Age-Dependent Alterations in Semen Parameters and Human Sperm MicroRNA Profile

Abstract

The trend to delay parenthood is increasing, impacting fertility and reproductive outcomes. Advanced paternal age (APA), defined as men’s age above 40 years at conception, has been linked with testicular impairment, abnormal semen parameters, and poor reproductive and birth outcomes. Recently, the significance of sperm microRNA for fertilization and embryonic development has emerged. This work aimed to investigate the effects of men’s age on semen parameters and sperm microRNA profiles. The ejaculates of 333 Portuguese men were collected between 2018 and 2022, analyzed according to WHO guidelines, and a density gradient sperm selection was performed. For microRNA expression analysis, 16 normozoospermic human sperm samples were selected and divided into four age groups: ≤30, 31–35, 36–40, and >40 years. microRNA target genes were retrieved from the miRDB and TargetScan databases and Gene Ontology analysis was performed using the DAVID tool. No significant correlation was found between male age and conventional semen parameters, except for volume. Fifteen differentially expressed microRNAs (DEMs) between groups were identified. Enrichment analysis suggested the involvement of DEMs in the sperm of men with advanced age in critical biological processes like embryonic development, morphogenesis, and male gonad development. Targets of DEMs were involved in signaling pathways previously associated with the ageing process, including cellular senescence, autophagy, insulin, and mTOR pathways. These results suggest that although conventional semen parameters were not affected by men’s age, alterations in microRNA regulation may occur and be responsible for poor fertility and reproductive outcomes associated with APA.