First study to evaluate possible effects of in utero DES exposure on genome-wide DNA methylation in humans
2015 Study Abstract
In utero exposure to diethylstilbestrol (DES) has been associated with increased risk of adverse health outcomes such as fertility problems and vaginal as well as breast cancer. Animal studies have linked prenatal DES exposure to lasting DNA methylation changes. We investigated genome-wide DNA methylation and in utero DES exposure in a sample of non-Hispanic white women aged 40–59 years from the Sister Study, a large United States cohort study of women with a family history of breast cancer. Using questionnaire information from women and their mothers, we selected 100 women whose mothers reported taking DES while pregnant and 100 control women whose mothers had not taken DES. DNA methylation in blood was measured at 485,577 CpG sites using the Illumina HumanMethylation450 BeadChip. Associations between CpG methylation and DES exposure status were analyzed using robust linear regression with adjustment for blood cell composition and multiple comparisons. Although four CpGs had p<105, after accounting for multiple comparisons using the false discovery rate (FDR), none reached genome-wide significance. In conclusion, adult women exposed to DES in utero had no evidence of large persistent changes in blood DNA methylation.
Previously reported candidate genes affected by DES
We also specifically report on nine genes (EMB, WNT11 TGFB1, ERBB2, EGFR, LTF, EGF, FOS, and JUN) that have previously been shown to have differential gene expression in mice uterine or vaginal tissue following pre or perinatal exposure to DES :
- Expression of estrogen receptor and proto-oncogene messenger ribonucleic acids in reproductive tracts of neonatally diethylstilbestrol-exposed female mice with or without post-puberal estrogen administration.
- Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes.
- Developmental exposure to diethylstilbestrol elicits demethylation of estrogen-responsive lactoferrin gene in mouse uterus.
- Persistent gene expression in mouse vagina exposed neonatally to diethylstilbestrol.
- Effects of neonatal diethylstilbestrol on c-fos and c-jun protooncogene expression in the mouse uterus.
- Developmental exposure to diethylstilbestrol alters uterine gene expression that may be associated with uterine neoplasia later in life.
- Wnt family genes and their modulation in the ovary-independent and persistent vaginal epithelial cell proliferation and keratinization induced by neonatal diethylstilbestrol exposure in mice.
We compared the mean β-value for 75 CpGs in the 5’ region (as annotated by Illumina) of these genes for women with and without DES exposure histories.
Epigenome wide study
In our primary model, adjusted for multiple testing and blood cell composition, four CpGs in two different genes (KIFC3, and DCAKD) had nominal p values < 10-5, and an additional 18 CpGs in 18 different genes had nominal p values < 10-4 (featured image). None of these 22 CpGs achieved genome-wide significance after considering multiple comparisons (q<0.05). Additional adjustments for age at menarche, BMI and parity did not affect the results.
In this study we used DNA from blood rather than from a known target tissue such as vagina, cervix or endometrium. Histologic changes in target tissues, including vaginal epithelium, are known to correlate with dose of DES exposure and with increased risk of adverse outcomes such as vaginal carcinoma. Although it may be possible to compare methylation in target tissues using archival formalin-fixed paraffin-embedded (FFPE) samples, it is difficult to find adequate FFPE samples from DES-exposed women. In addition, methylation analysis of degraded and cross-linked FFPE samples present substantial technical challenges.
In conclusion, although we cannot rule out the possibility of effects at younger ages or in other tissues, our study finds no evidence of large persistent effects of in utero DES exposure on blood DNA methylation.
Sources and more information
- Full text (free access) : In Utero Exposure to Diethylstilbestrol and Blood DNA Methylation in Women Ages 40–59 Years from the Sister Study, PLOS one, doi.org/10.1371/journal.pone.0118757, March 9, 2015.
- Log10 transformed DES association p-values for individual CpGs are plotted in relation to their chromosome location featured image credit journal.pone.0118757.