Sergeyev Oleg, Bezuglov Vitalik, Soloveva Natalia, Smigulina Luidmila, Denisova Tatiana, Dikov Yury, Shtratnikova Victoria, Vavilov Nikita, Williams Paige L, Korrick Susan, Lee Mary M, Zgoda Victor, Hauser Russ, Suvorov Alexander
Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.
Laboratory of Genetics of Reproductive Disorders, Research Centre for Medical Genetics, Moscow, Russia.
Andrology. 2025 May;13(4):840-859. doi: 10.1111/andr.13739. Epub 2024 Sep 4.
Within-subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood.
To investigate intraindividual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men.
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 CV < 15% and ICC > 0.90, respectively.
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.
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.
These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health.
年轻男性精液参数和射精分子成分的个体内变异性仍知之甚少。
研究年轻男性多次射精中精液参数和分子标志物的个体内变异性（IIV）。
在俄罗斯儿童研究中，对164名18 - 19岁的前瞻性队列参与者每隔6 - 8天采集的精液样本进行参数评估。配对样本的子集用于精浆（SP）和精液细胞外囊泡（EVs）中蛋白质的无标记定量和靶向质谱分析，以及使用RNA测序对EVs和精子中的小非编码RNA（sncRNA）进行分析。使用两次射精之间的平均差异、个体内变异、组内相关系数和一致性相关系数来评估所有参数的IIV。如果CV < 15%和ICC > 0.90，则分别认为具有低变异性、高重复性和高可靠性。
在技术重复中，所有研究参数的分析变异性都很低。对基本精液参数以及SP和EVs中的蛋白质进行了IIV评估：分别使用非靶向分析评估了319种和777种蛋白质；使用靶向定量评估了9种和10种蛋白质。我们还描述了EVs（409种sncRNA和78种tsRNA）和精子（265种sncRNA和15种tsRNA）中sncRNA的IIV，包括微小RNA、piwi相互作用RNA、转运RNA和转运RNA衍生的小RNA（tsRNA）。我们分别在SP和EVs中鉴定出22种和27种不重叠的蛋白质，以及在精液EVs和精子中分别鉴定出46种和9种sncRNA，包括5种和0种tsRNA，其变异性较低。在靶向蛋白质定量中，脂肪酸合酶（FAS）在两种介质中的IIV最低。
我们在之前被认为是男性生育力和生殖结果生物标志物的111种蛋白质、176种sncRNA和12种tsRNA中，鉴定出了许多变异性较低的蛋白质和sncRNA：乳铁蛋白、富含半胱氨酸的分泌蛋白3、α-1-抗糜蛋白酶、附睾精子结合蛋白1、谷胱甘肽S-转移酶Mu 3、α-1-酸性糖蛋白2、血清淀粉样P成分、氨肽酶N、中性肽链内切酶、FAS，以及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。
这些分子具有低IIV，可能是男性生育力和生殖健康有前景的候选生物标志物。
