Folic acid supplementation in pregnancy and implications in health and disease

Abstract

Maternal exposure to dietary factors during pregnancy can influence embryonic development and may modulate the phenotype of offspring through epigenetic programming. Folate is critical for nucleotide synthesis, and preconceptional intake of dietary folic acid (FA) is credited with reduced incidences of neural tube defects in infants. While fortification of grains with FA resulted in a positive public-health outcome, concern has been raised for the need for further investigation of unintended consequences and potential health hazards arising from excessive FA intakes, especially following reports that FA may exert epigenetic effects. The objective of this article is to discuss the role of FA in human health and to review the benefits, concerns and epigenetic effects of maternal FA on the basis of recent findings that are important to design future studies.

Full text

R E V I E W Open Access
Folic acid supplementation in pregnancy and
implications in health and disease
Subit Barua
*
, Salomon Kuizon and Mohammed A Junaid
Abstract
Maternal exposure to dietary factors during pregnancy can influence embryonic development and may modulate
the phenotype of offspring through epigenetic programming. Folate is critical for nucleotide synthesis, and
preconceptional intake of dietary folic acid (FA) is credited with reduced incidences of neural tube defects in
infants. While fortification of grains with FA resulted in a positive public-health outcome, concern has been raised
for the need for further investigation of unintended consequences and potential health hazards arising from
excessive FA intakes, especially following reports that FA may exert epigenetic effects. The objective of this article is
to discuss the role of FA in human health and to review the benefits, concerns and epigenetic effects of maternal
FA on the basis of recent findings that are important to design future studies.
Keywords: Folic acid, DNA methylation, Epigenetic, Imprinting, Prenatal nutrition, Neural tube defects, Autism
Review
Introduction
The emerging view of epidemiological studies indicates
the importance of the intrauterine environment in early
fetal development. With increased understanding of the
fundamental mechanism, appropriate DNA methylation
including the proper functioning of the epigenetic ma-
chinery is highlighted to be essential for embryogenesis
and adult health [1-3]. Thus, FA has gained considerable
attention because of its promising role in modulating di-
verse clinical conditions, whereas folate deficiency has
been linked with a variety of disorders including birth
defects and defects in the development of neural tube
closure [4-6]. As stated by Hippocrates nearly 2,500 years
ago: “Let food be thy medicine and medicine be thy food”
the mandatory FA fortification appears to be a first mod-
ern attempt to design a strategy for using food for the
prevention or treatment of developmental defects. How-
ever, this mandate has resulted in an increase in folate
level in the serum to approximately 19 ng/ml, which is
above the normal range of the serum folate concentra-
tions in humans i.e. 2.7-17 ng/ml [7]. Epidemiological
studies have shown that a significant number of women
who took FA supplements during pregnancy exceeded
the Institute of Medicine's recommended tolerable
upper limit of 1,000 μg/day. In addition, studies also
reported consumption of 400 μg/day of natural food
folate plus FA-containing prenatal supplements resulted
in supra-nutritional folate status with the greatest in-
creases in pregnant women followed by lactating and
non-pregnant women [8,9]. Concern has been raised if
such exposure as a result of FA fortification will have
any detrimental effects in the general population if not
overtly benefit. This review summarizes the beneficial
role of folate in human health, the metabolic pathway,
epigenetic mechanism and potential concerns based on
recent findings.
