Abstract
BackgroundWe analyzed placental DNA methylation levels at repeated sequences (LINE1 elements) and all CCGG sites (the LUMA assay) to study the effect of modifiable clinical or laboratory procedures involved in in vitro fertilization. We included four potential modifiable factors: oxygen tension during embryo culture, fresh embryo transfer vs frozen embryo transfer, intracytoplasmic sperm injection (ICSI) vs conventional insemination or day 3 embryo transfer vs day 5 embryo transfer.ResultsGlobal methylation levels differed between placentas from natural conceptions compared to placentas conceived by IVF. Placentas from embryos cultured at 20% oxygen showed significant differences in LINE1 methylation compared to in vivo conceptions, while those from embryos cultured at 5% oxygen, did not have significant differences. In addition, placentas from fresh embryo transfer had significantly different LINE1 methylation compared to placentas from in vivo conceptions, while embryos resulting from frozen embryos were not significantly different from controls. On sex-stratified analysis, only males had significant methylation differences at LINE1 elements stratified for the modifiable factors. As expected, LINE1 methylation was significantly different between males and females in the control population. However, we did not observe sex-specific differences in the IVF group. We validated this sex-specific observation in an additional cohort and in opposite sex IVF twins.ConclusionWe show that two clinically modifiable factors (embryo culture in 5 vs 20% oxygen tension and fresh vs frozen embryo transfer) are associated with global placental methylation differences. Interestingly, males appear more vulnerable to such treatment-related global changes in DNA methylation than do females.
Highlights
IntroductionWe analyzed placental DNA methylation levels at repeated sequences (LINE1 elements) and all CCGG sites (the Luminometric methylation assay (LUMA) assay) to study the effect of modifiable clinical or laboratory procedures involved in in vitro fertilization
We analyzed placental DNA methylation levels at repeated sequences (LINE1 elements) and all CCGG sites to study the effect of modifiable clinical or laboratory procedures involved in in vitro fertilization
Given the significant difference in parental age, we stratified each population to test for methylation differences associated with parental age (Additional file 1), based on a prior definition of “advanced parental age” (>35 vs ≤35 years; [25])
Summary
We analyzed placental DNA methylation levels at repeated sequences (LINE1 elements) and all CCGG sites (the LUMA assay) to study the effect of modifiable clinical or laboratory procedures involved in in vitro fertilization. We have recently demonstrated, using an analysis of placentas from donor oocyte These observations suggest strongly that some of the clinical or laboratory procedures used in ART are responsible for altering DNA methylation levels at multiple sites in the genome. Given this likelihood, we chose to compare modifications of several ART procedures, individually, for an association with DNA methylation differences. We used surrogate measures of genome-wide DNA methylation as biomarkers of whether exposure to individual clinical or laboratory protocols used in ART might alter epigenetic marks in the placenta. Searching for procedure-specific, site-specific methylation differences using array-based epigenome-wide profiling methods requires significant statistical penalties because of the requirement of correction for multiple testing
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