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 6 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.