Folic acid and neural tube defects
Birth defects are one of the major burdens in the human
public health with estimates from Centers for Disease
Control and Prevention (CDC) approaching 1 in every
33 newborns in the US and accounting for more than
20% of all infant mortalities [10,11]. Neural tube defects
(NTDs) are common complex multifactorial disorders in
the neurulation of the brain and spinal cord that occurs
between 21 and 28 days after conception in humans
[12]. Worldwide depending on the ethnic grouping and
geographical location, the prevalence has been reported
to vary widely between 1and 10 in every 1000 births or
* Correspondence: subitbarua@gmail.com
Department of Developmental Biochemistry, New York State Institute for
Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten
Island, NY 10314, USA
© 2014 Barua et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Barua et al. Journal of Biomedical Science 2014, 21:77
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established pregnancies [13]. While we are beginning to
understand the underlying etiologies, evidence gathered
so far implicates both -genetic and non-genetic factors
such as maternal nutritional status or maternal obesity
in the onset of NTDs [14-16]. Over the years, numerous
studies including community-based trials often sug-
gested NTDs as vitamin deficiency disorders and have
shown that the exogenous or periconceptional supple-
mentation of maternal FA can reduce the risk of NTDs
in offspring [17]. Indeed, research spanning decades sug-
gests folate deficiency as a risk factor of NTDs; however
the involvement of whole methylation metabolism has
also been linked with the etiology of NTDs [16,18,19].
Arguing against the maternal folate deficiency model
alone, some studies also reported normal concentrations
of folate in the mothers of human fetuses with NTDs.
Supporting this, studies in cultured rat embryos or FA
deficient mice were reported not to be affected by NTDs
as a result of FA deficiency [20-24]. In contrast, studies
also reported that exogenous FA and thymidine in the
homozygous splotch (Pax3) mouse embryos prevented
NTDs and corrected biosynthetic defects [25]. There-
fore, no consensus has been reached based on the pub-
lished data to date. However, as FA deficiency may be a
risk factor for NTDs additional studies will be required
to determine the mechanistic role of the FA pathway in
the onset of neural tube defects.
History and impact of folic acid on public health
A possible relationship between apparent folate defi-
ciency and increased incidence of prematurity was sug-
gested as early as 1944 by Callender [26]. This was later
confirmed by Gatenby and Lillie [27], and in 1960s,
Richard Smithells and Elizabeth Hibbard hypothesized
that the under nutrition or impaired folate status could
be an important factor in the origin of NTD based on
significant observations, that women who had given
birth to the children with birth defects i.e. anencephaly
and spinabifida have an altered formiminoglutamic acid
compared to the women with unaffected children [28].
To test this hypothesis Smithells and his group con-
ducted an intervention trial with supplementation of
a multivitamin containing diet with FA 0.36 mg/day
during the periconceptional period to the participating
women who previously had infants with NTD. In-
contrast, women who were already pregnant without
vitamin supplementation were considered as controls. In
the1980’s, they published the results of this multi-center
intervention study that revealed about 83-91% reduction
in NTD recurrence in supplemented women compared
to that of unsupplemented women [29-32]. These re-
sults first highlighted that multivitamin or FA supple-
mentation may play a significant role in gestation and
may reduce the recurrence of NTD. Later in 1991, after
a randomized control trial (RCT) conducted at 33 cen-
ters in seven countries, the British Medical Research
Council suggested, for women with a previous history of
NTD-affected -pregnancies, the daily supplementation
of400microgramsofFAiseffectiveinpreventingthe
recurrence of NTDs by 70% [33]. This was further sup-
ported by the results of a -RCT conducted in Hungary
in 1992 that reported a daily intake of 0.8 mg of FA
during the periconceptional period significantly reduced
the incidence of a first occurrence of NTD [34]. In
1991, the CDC recommended a daily intake of 4000 μg
of FA before and throughout the period of pregnancy
for women with prior history of NTD-affected preg-
nancy [35]. Later in 1998, based on the evidence and
recommendation from the wider medical community,
the U.S. Public Health Service and Food and Drug
Administration recommended mandatory fortifications
of FA in flour and grains to prevent NTD and birth de-
fects [36]. In 2007, the Canadian recommendations also
included obesity (BMI >35) as a health risk, and recom-
mended “thehigherdoseFAstrategy(5mg)”in patients
with a history of poor compliance with medications
and additional lifestyle issues of variable diet, no con-
sistent birth control, alcohol, tobacco, and recreational
non-prescription drugs use. Furthermore, to prevent
the occurrence of NTDs in epileptic and diabetic
mothers the recommendation is to take a higher dose of
FA, 4–5 mg/day [37-39].
