Conserved shifts in sperm small non-coding RNA profiles during mouse and human aging

Sperm aging impacts male fertility and offspring health, highlighting the need for reliable aging biomarkers to guide reproductive decisions. However, the molecular determinants of sperm fitness during aging remain ill-defined. Here, we profiled sperm small non-coding RNAs (sncRNAs) using PANDORA-seq, which overcomes RNA modification–induced detection bias to capture previously undetectable sncRNA species associated with mouse and human spermatozoa throughout the lifespan. We identified an “aging cliff” in mouse sperm RNA profiles—a sharp age-specific transition marked by significant shifts in genomic and mitochondrial tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs). Notably, rsRNAs in mouse sperm heads exhibited a transformative length shift, with longer rsRNAs increasing and shorter ones decreasing with age, suggesting altered biogenesis or processing with age. Remarkably, this sperm head-specific shift in rsRNA length was consistently observed in two independent human aging cohorts. Moreover, transfecting a combination of tsRNAs and rsRNAs resembling the RNA species in aged sperm was able to induce transcriptomic changes in mouse embryonic stem cells, impacting metabolism and neurodegeneration pathways, mirroring the phenotypes observed in offspring fathered by aged sperm. These findings provide novel insights into longitudinal dynamics of sncRNAs during sperm aging, highlighting an rsRNA length shift conserved in mice and humans. The molecular determinants of sperm fitness during aging remain ill-defined. Here, advanced sequencing to profile sperm small non-coding RNAs (sncRNAs) during mouse aging uncovers a sperm-head–specific shift in RNA length, which is conserved in humans and functionally reprograms mouse embryonic stem cell (mESC) gene expression. PANDORA-seq reveals a non-linear transition in sperm tRNA-derived small RNA (tsRNA) and rRNA-derived small RNA (rsRNA) composition between middle and advanced age, referred to as “aging cliff”, which is not seen by conventional sequencing. The aging-induced, sperm-head–specific rsRNA length shift is a conserved aging signature found in mice and two independent human cohorts. Mitochondrial tsRNAs and rsRNAs detected in sperm heads effectively distinguish age groups, suggesting mitochondria-to-nucleus signaling in sperm aging. tsRNA/rsRNA mixtures mimicking aged sperm profiles can reprogram the transcriptome of mESCs to alter metabolic and neurodegenerative pathways, mirroring offspring phenotypes from aged sperm. PANDORA-seq reveals a non-linear transition in sperm tRNA-derived small RNA (tsRNA) and rRNA-derived small RNA (rsRNA) composition between middle and advanced age, referred to as “aging cliff”, which is not seen by conventional sequencing. The aging-induced, sperm-head–specific rsRNA length shift is a conserved aging signature found in mice and two independent human cohorts. Mitochondrial tsRNAs and rsRNAs detected in sperm heads effectively distinguish age groups, suggesting mitochondria-to-nucleus signaling in sperm aging. tsRNA/rsRNA mixtures mimicking aged sperm profiles can reprogram the transcriptome of mESCs to alter metabolic and neurodegenerative pathways, mirroring offspring phenotypes from aged sperm. Longitudinal PANDORA-seq profiling of small non-coding RNAs in spermatozoa uncovers unexpected dynamics during lifespan with functional relevance for gene expression.