Trends in

Endocrinology & Metabolism

Review

One carbon metabolism and early

development: a diet-dependent destiny
Hunter W. Korsmo1,2 and Xinyin Jiang1,2,*

One carbon metabolism (OCM) is critical for early development, as it provides Highlights
one carbon (1C) units for the biosynthesis of DNA, proteins, and lipids and epige- One carbon metabolism (OCM) exerts its
netic modification of the genome. Epigenetic marks established early in life can impacts on offspring development by
participation in biosynthesis, epigenetic
be maintained and exert lasting impacts on gene expression and functions
modification, and remodeling of placen-
later in life. Animal and human studies have increasingly demonstrated that tal function.
prenatal 1C nutrient deficiencies impair fetal growth, neurodevelopment, and
cardiometabolic parameters in childhood, while sufficient maternal 1C nutrient Dysregulation in maternal OCM impairs
early development, leading to abnormal-
intake is protective against these detrimental outcomes. However, recent studies ities in growth, body composition, and
also highlight the potential risk of maternal 1C nutrient excess or imbalance cardiometabolic and neurocognitive
in disrupting early development. Further studies are needed to delineate the functions in the offspring.
dose–response relationship among prenatal 1C nutrient exposure, epigenetic
Growing evidence from animal and
modifications, and developmental outcomes. human studies suggests that sufficient
maternal supply of one carbon (1C) nutri-
Importance of OCM for early development ents may overcome the adverse influ-
ence of other intrauterine exposures,
The prenatal period is characterized by rapid cellular proliferation and differentiation, which makes
such as maternal obesity and alcohol in-
it susceptible to maternal nutrient supply. OCM, including the folate cycle, methionine cycle, and take, on early development.
transsulfuration pathway, provides one carbon (1C) units that are used for the biosynthesis of
nucleic acids, proteins, and lipids required for growth and epigenetic modifications that regulate Recent research suggests the poten-
tial risk of maternal 1C nutrient ex-
gene expression (Box 1 and Figure 1) [1]. The influence of OCM on early development and fetal cess and imbalance on growth and
programming of metabolic and cognitive health later in life is studied in various animal models neurodevelopment. Further studies
and is increasingly observed in human studies (Figure 2, Key figure). to delineate the dose–response
relationship between maternal 1C
nutrient intake and offspring outcome
Nutrients, including methionine, folate (see Glossary), choline and its oxidized metabolite are urgently needed.
betaine, vitamin B12, vitamin B2, and vitamin B6, participate in OCM as methyl donors or cofactors.
While sufficient supplies of these nutrients are critical for metabolic and cognitive development,
recent research has raised the concern of excess 1C metabolites leading to growth and
neurodevelopmental abnormalities [2–7]. This review provides an overview on the mechanisms
by which OCM impacts early development and recent findings on the time-specific and dose-
dependent influence of maternal OCM on offspring developmental outcomes.

Factors that influence OCM in the prenatal period 1

PhD Program in Biochemistry, The
OCM in the prenatal period is influenced by the supply of substrates and activity of enzymes. Graduate Center CUNY (City University
Increasing maternal intake of folate and vitamin B12 during pregnancy increases maternal and of New York), New York, NY 10016, USA
2
Department of Health and Nutrition
cord blood status of these nutrients [8,9]. Increasing maternal choline intake leads to higher
Sciences, Brooklyn College of the City
plasma choline levels and enhanced oxidation of choline to betaine [10]. Since these nutrients University of New York, Brooklyn,
are interrelated in OCM, disturbances in one nutrient influence the status of others. Pups from NY 11210, USA
folate-deficient mouse dams had lower betaine levels in the brain [11], while supplementing cho-
line to these dams partially preserved neurogenesis [12]. Since the enzyme methionine synthase
(MTR) that mediates methyl group donation from 5-methyl-tetrahydrofolate (5mTHF) to homo-
*Correspondence:
cysteine (Hcy) requires B12 as a cofactor, a folate excess and B12-deficient condition leads to XinyinJiang@brooklyn.cuny.edu
the so-called ‘methyl folate trap’ that impedes OCM [1]. (X. Jiang).