Folate metabolism
FA is central to folate-requiring one-carbon metabolism
which play key roles in numerous cellular reactions.
These involve amino acid metabolism, biosynthesis
of purine and pyrimidine; (the building blocks for
DNA and RNA synthesis), and formation of primary
methylating agent S-adenosyl-methionine (SAM), which
is the universal methyl donor for DNA, histones, pro-
teins and lipids [10]. Mechanistically, the transport of
transmembrane folate is facilitated by both receptors
and specific carriers active across cell membranes [40].
Under normal circumstances, natural dietary folate is
absorbed in the intestine and/or liver and metabolized
primarily to 5-methyl tetrahydrofolate (5-methylTHF)
and subsequently gets polyglutamated for cellular reten-
tion (Figure 1). However, FA consumed in fortified
foods/supplements is reduced primarly to dihydrofolate
by the enzyme dihydrofolate reductase in the liver and
finally converted to the tetrahydrofolate (THF), the sub-
strate for polyglutamate synthetase. The polyglutamyl
form of tetrahydrofolate (THF) formed either from FA
or normal dietary folate is the central folate acceptor mol-
ecule in the one-carbon cycle. Next, THF is converted
to 5,10-methyleneTHF by vitamin B6 dependent serine
hydroxymethyltransferase and then reduced irreversibly to
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5-methylTHF by methylenetetrahydrofolate reductase
(MTHFR). 5-Methyl-THF acts as a primary methyl donor
for the remethylation of homocysteine to methionine.
Methonine is a key substrate for S-adenosylmethionine
(SAM) which plays a central role in methylation reactions
catalyzed by DNA methyltransferases (DNMTs) forming
5-methylcytosine [41-43]. Thus, the entire FA metabolism
is modulated by several folate coenzymes. Mechanistically,
the central role of this coenzyme is to modulate the meta-
bolic pathway by accepting or donating one-carbon units
[44]. Key genes in this pathway that are involved in trans-
ferring the methyl group to homocysteine, and have been
most extensively studied include methylenetetrahydrofo-
late reductase (MTHFR), methionine synthase reductase
(MTRR), reduced folate carrier (RFC), along with vitamin
B12- dependent methionine synthase (MTR) [45]. Intri-
guingly, the etiology of NTDs has long been genetically
associated with the dysregulation of the major folate path-
way or methionine synthase genes, and single-nucleotide
polymorphisms (SNPs) such as 677C > T in the MTHFR
gene in humans [25,46]. Thus, further studies on FA in-
duced methylation and detailed analysis of the folate path-
way and its role in mammalian neural tube closure could
give us more insights in coming years.
Implications of FA in epigenetic regulation
The possible impact of nutritional supplements, for
example FA, on the mammalian genome can have long
lasting effects in human health without any underlying gen-
etic change. The availability of many dietary components
involved in one-carbon metabolism including vitamin B6,
choline, betanine, methionine, vitamin B12 and folate can
result in alterations in the DNA methylation and histone
modification. Mechanistically, the modulation of methyla-
tion patterns depends on the level of two metabolites of
one-carbon metabolism: S-adenosylmethionine (SAM), a
methyl donor and S-adenosylhomocysteine (SAH), a prod-
uct inhibitor of methyltranferases [47-49]. Thus, nutrient
epigenetic factors such as FA, a cofactor in one-carbon me-
tabolism during gestation can affect the fetal programming
and may modulate the genome-wide methylation pattern of
DNA and cause dysregulation in the expression of genes
[50]. The epigenetic impact of FA along with other one-
carbon metabolites is best studied in the agouti mouse (A
vy
)
experiment that has shown that the dietary methyl donors,
including FA, has no affect on the A
vy
methylationinthe
mother but clearly affected the A
vy
methylation and pheno-
type of developing offspring [51]. Similar to the animal
study, a study in young children from mothers having peri-
conceptional FA of 400 μgperdaywasshowntohaveen-
richment in the methylation of maternally imprinted
insulin-like growth factor 2 gene (IGF2) compared to those
with no periconceptional maternal FA [52]. In addition,
several epidemiologic and molecular evidences also link fol-
ate supplementation and epigenetic alteration by DNA
methylation with neural growth and recovery, including the
activation of folate receptor (Folr1), in spinal cord regener-
ation [53,54]. In an attempt to understand the FA induced
epigenetic mechanism to rescue neural tube closure, a re-
cent study in Splotch embryos (Sp−/−)has also shown that
Figure 1 Summary of folate metabolism (simplified). The schematic diagram shows that after entry of synthetic FA or natural dietary folates
through receptors/carriers in cell membrane, the intracellular folate/ FA pass through series of biochemical reactions, and alter DNA methylation.
