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

Abstract

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.

Germ Cell Drivers: Transmission of Preconception Stress Across Generations

“…Even as some evidence has confirmed that epigenetic modifications identified in sperm alter offspring development in a meaningful way, the biological mechanism remains unclear for how these epigenetic changes impact offspring development. The prevailing thought is that sncRNA alter the developmental trajectory of the offspring by initiating a cascade of molecular events that propagate the stress signal, shifting the timing of developmental events and guiding development toward a set of neurobiological, physiological, and behavioral traits that are more adaptive in a stressful environment (Morgan et al, 2019(Morgan et al, , 2020; see Figure 3).…”

“…Even relatively minor modifications in sperm sncRNA content could shift timing of developmental FIGURE 3 | Hypothesized model of the impact of stress on sperm small non-coding RNA content on developmental trajectories, highlighting the fact that relatively minor changes in sperm small non-coding RNA (as indicated by α1 or α2) could shift the timing of developmental events (e.g., embryo division and implantation) in ways that produce significant differences over time. Reprinted from Morgan et al (2020). Repeated sampling facilitates within-and between-subject modeling of the human sperm transcriptome to identify dynamic and stress-responsive sncRNAs.…”

“…In human studies of male preconception stress on sperm epigenetic changes, research suggests that trauma in childhood and perceived stress in adulthood are associated with changes in the sncRNA content of sperm ( Dickson et al, 2018 ; Morgan et al, 2020 ). In a cross-sectional study, a higher number of adverse childhood experiences (ACEs; e.g., abuse, neglect, family dysfunction in childhood) correlated with lower levels of multiple miRNA in sperm ( Dickson et al, 2018 ).…”

“…In a cross-sectional study, a higher number of adverse childhood experiences (ACEs; e.g., abuse, neglect, family dysfunction in childhood) correlated with lower levels of multiple miRNA in sperm ( Dickson et al, 2018 ). In the first longitudinal study to measure changes in sperm sncRNA populations over time in healthy adult men, each month for 6 months sperm samples were collected and perceived stress was assessed ( Morgan et al, 2020 ). Five miRNA and four tRNA in sperm fluctuated with perceived stress in a dynamic manner, suggesting that they were responsive to stress.…”

“…Unlike DNA methylation, sncRNA are not required to be a self-perpetuated signal following fertilization or during development as they have an immediate function at conception and, therefore, have emerged as causal agents of stress signal transmission ( Meikar et al, 2011 ; Morgan et al, 2019 , 2020 ; Chan et al, 2020 ). In brief, sncRNA are short RNA that are less than 200 nucleotides in length and are considered “non-coding” because they typically are not translated into proteins ( Marcho et al, 2020 ).…”