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Box 1. OCM: participating nutrients, pathways, and enzymes

Glossary
The folate cycle
Cardiometabolic diseases: diseases
In the folate cycle, tetrahydrofolate (THF) is reversibly converted to 5,10-methylenetetrahydrofolate (5,10-CH2-THF) by the that are characterized by a cluster of
B6-dependent serine hydroxymethyltransferase (SHMT) and provides 1C units for thymidylate synthesis. 5,10-CH2-THF metabolic risk factors, including insulin
can be converted back to THF via methylenetetrahydrofolate dehydrogenase (MTHFD). Both 5,10-CH2-THF and THF resistance and impaired glucose
can be converted to 10-formyl-THF (10fTHF) required for purine synthesis. 5,10-CH2-THF is also reduced to 5-methyl- tolerance, dyslipidemia, hypertension,
THF (5mTHF) via the B2-dependent methylenetetrahydrofolate reductase (MTHFR), which commits folate-derived methyl and abdominal adiposity.
groups to the methionine cycle [1]. Choline: choline is a semi-essential
nutrient found in foods, such as egg
The methionine cycle yolks, meat, and cruciferous vegetables.
In the methionine cycle, methionine is converted to S-adenosylmethionine (SAM) by methionine adenosyltransferase It contains three labile methyl groups that
(MAT). SAM serves as the universal methyl donor for various methyltransferases. SAM turns into S-adenosylhomocysteine are donated for methylation reactions.
(SAH) after transmethylation. The SAM:SAH ratio is often used as an indicator of cellular methylation potential [27]. SAH is Choline deficiency causes nonalcoholic
reversibly hydrolyzed to homocysteine (Hcy) by S-adenosyl-L-homocysteine hydrolase (AHCY). Hcy can be remethylated fatty liver disease.
to methionine via two independent pathways using folate or choline. The folate-dependent pathway requires 5mTHF to Epigenetic programming: the
transfer its methyl group to Hcy by the B12-dependent methionine synthase (MTR) and methionine synthase reductase intrauterine environment modifies the
(MTRR). The choline-dependent pathway requires oxidation of choline to betaine via choline dehydrogenase (CHDH). epigenetic marks of the fetus. These
Betaine transfers its methyl group to Hcy via betaine homocysteine S-methyltransferase 1 (BHMT1). In addition to partic- epigenetic modifications can be
ipating in the methionine cycle, choline is also acetylated to the neurotransmitter acetylcholine via choline acetyltransferase maintained after birth and into
(ChAT) and converted to phosphatidylcholine (PC) via the cytidine diphosphate (CDP)-choline pathway [44]. PC can also adulthood, thereby influencing gene
be made from the de novo pathway from sequential methylation of phosphatidylethanolamine (PE) by phosphatidyletha- expression and associated physiological
nolamine N-methyltransferase (PEMT). Interestingly, PEMT is a major consumer of methyl groups provided by SAM. As functions in the long term. Therefore, the
such, a methyl group originated from choline for SAM synthesis can also be used for the de novo synthesis of choline metabolic phenotypes later in life are
[10]. This seemingly futile cycle of choline metabolism may be important for the biosynthesis of specific species of PC, programmed by epigenetic changes
since PC produced by the PEMT pathway is enriched with long-chain polyunsaturated fatty acids (PUFAs), such as early in life.
