The key finding of our study is that periconceptional folic acid use of the mother is related to an increased methylation of the IGF2
DMR of the child. The reported stability of IGF2
DMR methylation up to middle age 
supports the interpretation that the IGF2
methylation changes we observed are explained by periconceptional folic acid exposure. The difference in DNA methylation associated with folic acid exposure is remarkably similar to our previous observation of a 5.2% reduced IGF2
methylation after periconceptional exposure to famine 
. The opposite direction of the associations suggests that the availability of methyl donors during the periconceptional period may affect IGF2
DMR methylation. We further hypothesized that changes in IGF2
DMR methylation would influence intrauterine growth. Our study indeed indicated an association between IGF2
DMR methylation and birth weight as surrogate for intrauterine growth, but not between periconceptional folic acid use and birth weight.
Compared to our findings in humans, the size of the effects on DNA methylation of prenatal exposures have found to be comparable in sheep but are larger in rodents 
. In this comparison we have to emphasize that human populations are inevitably more heterogeneous than the inbred rodents kept at the same, well-controlled environmental conditions. The different effects in animal studies can also be due to the common use of a combined intervention consisting of multiple methyl donors and/or protein deficiency instead of folic acid only. Lastly, the larger effects in rodents are shown in other tissues than in blood, which are not readily accessible from human study subjects.
In addition, our study indicated an association between higher IGF2
methylation and lower birth weight. This inverse association is compatible with a relative intrauterine silencing of the embryonic growth factor IGF2
resulting in reduced growth 
. This links our data to the finding that IGF2
loss of imprinting leads to somatic overgrowth (Beckwith-Wiedemann syndrome) 
and possibly colorectal cancer 
although we cannot exclude the explanation that the change in IGF2
methylation marks possible greater changes elsewhere in the genome that underlie the association observed. Periconceptional folic acid use may contribute to the restoration of a loss of imprinting. It remains to be established whether DNA methylation changes contribute to the adverse effects reported for periconceptional folic acid use 
. Studies are required to establish optimal timing, dose and type (natural folate or synthetic folic acid) to prevent birth defects and at the same time minimize adverse effects later in life.
From this study reveals that periconceptional folic acid use is associated with epigenetic changes in IGF2. However, in contrast with large mother-child cohorts, we did not find a positive association between periconceptional folic acid use and higher birth weight 
. It is known that women using periconceptional folic acid supplements are generally more health conscious and higher educated. Furthermore, the women exposed to the Dutch famine were not only deprived of folate, but also of other essential macro- and micronutrients that serve as methyldonors, e.g., methionine. Thus, it should be emphasized that many factors together including other genes besides periconceptional folic acid contribute to birth weight. This may explain the absent association between periconceptional folic acid use and birth weight in our study.
Periconceptional use of folic acid did not affect average levels of the biomarkers SAM, SAH or SAM/SAH measured at 17 months in the mother and in the child. However, we found a significant correlation between the maternal SAM concentration at the study moment and IGF2 methylation of the child. The developmental hypothesis of health and disease states that periconceptional exposures may affect metabolic imprinting of the child. This is in line with our finding that periconceptional folic acid use can affect the metabolic imprinting of the methylation pathway of the child. Of note, this association was not influenced by fortification of food with folic acid which is absent in The Netherlands, UK and other European countries, which may have strengthened the observed associations.
Although we did not measure levels of biomarkers of methylation in the periconception period, their levels will have been comparable to those we measured 17 months after delivery. This is substantiated by Nurk et al. showing that the biomarkers of methylation show a limited variability in the periconception period and over a subsequent period of 1–2 years 
. Furthermore, there are no substantial differences in preconceptional maternal dietary habits and lifestyles and those 1 to 1.5 year post partum which affect these biomarkers 
A limitation of both our study and others is that they relied on genomic DNA extracted from whole blood so that heterogeneity in cell populations may have contributed to the outcomes 
. Our study likely is less sensitive to such heterogeneity because the epigenetic state of imprinted loci is less dependent on cell differentiation and, importantly, a previous study showed that when demethylation of IGF2
DMR was observed in peripheral blood lymphocytes of an individual, this was also found in colon tissue 
, which has a distinct embryologic origin (endoderm and mesoderm, respectively). The finding that the common epigenetic variation in IGF2
DMR might influence birth weight is intriguing but should be interpreted with care because the sample size was relatively small and other (non)genetic factors are also involved. It is currently unknown whether modestly increased IGF2
DMR methylation upregulates IGF2
expression. Furthermore, it has not been established whether such quantitative differences measured in lymphocytes mark a soma-wide phenomenon as was suggested for loss-of-imprinting of the IGF2
. Therefore, in future human studies the sampling of different tissues should be performed.
Our study provides the first evidence that periconceptional folic acid use may be related to DNA methylation in the child. Moreover, such DNA methylation changes may have phenotypic consequences as illustrated by the association between higher IGF2 methylation and decreased birth weight. A simple preventive strategy as periconceptional folic acid use may affect epigenetic control and as such may link the prevention of intrauterine development, i.e., birth defects such as neural tube defects, and growth due to a loss of imprinting with the risk of chronic diseases in these children throughout life. It has to be established how folic acid intake affects the epigenetic regulation of sets of relevant genes and whether adverse effects are to be expected from an altered methylation at such loci. Given the ongoing exposure it is timely to monitor the (long term) effect on DNA methylation also of other lifestyles and environmental influences, such as overnutrition, fortification of food, smoking, stress and the use of assisted reproductive techniques.