Normal spermatogenesis in older men is associated with compensatory transcriptome changes.

Male reproductive ageing is a complex process involving progressive and detrimental histological and physiological alterations to the testis and beyond. Age-related morbidities often confound reproductive function, making it difficult to disentangle systemic from reproductive male ageing. We have previously shown that healthy ageing is associated with full spermatogenesis, normal sperm production and hormonal secretion. However, the molecular mechanisms allowing the human testis to age without major loss of function remained elusive. In this study, we investigated the transcriptomic dynamics of the ageing human testis using bulk RNA sequencing of testicular samples with full spermatogenesis from young (24-31 years, n = 4), middle-aged (41-45 years, n = 3), and aged (54-75 years, n = 6) men. We found that, in healthy human testis, ageing is associated with widespread alternative splicing events, affecting genes involved metabolic pathways and DNA repair. Moreover, we identified significant transcriptional changes during ageing, particularly associated with inflammation and oxidative stress. Importantly, a subset of genes showing age-dependent expression patterns was involved in the formation of double-strand breaks (DSBs) and DNA repair and was expressed during early meiosis. Quantification of γH2AX, a marker of DSBs, in an independent validation cohort, did not show age-related abnormal accumulation of DSBs in the germline. These findings provide a comprehensive view of the transcriptional changes occurring during healthy ageing in the human testis, and we hypothesise that these reflect compensatory mechanisms that help preserve reproductive capacity over time in human males.