docosahexaenoic acid (DHA). Upregulation of PEMT activity may enhance PC–DHA delivery and enrichment in the fetal Epigenome: the nonhereditary
brain [10]. regulatory modifications of the genome
that directly impact the process of
The transsulfuration pathway transcription. This encompasses DNA
modifications, histone modifications,
Hcy leaves the methionine cycle via the transsulfuration pathway. It is first converted to cystathionine by cystathionine beta
and noncoding RNAs.
synthase (CBS), which is B6 dependent [1]. Cystathionine is converted to cysteine by cystathionine gamma-lyase (CTH),
Folate: folate (vitamin B9) is a water-
which is also B6 dependent. Cysteine is used to make glutathione and thus links the transsulfuration pathway with redox
soluble vitamin naturally found in foods,
balance [1].
such as green leafy vegetables. Folic
acid (FA) is the most oxidized and stable
form of folate that is manufactured for
use in supplements and fortified foods.
Various SNPs have been discovered in genes encoding 1C metabolic enzymes, such as meth- FA is converted to dihydrofolate (DHF)
ylenetetrahydrofolate reductase (MTHFR), methylenetetrahydrofolate dehydrogenase (MTHFD) and then to tetrahydrofolate (THF) by
dihydrofolate reductase (DHFR) in the
1, MTR, PEMT, and BHMT1, influencing 1C metabolite status in the maternal–fetal dyad and liver to participate in the folate cycle.
the risk of adverse pregnancy and neonatal outcomes, such as preeclampsia, preterm birth, Hyperhomocysteinemia:
and neural tube defects (NTD) [13,14]. One carbon metabolic enzymes are also regulated characterized by abnormally high level of
homocysteine (>15 μmol/L) in the blood.
by hormonal changes during pregnancy. For example, PEMT, the enzyme that mediates
It can be caused by disturbances in
de novo choline synthesis, contains three estrogen response elements in the promoter, and its OCM and indicates deficiencies of folate,
expression is increased by 17-β estradiol [15] and thus has an increase in activity during preg- vitamin B12, or other 1C nutrients.
nancy [10]. Hyperhomocysteinemia is a risk factor of
cardiometabolic diseases.
Insulin-like growth factor 2 (IGF2): a
OCM, placental function, and biosynthesis during early development growth-promoting hormone that is
typically elevated during gestation. This
The folate cycle provides 1C units for nucleotide synthesis in the growing fetus. Moreover, OCM is protein has an essential role in
interrelated with macronutrient biosynthesis and transport. Phosphatidylcholine (PC) is an essential promoting cellular growth and
component of very low-density lipoprotein (VLDL) that mediates endogenous lipid transport [16]. proliferation in many tissues before birth.
Expression of the IGF2 gene is parentally
Both choline and folate deficiencies lead to steatosis by impacting PC availability [17]. The 1C
imprinted by DNA methylation, with only
nutrients also modify DNA methylation and expression of metabolic genes, which may then exert the paternally inherited copy being
lasting influence on macronutrient metabolism of offspring [18,19]. active.
Mechanistic target of rapamycin
(mTOR): mTOR regulates several
The critical role of 1C nutrients on placental functions, which regulate macronutrient transport to
cellular processes, such as protein
the growing fetus, has been recently revealed (Figure 2). Higher maternal choline intake during

