Background: There is growing interest on the epigenetic effects of in-utero environmental exposures on DNA methylation and its relation to childhood or adult-life health outcomes. The placenta is a well-established target of environmental toxicants that can be easily collected in epidemiological studies. However, no study has yet analyzed to what extent storage conditions of placental tissue after delivery affect DNA methylation. We designed a study to address this issue providing informative results for on-going and future cohort studies. Methods: Two full term placentas were biopsied in three different regions of the villous parenchyma. Biopsies were sequentially stored at -80°C after standing at room temperature (RT) for 30 minutes, 1 hour, 2 hours, 6 hours and 24 hours. Global DNA methylation was measured by two common techniques: analysis of retrotransposons by pyrosequencing (10 different repetitive elements in LINE, Alu and HERV families) and the Luminometric Methylation Assay (LUMA). Time-to storage at RT, technique-related and regional variability in methylation levels were compared by coefficients of variation (COV). Results: The time-to storage COV was 2.49% (placenta 1) and 2.86% (placenta 2) 5-methylcytosine (5mC), similar to the mean technical variation we observed for the pyrosequencing technique (COV=1.91% and 1.51% for each placenta, respectively). LUMA yielded more variable results, both among time points and as well as between technical replicates. Conclusion: DNA methylation levels are not affected by storage delays at RT. DNA methylation studies using samples stored in unknown or suboptimal conditions should avoid using techniques sensitive to DNA degradation such as the LUMA Assay.
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