This observational study will evaluate whether sperm cryopreservation can help preserve sperm quality and molecular characteristics that may be affected by increasing paternal age. Men aged 18 years and older will provide a semen sample and a blood sample for laboratory analysis. Each semen sample will be divided into fresh, slow-frozen, and vitrified aliquots. The study will compare sperm quality (as assessed by computer-assisted sperm analysis), DNA methylation profiles, DNA fragmentation, sperm telomere length before and after cryopreservation. The effects of age on these parameters will also be assessed. The impact of age on seminal plasma extracellular vesicle composition and blood cell telomere length will also be analysed. The goal is to determine whether sperm cryopreservation may represent a strategy for preserving sperm characteristics associated with younger paternal age.
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.
Study Type
OBSERVATIONAL
Enrollment
45
IVI Murcia
Murcia, Murcia, Spain
RECRUITINGSperm DNA methylation profile
DNA methylation levels of a predefined nine-gene panel measured by pyrosequencing with duplicate measurements and compared between fresh, slow-frozen, and vitrified sperm samples.
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Sperm concentration
Sperm concentration (million sperm/mL) measured using computer-assisted sperm analysis (CASA).
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Total sperm motility
Percentage of motile sperm measured using CASA.
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Progressive sperm motility
Percentage of progressively motile sperm measured using computer-assisted sperm analysis (CASA).
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Normal sperm morphology
Percentage of sperm with normal morphology assessed by microscopic evaluation of stained slides according to World Health Organization (WHO) criteria.
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Sperm DNA fragmentation
DNA fragmentation measured in fresh, slow-frozen, and vitrified sperm samples.
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Sperm telomere length
Sperm telomere length measured in fresh, slow-frozen, and vitrified sperm samples.
Time frame: Baseline (study semen collection visit) and after thawing of cryopreserved semen samples, through study completion (up to 9 months).
Seminal plasma extracellular vesicle composition
Characterization of seminal plasma extracellular vesicle composition in fresh semen samples.
Time frame: Baseline (study semen collection visit).
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