Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.