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synthesis, anabolism, and senescence,

by integrating input from insulin, growth
factors, and amino acids in the
upstream. It is an important sensor of
cellular nutrients, oxygen, and energy
levels. mTOR forms two complexes
mTORC1 and mTORC2 with other
proteins. Rapamycin is an inhibitor of
mTORC1. mTOR dysregulation is
observed in cardiometabolic diseases.
Metastable epiallele (ME): genomic
loci that vary in methylation among
individuals yet correlate across tissues
from the three germ layers within an
individual.
SAM:SAH ratio: often used as an
assessment of methylation potential.
Glycine N-methyltransferase (GNMT) is a
major user of SAM-derived methyl
groups and regulates the intracellular
SAM:SAH ratio.
Neural tube defect (NTD): severe
congenital malformation related to the
improper closure of the neural tube
between days 17 and 28 after
fertilization. The two most common
NTDs are spina bifida and anencephaly.
SNP: a single nucleotide variation in the
genome that can directly impact the
functional role of a protein.
Vitamin B12: vitamin B12 is a cobalt-
containing vitamin exclusively found in
animal and microbial foods. Therefore,
Trends in Endocrinology & Metabolism vitamin B12 deficiency during pregnancy
Figure 1. One carbon metabolism metabolic pathways. Folate cycle metabolites and enzymes are shown in is often more prevalent in populations
green boxes. Methionine cycle metabolites and enzymes are shown in red boxes. Choline metabolites and consuming a mostly plant-based diet.
enzymes are shown in purple boxes. Transsulfuration pathway metabolites and enzymes are shown in orange
boxes. Key methyltransferase enzymes are shown in blue boxes. Coenzymes are shown in gray circles: vitamin B2,
B 6 , and B 12 . *Enzymes mainly expressed in the liver; #BHMT1 is also expressed in the kidney. Abbreviations:
5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 5,10-CH=THF, 5,10-methenyl-tetrahydrofolate; 5mTHF, 5-methyl-
tetrahydrofolate; 10fTHF, 10-formyl-tetrahydrofolate; ACh, acetylcholine; AHCY; S-adenosyl-L-homocysteine
hydrolase; BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine β-synthase; CCT, CTP:
phosphocholine cytidylyltransferase; CDP-choline, cytidine diphosphate-choline; ChAT, choline acetyltransferase;
CHDH, choline dehydrogenase; CK, choline kinase; CPT, cholinephosphotransferase; Cth, cystathionine;
CTH, cystathionine γ-lyase; Cys, cysteine; DHF, dihydrofolate; DHFR, dihydrofolate reductase; DMG, dimethylglycine;
DNMTs, de novo and maintenance DNA methyltransferases; dTMP, thymidine monophosphate; dUMP, deoxyuridine
monophosphate; Gly, glycine; GNMT, glycine N-methyltransferase; Hcy, homocysteine; HMT, histone
methyltransferase; Hse, homoserine. MATI/III, methionine adenosyltransferase; Met, methionine; MTR, methionine
synthase; MTRR, methionine synthase reductase; MTHFD1/2, methylenetetrahydrofolate dehydrogenase;
MTHFR, 5,10-methylenetetrahydrofolate reductase; PC, phosphatidylcholine; pCholine, phosphocholine; PE,
phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; PLD, phospholipase D; PRMT,
protein arginine methyltransferase. SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; Sar, sarcosine; Ser,
serine; SHMT1, serine hydroxymethyltransferase 1; TYMS, thymidylate synthase.

pregnancy suppressed the expression of the antiangiogenic gene soluble fms-like tyrosine kinase
1 (sFLT1) in the placenta in humans and promoted placental angiogenesis in mice [20,21]. Better
placental angiogenesis facilitates macronutrient transport to and growth of the fetus [22]. By
contrast, maternal B12 deficiency reduced placental expression of fatty acid transporters [23],
while maternal folate deficiency inhibited placental mechanistic target of rapamycin (mTOR)
signaling and amino acid transporter expression, thereby resulting in fetal growth restriction in
rodents [24]. Interestingly, under the condition of energy excess in a mouse model of high-fat

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Key figure
Potential mechanisms by which one carbon (1C) metabolism influences
early development

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Figure 2. Maternal intake of 1C nutrients may affect the status of 1C nutrients in the fetus, thereby influencing biosynthesis of
nucleic acids, proteins, and lipids and epigenetic regulation, eventually affecting cellular growth and metabolism. Maternal 1C
nutrient status may also exert its influence via the placenta by modifying mTOR pathway activity and subsequently affecting
macronutrient transport to the fetus as well as altering placental epigenetic marks, resulting in secondary epigenetic changes
in pathways that regulate growth and stress response in the fetus. These alterations in placental functions by 1C nutrients

(Figure legend continued at the bottom of the next page.)

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feeding, choline supplementation reduced fetal overgrowth, potentially by downregulating glucose

and lipid transporters and activity of the mTOR pathway in the placenta [25]. Overall, these findings
suggest a modulatory role of 1C nutrients in normal fetal growth by maintaining homeostasis of
placental metabolism.

