Repeated sampling facilitates within- and between-subject modeling of the human sperm transcriptome to identify dynamic and stress-responsive sncRNAs

Epidemiological studies from the last century have drawn strong associations between paternal life experiences and offspring health and disease outcomes. Recent studies have demonstrated sperm small non-coding RNA (sncRNA) populations vary in response to diverse paternal insults. However, for studies in retrospective or prospective human cohorts to identify changes in paternal germ cell epigenetics in association with offspring disease risk, a framework must first be built with insight into the expected biological variation inherent in human populations. In other words, how will we know what to look for if we don’t first know what is stable and what is dynamic, and what is consistent within and between men over time? From sperm samples from a ‘normative’ cohort of healthy human subjects collected repeatedly from each subject over six months, 17 healthy male participants met inclusion criteria and completed donations and psychological evaluations of perceived stress monthly. sncRNAs (including miRNA, piRNA, and tRNA) isolated from mature sperm from these samples were subjected to Illumina small RNA sequencing, aligned to subtype-specific reference transcriptomes, and quantified. The repeated measures design allowed us to define both within- and between-subject variation in the expression of 254 miRNA, 194 tRNA, and 937 piRNA in sperm over time. We developed screening criteria to identify a subset of potential environmentally responsive ‘dynamic’ sperm sncRNA. Implementing complex modeling of the relationships between individual dynamic sncRNA and perceived stress states in these data, we identified 5 miRNA (including let-7f-5p and miR-181a-5p) and 4 tRNA that are responsive to the dynamics of prior stress experience and fit our established mouse model. In the current study, we aligned repeated sampling of human sperm sncRNA expression data with concurrent measures of perceived stress as a novel framework that can now be applied across a range of studies focused on diverse environmental factors able to influence germ cell programming and potentially impact offspring development. Six sperm samples were collected monthly from 17 healthy male participants. sncRNA (including miRNA, piRNA, and tRNA) were isolated from mature sperm from these samples, subjected to Illumina small RNA sequencing, aligned to subtype-specific reference transcriptomes, and quantified.

上个世纪的流行病学研究已明确证实，父代生活经历与子代健康及疾病转归之间存在显著关联。近期研究表明，精子小非编码RNA（small non-coding RNA, sncRNA）的组成会随父代受到的不同应激刺激发生变化。然而，若要通过回顾性或前瞻性人类队列研究，识别与子代疾病风险相关的父代生殖细胞表观遗传改变，首先需要基于人群固有潜在生物学变异的认知，构建相应研究框架。换言之，若我们尚未明确哪些分子表达稳定、哪些具有动态变化，以及不同男性个体间及其随时间推移的表达一致性规律，又该如何确定目标研究对象？本研究的样本来自一项针对健康人群的“规范性”队列：在6个月内对每名受试者重复采集精子样本，最终共有17名符合纳入标准的健康男性参与者完成了每月一次的样本捐献与感知压力心理评估。从上述样本的成熟精子中分离得到的sncRNA（包含microRNA[miRNA]、piwi相互作用RNA[piRNA]与转运RNA[tRNA]），通过Illumina小RNA测序技术进行测序，将测序数据比对至亚型特异性参考转录组并完成表达定量。本次研究采用重复测量设计，得以明确254种miRNA、194种tRNA与937种piRNA在精子中的表达随时间推移产生的个体内与个体间变异情况。我们制定了筛选标准，以识别出潜在受环境调控的“动态”精子sncRNA子集。基于上述数据，通过对单种动态sncRNA与感知压力状态间的关联进行复杂建模分析，我们最终筛选出5种miRNA（包括let-7f-5p与miR-181a-5p）与4种tRNA，它们可响应既往压力经历的动态变化，且契合我们已建立的小鼠模型研究结果。本研究将人类精子sncRNA表达的重复采样数据与同期感知压力测量结果进行关联分析，构建了一套全新的研究框架，该框架可应用于一系列旨在探究能够影响生殖细胞编程、并潜在改变子代发育结局的各类环境因素的相关研究。本研究每月从17名健康男性参与者处采集6份精子样本，从上述样本的成熟精子中分离得到sncRNA（包含miRNA、piRNA与tRNA），通过Illumina小RNA测序技术进行测序，比对至亚型特异性参考转录组并完成表达定量。