Abbreviations: DHFR, dihydrofolate reductase; DHF, dihydrofolate; THF, tetrahydrofolate; SHMT, serine-hydroxymethyltransferase; 5,10-methenyl- THF,
5,10-methenyl-tetrahydrofolate; MTHFR, 5,10-methylenetetrahydrofolate reductase; 5-methyl THF, 5-methyltetrahydrofolate; MS, methionine synthase;
B12, vitamin B-12; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; DNMT, DNA methyltransferase; MT, methyltransferases.
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maternal intake of folate prior to conception decreases the
H3K27 methylation marks and remodels the chromatin on
Hes1 and Neurog2 promoters, genes that are essential for
neural tube development [55]. Recently, our study has
shown that FA supplementation dysregulates expressions of
several genes including FMR1 in lymphoblastoid cells [56],
and a follow up study in a mouse model has also identified
widespread alteration in the methylation pattern of the
brain epigenome in offspring from high maternal FA during
gestation [57]. The alterations in the methylation pattern
were exhibited both in CpG and non-CpG regions resulting
in differences in the expression of several key developmen-
tal and imprinted genes. In addition, we also found that the
methylation and expression of several genes are altered in a
gender-specific manner. Thus, it is clear that folate plays a
key role in epigenetic regulation of fetal developmental pro-
gramming. In the future, more studies on the role of folate
deficiency or over supplementation on epigenetic alter-
ations will establish causality of the amount of FA and
DNA methylation in diseases.
Folate intake and concern about potential adverse effect
The clinical significance of the chronic or high intake of
FA is not well established. Post fortification epidemio-
logical studies have reported an increase of approxi-
mately twice the amount in the intake of FA than
previously projected. Concern has been raised regarding
the potential health effects, since in addition to the forti-
fied products there is prevalence of using widespread
supplementation including over-the counter prenatal vi-
tamins as well as energy drinks which are substantially
enriched with various vitamins [58,59]. Recently, our
study in the mouse model has found that ten-fold in-
crease in maternal FA supplementation during gestation
altered the expression of several genes in the frontal cor-
tex of day old pups [60]. Moreover, continuation of such
higher amounts of FA throughout the post-weaning
period exhibited alterations in behaviors compared to
offspring from mothers having lower doses of gestational
FA supplementation. Mechanistically, such changes of
behavioral outcomes may possibly result from alterations
of gene expression as a result of aberrant methylation.
Intriguingly, results from several studies also sug-
gested that folate supplementation can induce aberrant
patterns of DNA methylation, and mechanistically may
play a dual role in carcinogenesis. FA supplementation
may prevent the early lesions, or potentially harm by en-
hancing the progression of established preneoplastic le-
sions [61]. Studies in rodent models -have shown that
supplementation of FA promotes the progression of mam-
mary tumor, and supporting this view a study in a genetic-
ally engineered mouse model of a human cancer has
shown that FA deficiency during the peri-gestational
period protects or decreases medulloblastoma formation
[62,63]. However, a meta-analysis of data conducted on
50,000 individuals to assess the effects of FA found that
FA supplementation does not substantially increase or
decrease incidence of site-specific cancer during the
first 5 years of treatment (RR = 1.06, 95% CI 0.99-1.13)
in comparison to placebo [64]. Moreover, a study from
children participating in the Northern California Child-
hood Leukemia Study (NCCLS) further revealed no sig-
nificant association of folate concentration at birth with
childhood acute myeloid leukemia (Additional file 1:
Table S1). In contrast, several RCT and meta-anlayis
have reported that prenatal multivitamins containing
FA -are associated with a significant protective effect
on pediatric cancers: leukemia, pediatric brain tumors
and neuroblastoma [65], (Additional file 1: Table S1).