OCM and epigenetic programming in early life

Maternal exposures during the prenatal period, such as famine, nutrient excess or deficiency,
seasonality, pollutants, and stress, have been associated with alterations in growth and
development as well as long-term health outcomes in the offspring [26]. It is suggested that
these maternal exposures program the epigenetic marks, such as DNA methylation and histone
modifications, in the growing fetus [26]. As these epigenetic marks persist after birth, they exert
lasting impacts on the health and development of the offspring (Box 2). OCM regulates the supply
of methyl groups for epigenetic modifications and thus plays a critical role in epigenetic
programming in early life.

Studies have investigated whether the maternal S-adenosylmethionine:S-adenosylhomocysteine

(SAM:SAH) ratio is related to DNA methylation in offspring. A study in Gambia demonstrated
that women who conceived in the rainy season (versus dry season) had higher 1C nutrient status
and serum SAM:SAH ratios in early pregnancy [27]. Accordingly, there was higher total methyla-
tion of 50 metastable epialleles (MEs) in their children at 3 months [27]. While these results
were consistent with the notion that better maternal 1C nutrient status is associated with higher
‘methylation potential’ of the offspring, it should be cautioned that the SAM:SAH ratio may be
tissue specific and subjected to homeostatic regulation [28]. Therefore, the use of serum SAM:
SAH to predict methylation changes in other tissues and between the maternal and fetal dyad
needs to be further validated. Alterations in DNA methylation in fetal or offspring tissue by
maternal 1C nutrient intake may not always be accompanied with tissue SAM:SAH ratio changes.
In a randomized controlled trial (RCT) (n = 24) among pregnant women in their third trimester, the
higher choline intake group had higher global DNA methylation in the placenta than the lower
intake group; however, the placental SAM:SAH ratio did not differ [29].

Influences of maternal 1C nutrient intake on the offspring epigenome and phenotypes are clearly
demonstrated in studies of viable yellow agouti mice. Supplementing 1C nutrients to dams
increased DNA methylation of the intracisternal A particle (IAP) retrotransposon at the agouti
locus, thereby reducing its expression, which in turn obviates the influence of this gene on
offspring fur coat color, food intake, and obesity [30].

Human studies have identified associations between maternal OCM and epigenetic modifications
of genes in pathways related to growth (e.g., IGF, H19, and RXRA), brain development
(e.g., BDNF), obesity and cardiometabolic function (e.g., POMC and LEP), and stress response
(e.g., NR3C1 and CRH) [18,26,29,31–34]. The relationship between maternal 1C nutrient intake
and offspring DNA methylation seems to be time specific; for example, cord blood LEP (encoding
leptin) methylation was positively associated with periconceptional folic acid (FA) supplementation
but was inversely related to second trimester folate intake [32]. The influence of maternal 1C
nutrients may also be sexually dimorphic. Caffrey et al. demonstrated in an RCT (n = 86) that

eventually affect fetal growth and metabolism. Taken together, maternal 1C metabolism programs developmental and
metabolic pathways early in life, thereby exerting lasting impacts on functional outcomes, such as neurocognitive
development, body size, and cardiometabolic differences. Abbreviations: CH3, methyl group; CRH, corticotropin-releasing
hormone; FATP, fatty acid transport protein; GLUT, glucose transporter; IGF2, insulin-like growth factor 2; mTOR,
mechanistic target of rapamycin; SAM, S-adenosylmethionine; SNAT, sodium-coupled neutral amino acid transporter.

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Box 2. Epigenetic modifications in early life

Epigenetics refers to the heritable changes in gene expression that do not alter DNA sequence. DNA methylation is a
common epigenetic modification that requires SAM-derived methyl groups (Figure I). It involves covalent attachment of
a methyl group at the C5 position of cytosine by DNA methyltransferases. Histone methylation is another SAM-dependent
epigenetic modification, which includes methylation of the lysine or arginine residues of histone tails by histone
methyltransferases.

