Reproductive factors have been linked to both breast cancer and DNA methylation, suggesting methylation as an important
mechanism by which reproductive factors impact on disease risk. However, few studies have investigated the link between
reproductive factors and DNA methylation in humans. Genome-wide methylation in peripheral blood lymphocytes of 376
healthy women from the prospective EPIC study was investigated using LUminometric Methylation Assay (LUMA). Also,
methylation of 458877 CpG sites was additionally investigated in an independent group of 332 participants of the EPIC-Italy
sub-cohort, using the Infinium HumanMethylation 450 BeadChip. Multivariate logistic regression and linear models were
used to investigate the association between reproductive risk factors and genome wide and CpG-specific DNA methylation,
respectively. Menarcheal age was inversely associated with global DNA methylation as measured with LUMA. For each
yearly increase in age at menarche, the risk of having genome wide methylation below median level was increased by 32%
(OR:1.32, 95%CI:1.14–1.53). When age at menarche was treated as a categorical variable, there was an inverse dose-response
relationship with LUMA methylation levels (OR12–14vs.#11 yrs:1.78, 95%CI:1.01–3.17 and OR$15vs.#11 yrs:4.59, 95%CI:2.04–
10.33; P for trend,0.0001). However, average levels of global methylation as measured by the Illumina technology were not
significantly associated with menarcheal age. In locus by locus comparative analyses, only one CpG site had significantly
different methylation depending on the menarcheal age category examined, but this finding was not replicated by
pyrosequencing in an independent data set. This study suggests a link between age at menarche and genome wide DNA
methylation, and the difference in results between the two arrays suggests that repetitive element methylation has a role in
the association. Epigenetic changes may be modulated by menarcheal age, or the association may be a mirror of other
important changes in early life that have a detectable effect on both methylation levels and menarcheal age.