Background: Almost all species show sexual discordance in many traits and diseases. DNA methylation is known to contribute to these differences through well-established mechanisms including X-inactivation in females, imprinting and parent-of-origin effects.Sex hormones also appear to influence sexual discordance by interacting directly with the DNA methylation pathways. However, many of the population studies reporting differences in DNA methylation patterns between sexes do so as a secondary observation. More importantly, there is little agreement about the extent or strength of the discordances, or whether they change over the life course or in response to exposures. Methods: In a sample of 700 individuals from the Avon Longitudinal Study of Parents and Children, we used multi-level regression models to identify methylation differences between sexes at birth, childhood and late adolescence, and to identify changes in these differences over time. CpG sites on the sex chromosomes were excluded, along with CpGs in autosomal regions homologous with sex chromosomal regions or near common SNPs. Results: We show that autosomal sex-discordant methylation is widespread, affecting approximately 12,000 CpG sites at each time point, and stable; at least 8,500 sites are consistently different across all time points. Just over 1,000 methylation differences change from birth to late adolescence, 90% of these between birth and around age seven. Sexually discordant CpG sites are enriched in genomic loci containing androgen but not estrogen targets and in genes involved in tissue development but not housekeeping functions. There are large overlaps with sex-discordant loci in both fetal and adult brain cortex. Variance at these sites tends to be greater in males than in females. A methylation-derived sex score capturing the variance was calculated at each time point and found to be highly correlated between time points, with higher correlation in females than in males. This score is nominally associated with sex hormone levels in childhood as well as some phenotypes previously linked to sex hormone levels. Conclusions: These findings suggest that sex- discordant autosomal DNA methylation is widespread throughout the genome, likely due to the first androgen exposures in utero. It is then stably maintained from birth to late adolescence despite dramatically increased sexual dimorphism in the levels of the very same hormones. Methylation variation at sex-discordant sites within the sexes, as summarized by the methylation sex score, likely reflects in utero androgen exposure which is relevant to human health.

Sex-associated autosomal DNA methylation differences are wide-spread and stable throughout childhood