Interindividual variability of sperm DNA methylation is associated with reduced fecundability in couples attempting natural conception.

STUDY QUESTION: Is sperm DNA methylation variability, referred here as sperm Epigenetic Variability Outliers and Stochasticity (EVOS), associated with time-to-pregnancy (TTP) and pregnancy outcomes in couples attempting to conceive naturally? SUMMARY ANSWER: Higher sperm EVOS is linked to reduced fecundability in couples attempting to conceive naturally. WHAT IS KNOWN ALREADY: Sperm methylation variability in promoter regions has been linked to poor reproductive outcomes in the clinical population. STUDY DESIGN, SIZE, DURATION: The study was conducted among couples from the Longitudinal Investigation of Fertility and the Environment (LIFE) Study (n = 372), which comprised couples attempting natural conception. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm EVOS was calculated as the sum of sperm DNA methylation CpG outliers on the Illumina EPIC (v1) array. The association of log-transformed sperm EVOS and TTP was examined using Cox proportional hazards model (CoxPH) and Kaplan-Meier survival analysis (KM), and by comparing pregnancy rates at 3, 6, and 12 months of follow-up. Associations between sperm EVOS and pregnancy outcomes (gestational age, live birth, and newborn metrics) were also investigated using multivariable linear or logistic regression. Models were performed with and without adjustments for potential confounders, including male and female age, BMI, smoking status, and average methylation at the DLK1 region as an indicator for potential somatic cell contamination. Sensitivity analysis was restricted to couples with normospermic men based on 5th percentile values from the World Health Organization (WHO) 2010. The functional and biological relevance of CpGs identified with EVOS were evaluated using Metascape. MAIN RESULTS AND THE ROLE OF CHANCE: Sperm EVOS displayed a right-skewed distribution, with median numbers of EVOS CpGs of 673. Men from non-pregnant couples had higher median sperm EVOS than men from pregnant couples at 3, 6, and 12 months (P < 0.05). In both crude and adjusted CoxPH, sperm EVOS was associated with reduced fecundability of couples attempting natural conception (adjusted fecundability odds ratio (FOR) = 0.79; 95% CI: 0.67, 0.94; P = 0.01), indicating a longer TTP. KM analysis revealed that couples in the highest quartile (Q4) of EVOS had longer times to pregnancy compared to those in the middle (Q2+Q3) and lowest (Q1) quartiles (P = 0.001), with respective 66% (95% CI: 53%, 75%), 81% (95% CI: 73%, 87%), and 86% (95% CI: 76%, 91%) predicted probabilities of conceiving within 12 months for each group. No association was found between sperm EVOS and live birth, gestational age or newborn metrics. Functional enrichment analysis revealed that most variable EVOS-associated sites were enriched in genes involved in glycolysis, carbohydrate metabolism, DNA damage checkpoint signaling, and ciliary landscape pathways. Sensitivity analysis restricted to couples of men classified as normospermic (n = 285) according to 2010 WHO criteria indicated a similar inverse association between sperm EVOS and couple fecundability (FOR = 0.82, 95% CI: 0.68, 0.99; P = 0.04). LARGE SCALE DATE: Sperm DNA methylation data are available at the GEO repository GSE185445. LIMITATIONS, REASONS FOR CAUTION: Identification of normospermic men was performed irrespective of sperm motility, which was measured after overnight transport on ice. WIDER IMPLICATIONS OF THE FINDINGS: Sperm EVOS captures inter-individual epigenetic variability characterized by the accumulation of deviations from normal site-specific DNA methylation patterns throughout the sperm methylome, which may reflect DNA damage and alterations in epigenetic maintenance. Our findings indicate that the accumulation of DNA methylation outliers in sperm is associated with lower pregnancy probabilities in the general population. Such findings suggest that sperm EVOS may represent a novel epigenetic biomarker of fecundability in couples attempting natural conception, including couples in which men are classified as having normal semen parameters. FUNDING: This work was funded in part by grants from the National Institutes of Health (R01-ES028298 J.R.P.) and the Robert J. Sokol, MD Endowed Chair of Molecular Obstetrics and Gynecology (J.R.P.). DISCLOSURES: The authors declare no competing financial interests or personal interests. TRIAL REGISTRATION NUMBER: N/A.