In addition, recent ecological studies provided support
for a decrease in Wilms tumour, neuroblastoma, primi-
tive neuroectodermal tumours and ependymomas after
Canadian and United States FA fortification [66,67],
(Additional file 1: Table S1).
Although controversial, over-supplementation is also re-
ported to be involved in certain chronic disease and found
not to reduce cardiovascular disease [68,69]. In addition,
acute folate intake is also found to result in significant
down-regulation of folate transporters in kidney, and thus
dysregulated the renal folate uptake process [70]. More-
over, several RCT and observational studies suggested that
maternal intake of multivitamins including FA during
pregnancy may modulate pregnancy related outcomes
[71-75] including developmental outcome of offspring
(Additional file 1: Table S1).
The causal link between the maternal FA supplemen-
tation and the development of childhood asthma has
been of interest as asthma is considered to be an inter-
action of both genetic and environmental risk factors,
and concern has arisen as epidemiological studies have
also shown that increased folate in pregnancy may influ-
ence poor respiratory health in children [76,77]. Several
studies, including RCT and observational studies were
conducted to reveal such associations, however conflict-
ing results were found in these studies. While some
studies found positive association between FA exposure
and increase in risk of childhood asthma, other studies
found no such association (Additional file 1: Table S1).
In addition, studies in humans, also reported to have
found higher blood folate concentration of unmetabo-
lized FA and naturally occurring folates [78].
To gain a better understanding if maternal supplemen-
tation of FA modulates pregnancy related outcomes,
much focus has been given to reveal the role of FA sup-
plementation in the increased incidence of dizygotic
twining. This followed after the report of a Swedish
study suggested a possible association of FA supplemen-
tation with the increase in the twining rate [79]. A meta-
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analysis using the Food Standards Australia New
Zealand (FSANZ) framework by Muggli et al. [80] has
suggested that the hypothesis of the increase in dizygotic
twins is still to be demonstrated (OR = 1.26, 95% CI
0.91-1.73 for pre-conceptional supplementation and di-
zygotic twins; OR = 1.02, 95% CI 0.85-1.24 for overall
twins), and, if true, it would only cause a very limited in-
crease. However, Berry et al. reported that the associ-
ation of FA with an increase in dizygotic twining as
reported by the Swedish study has probably led to false
findings based on the reported 40% misclassification of
the use of in vitro fertilization [81]. This was further
supported by a Norwegian study that found no evidence
for an association between preconceptional folate sup-
plements and twinning after exclusion of known in vitro
fertilization pregnancies, and accounting for under-
reporting of both in vitro fertilization pregnancies and
folate use [82].
Thus it is clear that FA intake during pregnancy and
during daily life plays a significant role in modulating
gene expression and disease related outcome. Consider-
ing the important role of FA in several cellular process,
including epigenetic modulation and reducing the inci-
dence of NTDs (Additional file 1: Table S1), the dose,
timing (pre-conceptional/peri-conceptional/in-pregnancy),
and source of folate intervention during pregnancy and
throughout the life time may be critical. In the future,
more clinical and basic studies to decipher the link be-
tween over supplementation and normal development will
help us to understand the discordances between benefit
and possible harm.
