Urinary concentrations of phthalate and phthalate alternative metabolites and sperm DNA methylation: A multi-cohort and meta-analysis of men in preconception studies

Phthalates are ubiquitous pollutants in the environment; however, the mechanisms of phthalate-associated reproductive disorders in men are not fully understood. The aim of this study is to investigate associations between urinary phthalate metabolite concentrations and sperm DNA methylation. The study was conducted on 697 men from three prospective pregnancy cohorts: Longitudinal Investigation of Fertility and the Environment (LIFE) Study, Sperm Environmental Epigenetics and Development Study (SEEDS), and Environment and Reproductive Health (EARTH) Study. Eighteen phthalate and two phthalate alternative metabolites were quantified by mass spectrometry in preconception urinary samples and sperm DNA methylation was measured via Illumina EPIC Array (v1). Regional methylation analyses were conducted to identify cohort-specific loci associated with urinary phthalate metabolites. Models were adjusted for age, body mass index (BMI), race, smoking status, urinary creatinine/specific gravity, and analytical batch for phthalate measurements. The cohort- specific results were meta-analyzed using METAL. Participants had an average age of 30 years, most (79.6 %) of whom had BMI>25 kg/m(2) and were non-smokers (90.1 %). A total of 7,979 differentially methylated regions (DMRs; 7,979 LIFE-specific DMRs, 72 SEEDS-specific DMRs, and 23 EARTH-specific DMRs) were associated with urinary MBzP, MiBP, MMP, MCNP, MCPP, MBP, and MCOCH. Meta-analysis identified fewer DMRs than cohort- specific models: 946 DMRs were associated with MBzP, 27 DMRs associated with MiBP, and 1 DMR associated with MEHP. The majority of cohort-specific and meta-analysis-derived DMRs displayed a positive association with phthalate metabolite concentrations and were enriched in genes associated with spermatogenesis, response to hormones and their metabolism, embryonic organ development and developmental growth. In conclusion, several preconception urinary phthalate metabolites were associated with increased DNA methylation patterns in sperm. These findings provide an epigenetic pathway by which environmental phthalate exposures can impact couples' reproductive outcomes.