Folic acid throughout pregnancy: too much?

In the United States and multiple other countries across the globe, like it or not, every mouthful of bread and morning cereal is fortified with folic acid. Folate, or vitamin B-9, is endogenously found in foods such as dark-green leafy vegetables, legumes, and organ meats. However, its synthetic form, folic acid, is now broadly used in vitamin supplementation and food enrichment around the world. In 1965, the link between folate deficiencies and neural-tube defects (NTDs) was first discovered. NTDs result from the failure of neural tube closing in early embryonic development, which leads to neural tissue damage (1–3). Since then, there has been a series of public health campaigns centered around increasing folic acid intake in the general community, with a focus on women of reproductive age. For example, in 1991, the CDC (4, 5) issued a recommendation that women who previously experienced a pregnancy with an NTD should consume 4000 μg folic acid as soon as they begin to try to conceive again. In 1992, the US Public Health Service followed up on the CDC's recommendation when they announced that all women of childbearing age should consume 400 μg folic acid/d, a recommendation that was again reinforced in 2009 (4–6). Between 1996 and 1998, mandatory fortification of food with folic acid was established, largely to ensure that women who unknowingly became pregnant (approximately half of pregnancies in the United States) would have sufficient folic acid intake for NTD prevention. This mandatory fortification protocol added 140 μg folic acid for every 100 g of cereal grain product. This provides an additional 100–200 μg folic acid/d to women of reproductive age. These interventions have proven to be successful at decreasing the incidence of NTDs globally.

What is the mechanistic basis for the need for folate? Folate plays an important role in many biological pathways, including one-carbon metabolism where folate serves as a coenzyme essential for the production of S-adenosylmethionine (SAM). SAM serves as a universal methyl donor and is a critical component of epigenetic regulation via DNA methylation. Epigenetic regulation of genes refers to the ability of gene expression to be altered, in a somatically heritable fashion, without changing the actual sequence of DNA. Environmental influences, including diet, can initiate epigenetic changes that may lead to altered regulation of genes. Studies are beginning to support that 1 link between NTDs and folate deficiency is through altered DNA methylation (7, 8). This was the impetus for additional studies that examined the link between maternal folate status and NTDs and cognitive status in offspring.

The article by Caffrey et al. (9) in this issue of the Journal was the first reported randomized controlled trial to assess potential causal links between maternal folate status during pregnancy and levels of DNA methylation in offspring in genes involved in brain development. Specifically, the authors sought to determine if there were any epigenetic consequences of continued folate supplementation in neurodevelopmental genes through the second and third trimesters of pregnancy. Current recommendations that include supplementation before and during the first trimester are most critical, because this is when the neural tube forms. Women from the Folic Acid Supplementation in the Second and Third Trimesters (FASSTT) (8) cohort were enrolled and randomly assigned to take either folate supplements or placebo, beginning in the second trimester. The FASSTT participants had all used the recommended dose of folic acid during the first trimester.

The authors focused attention on epigenetically regulated genes that are involved in brain development and function. They paradoxically found significantly lower levels of cord blood DNA methylation at long-interspersed nuclear element 1 (LINE-1), insulin-like growth factor 2 (IGF-2), and brain-derived neurotrophic factor (BDNF) from infants born to mothers who continued folic acid supplementation compared with the placebo controls. They also found sex-specific differences in methylation of these genes, with decreased methylation only in female offspring for IGF2 and only in males for BDNF. The underlying reason for this sex difference is currently unknown, although it is interesting given the sex-specific discrepancies that exist in many disorders associated with cognition and behavior. On the basis of the randomized controlled study design reported in this article, the authors were able to determine that these changes in DNA methylation were directly influenced by the amount of folic acid taken, and they were not just correlational. It is curious that prolonging the use of a supplement that is assumed to increase the availability of the universal methyl donor SAM would lead to an overall decrease in DNA methylation of the genes that were investigated in this study. Aside from deficiency of key enzymes in the one-carbon metabolism pathway leading to decreased methylation in the context of excess folate (10), mathematical modeling indicates that folate excess within cells is inhibitory to any folate-dependent enzymes (11), thus reducing the supply of available methyl donors. Reduced methylation at IGF2 gene regulatory regions with increased folate intake during pregnancy has also been reported by our group and others (12, 13).

The authors also analyzed the effects of vitamin B-12 and cesarean delivery status on DNA methylation. Vitamin B-12 also plays a role in one-carbon metabolism, ultimately contributing to the availability of methyl donors, whereas infants born via cesarean delivery were previously shown to have different levels of DNA methylation in their hematopoietic stem cells and white blood cells than infants delivered vaginally (14–16). They report that cesarean delivery was a significant determinant of DNA methylation levels of LINE-1 and BDNF genes in the offspring, whereas vitamin B-12 concentrations in cord blood were significantly associated with levels of DNA methylation of IGF2. Sex-specific effects for these correlations were not determined.

Although this study (9) was the first of its kind to assess a causative relation between maternal folate supplementation and DNA methylation in cord blood of offspring, there are some larger questions that remain unanswered about the practice of folate supplementation in general. What was the rationale for continuing folic acid supplementation beyond the first trimester? If the neural tube is finished forming early in the first trimester, are there unintended consequences of continuing supplementation on outcomes in children? It will be important for the children born to mothers who received extended folic acid supplementation to be followed for cognitive and social development.

Little is known about how altering the methylation status of genes critical to neuronal function and cognition changes gene expression. Examining this relation with the use of in vitro and in vivo models with varying amounts of folic acid is a critical next step to determine relevance and importance to downstream outcomes. In addition, we do not fully understand the consequences of population-level fortification from food-enrichment programs. There is conflicting evidence about the role that increased folic acid supplementation plays in colon cancer risk, which may be particularly relevant given the potential for reduced DNA methylation in an excess of folate. Loss of methylation throughout the genome is indeed one of the hallmarks of cancer. Folic acid supplementation during the first trimester of pregnancy has been a public health success through the reduction in the incidence of NTDs. However, there are still avenues to be explored when considering the long-term effects of increasing folic acid consumption in the population.

Notes

Supported by the National Institute of Environmental Health Sciences award P01ES022831, USEPA grant RD-83543701 and by the John Templeton Foundation.

Abbreviations used: BDNF, brain-derived neurotrophic factor; IGF2, insulin-like growth factor 2; NTD, neural-tube defect; SAM, S-adenosylmethionine.