Human Sperm Cryopreservation as an Alternative to the Decline of Sperm Quality With Aging

The aging process is associated with declining fertility and increased risks of adverse reproductive and offspring health outcomes. Although the effects of maternal age have been extensively studied, the impact of paternal aging on sperm quality, reproductive outcomes, and molecular characteristics of sperm remains incompletely understood.

The primary objective of this study is to evaluate whether sperm cryopreservation may serve as an alternative to the use of sperm collected at older ages in assisted reproduction treatments. The primary endpoint is the difference in sperm DNA methylation patterns between fresh ejaculated samples and samples subjected to cryopreservation. Cryopreservation will be considered a potentially valid strategy if DNA methylation patterns remain substantially preserved following the cryopreservation process.

At least 45 male participants will be recruited and grouped according to age. Participants will provide one semen sample and a peripheral blood sample. Following routine semen analysis, each semen sample will be divided into three aliquots: fresh, slow-frozen, and vitrified. Cryopreserved aliquots will be stored in liquid nitrogen and subsequently thawed or warmed for analysis.

Standard semen parameters, including volume, concentration, motility, sperm kinetics, and morphology, will be evaluated. Additional analyses will include sperm DNA methylation, DNA fragmentation, telomere length, and characterization of extracellular vesicles and their composition. Identical assessments will be performed on fresh and cryopreserved samples to compare the effects of slow freezing and vitrification.

Secondary objectives include evaluating age-related differences in semen quality and molecular biomarkers, identifying markers associated with biological gamete age, characterizing age-related differences in seminal extracellular vesicles, and exploring associations between sperm molecular characteristics and lifestyle. Participants will also complete validated questionnaires regarding lifestyle and dietary habits.

Inclusion criteria

• Male participants aged 18 to 65 years
• Attending IVI Murcia for semen analysis and/or assisted reproduction treatment
• Able and willing to provide written informed consent
• Willing to provide a semen sample and a blood sample for research purposes

Exclusion criteria

• Previous diagnosis of azoospermia or severe oligozoospermia (sperm concentration <1 million sperm/mL)
• Known chromosomal abnormality (abnormal karyotype)
• Known Y chromosome microdeletion
• Febrile illness within the previous 3 months
• Presence of varicocele
• Current use of vitamin or antioxidant supplements intended to improve semen quality