背景:年轻男性精液参数和射精分子成分的受试者内部变异性仍然知之甚少。
目的:研究年轻男性重复射精的精液参数和分子标记的个体内变异性(IIV)。
方法:从俄罗斯儿童研究的164名18-19岁参与者中收集6-8天的样本中评估精液参数,一个前瞻性队列。配对样品的子集用于精浆(SP)和精浆细胞外囊泡(EV)中蛋白质的无标记定量和靶向质谱,以及使用RNA-seq对电动汽车和精子中的非编码小RNA(sncRNA)进行分析。两次射精之间的平均差异,受试者内部变异,组内相关性,和一致性相关性用于评估所有参数的IIV。如果CVw<15%和ICC>0.90,则分别考虑低变异性、高再现性和高可靠性。
结果:技术重复中所有研究参数的分析变异性都很低。评估了SP和EV中的基本精液参数和蛋白质:319和777蛋白质,分别,使用非靶向分析;9和10蛋白质使用靶向定量。我们还描述了sncRNA的IIV,包括microRNA,piwi相互作用的RNA,tRNA,和电动汽车中的tRNA衍生的小RNA(tsRNA)(409sncRNA和78tsRNA)和精子(265sncRNA和15tsRNA)。我们在SP和EV中鉴定了22和27个非重叠蛋白,分别,和46和9sncRNA,精浆EV和精子中包括5和0个tsRNA,分别,低变异性。在靶向蛋白质定量中,脂肪酸合酶(FAS)在两种培养基中具有最低的IIV。
结论:我们鉴定了许多蛋白质和sncRNA,它们在111种蛋白质中具有低变异性,176sncRNA,和12个tsRNA,以前被认为是男性生育力和生殖结果的生物标志物:乳转铁蛋白,富含半胱氨酸的分泌蛋白3,α-1-抗胰凝乳蛋白酶,附睾精子结合蛋白1,谷胱甘肽S-转移酶Mu3,α-1-酸性糖蛋白2,血清淀粉样蛋白P成分,氨基肽酶N,Neprilysin,FAS,和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。
结论:这些分子具有低IIV,可能是男性生育力和生殖健康的有希望的候选生物标志物。
BACKGROUND: Within-subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood.
OBJECTIVE: To investigate intraindividual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men.
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 (EVs), and for small non-coding RNA (sncRNA) profiling in EVs and spermatozoa 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. We also described the IIV for sncRNA, including microRNA, piwi-interacting RNA, tRNA, and tRNA-derived small RNA (tsRNA) in EVs (409 sncRNA and 78 tsRNA) and in spermatozoa (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 0 tsRNA in seminal EVs and spermatozoa, respectively, with low variability. The fatty acid synthase (FAS) had the lowest IIV in both media in targeted protein quantification.
CONCLUSIONS: 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: lactotransferrin, cysteine-rich secretory protein 3, alpha-1-antichymotrypsin, epididymal sperm-binding protein 1, glutathione S-transferase Mu 3, alpha-1-acid glycoprotein 2, serum amyloid P-component, aminopeptidase N, neprilysin, 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.
CONCLUSIONS: These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health.