility in the evaluation of male fertility. Funding. Russian Foundation for Basic Research (RFBR) #20-015-00458 - proteome; Russian Science Foundation (RSF), #18-15-00202 - sncRNA; and the National Institute of Environmental Health Sciences (NIEHS), USA, #R01 ES014370 - parent RCS. Presentation: Saturday, July 12, 2025