Maternal folate intake and health outcomes in children - a
brief systemic review of recent cohorts study
For a better understanding of the effect of maternal FA,
we systematically reviewed recent published literature
(2011–2014) in order to assess the outcome of maternal
FA supplementation on the health of newborn infants
[83-134] (Additional file 1: Table S1). While results of sev-
eral cohort and observational studies in USA, Canada,
Chile, Australia, several countries in Europe and Asia have
reported the clinical significance of FA supplementation,
the direction of the beneficial effect was not in favorable
terms in all the cases. Therefore, several countries have
mandatory regulated FA fortifications, and despite its effi-
cacy there is no universal agreement based on the pub-
lished data to date [135]. The concern regarding the
appropriate dose and potential side effects are still a mat-
ter of debate [16,136]. As maternal FA can induce poten-
tial epigenetic effects on the genome of the offspring
which may vary with the metabolic ability of individual
race, sex, geographical locations or interactions with other
nutrients, one possible reason of inconsistency between
studies may be due to differences in the design of the
study. In the future there is definitely a need of global
collaboration to accumulate scientific evidence from a
clinical perspective, and to interpret these intervention
studies and potential effect in large cohorts.
Figure 2 A representative integrative model of possible epigenetic influence on pregnancy outcomes. Maternal intake of FA may result in
epigenetic modulation in the offspring brain methylome with overall or site specific alterations of methylation in genomic DNA, non-coding RNA
and histone modifications. These effects may alter gene expression of several imprinted, candidate autism susceptibility and key developmental
genes. Such changes may impact other biological processes, and associate with the overall developmental outcome. Scientific artwork adapted
from [137,138].
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Conclusion
The clinical application of FA supplementation/intake to
prevent NTDs has been well proven for the last 20–25
years (Additional file 1: Table S1). However considering
the concern with the level of folate concentration following
post-fortifications, it is of interest to explore if FA exposure
in significant sections of the population is influencing other
normal biological processes, such as the brain’s develop-
ment (Figure 2). Determining the level and distribution of
the methylation profile of the brain epigenome may reveal
the mechanism and downstream consequences of various
neuropsychiatric and imprinted disorders including autism.
Moreover as the level of folate status can influence methy-
lation, in the future more studies are needed to explore the
systemic differences in the DNA methylation profile in re-
lation to timing and dose between different populations
and between genders. Studies and careful monitoring of
the consequences of FA intake in global perspectives will
help clinicians to determine a proper therapeutic strategy
and the best preventive measures to improve the overall
public health, moreover to precisely differentiate the evalu-
ation of this vitamin in nutrition, in fortification and in
supplementation.
Additional file
Additional file 1: Table S1. Studies of maternal folate intake and
health outcomes in children.
Abbreviations
FA: Folic acid; NTD: Neural tube defect; CDC: Centers for disease control and
prevention; RCT: Randomized control trial; SAM: S-adenosyl- methionine;
SAH: S-adenosylhomocysteine; DNMTs: DNA methyltransferase;
5-methylTHF: 5-methyl tetrahydrofolate; THF: Tetrahydrofolate; MTHFR:
Methylenetetrahydrofolate reductase; MTRR: Methionine synthase reductase;
MTR: Methionine synthase; RFC: Reduced folate carrier; SNPs:
Single-nucleotide polymorphisms; IGF2: Insulin-like growth factor 2 gene;
BMI: Body mass index; TRoCA: Tabriz registry of congenital anomalies;
LiST: Live saved tool; PTB: Preterm birth; SGA-W: Small-for-gestational age for
weight; SGA-H: Small-for-gestational age for height; ALL: Acute lymphoblastic
leukemia; AML: Acute myeloid leukemia; CBT: Childhood brain tumors.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
SB conceptualized the content, and wrote the manuscript; SK assisted with
the revision of manuscript; MAJ conceptualized the content and critically
revised the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Financial support from the March of Dimes Research Foundation
(12-FY12-170) and the New York State Office for People With Developmental
Disabilities is gratefully acknowledged.
Received: 5 April 2014 Accepted: 11 August 2014
Published: 19 August 2014
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doi:10.1186/s12929-014-0077-z
Cite this article as: Barua et al.:Folic acid supplementation in pregnancy
and implications in health and disease. Journal of Biomedical Science
2014 21:77.
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