The drastic epigenetic remodeling renders the perinatal period a critical window for epigenetic programming by
environmental exposures, including maternal OCM [119]. The periconceptional period involves rapid demethylation of
the genome following fertilization, giving rise to cellular pluripotency. However, the imprinted genes that are expressed
in a parent-of-origin-specific manner (e.g., IGF2, H19) resist demethylation in this process. Remethylation occurs after
implantation and persists throughout pregnancy in a tissue-specific manner. The primordial germ cells in the developing
embryo undergo a second wave of genomic demethylation as they enter the genital ridge. Within this wave the imprint
marks are erased, followed by de novo methylation that establishes sex-specific imprints during gametogenesis [119].
Histone methylation, especially trimethylation at histone 3 lysine 4 (H3K4me3), helps to maintain proliferation and
pluripotency of embryonic stem cells [120].

Adequate intake of 1C nutrients influences gametogenesis and gamete stability and plays an integral role in zygogenesis
[1]. Reproductive success is known to be negatively impacted by deficiencies of 1C nutrients and can be defined by
elevated Hcy levels (≥20 mmol/L) in follicular fluid [1]. Oocyte maturation and zygotic implantation is negatively impacted
by hyperhomocysteinemia. Human embryonic stem cells require sufficient 1C nutrients to maintain H3K4me3 to remain
undifferentiated [120]

Figure I. Influence of one

carbon metabolism (OCM)
on epigenetic regulation. OCM
nutrients provide methyl groups
for the universal methyl donor
S-adenosylmethionine (SAM) to
be used by DNA and histone methyl-
transferases, thereby influencing
DNA and histone methylation.
Abbreviations: 1C, one carbon;
CH3, methyl group; DNMT, DNA
methyltransferase; HMT, histone
methyltransferase; SAH, S-
adenosylhomocysteine; SAM, S-
adenosylmethionine.
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IGF2 methylation was lower in female offspring of women supplemented with 400 μg/day FA
through the second and third trimesters while BDNF methylation was lower only in the male off-
spring [31]. Although 1C nutrients provide the methyl group for DNA methylation, maternal 1C nu-
trient intake or status can be either positively or inversely associated with, or unrelated to, global
or site-specific DNA methylation (e.g., the imprinted gene IGF2 encoding insulin-like growth
factor 2) in the offspring, suggesting regulating mechanisms other than substrate provision
[18,33,34]. One potential mechanism is related to methylation of DNA methyltransferase 1
(DNMT1) by 1C nutrients, which may lower its expression and activity. Offspring DNMT1
methylation was inversely associated with FA intake up to the second trimester, yet was positively
associated with FA use that extends into the third trimester [18]. In addition, the influence of
maternal OCM is partially mediated through the placenta (Figure 2). An RCT showed that higher
maternal choline intake (930 versus 480 mg/d) in the third trimester increased NR3C1 and CRH
DNA methylation (encoding glucocorticoid receptor and corticotropin-releasing hormone,
respectively) and thus lowered corticotrophin-releasing hormone (CRH) expression in the placenta.
The decrease in placental CRH transport to the fetus may have subsequently caused decreases in
methylation of these genes in the fetal cord blood [29]. In summary, although there is a growing

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body of work interrogating the relationship between maternal OCM and offspring epigenetic
programming in humans, results are not consistent. Most of the studies are observational, with
other lifestyle factors as potential confounders, precluding the possibility to draw a causative
conclusion. There are also challenges in the accurate assessment of 1C intake using food
frequency questionnaires, while measurements of serum 1C metabolite levels may also be affected
by hemodilution during pregnancy and tissue uptake. Current studies mainly examine methylation
of a few selected loci in the genome and thus may not represent changes in the whole methylome.
Further, DNA methylation is tissue specific and subjected to dynamic changes during early
development. Whether cord blood or buccal cell DNA methylation, as measured in many studies,
reflects methylation changes in other tissues remains to be ascertained.

Maternal OCM and offspring functional outcomes

Congenital defects
OCM affects the risk of congenital defects, such as NTDs, congenital heart disease, and oral
clefts [35]. Clinical trials conducted in various countries established the effect of FA supplementa-
tion on reducing the risk of NTDs [36,37]. As a result, mandatory FA fortification in staple foods
has been adopted in over 80 countries [38].

Intake and status of other nutrients in OCM, including vitamin B12 and choline, are also associated
with NTD risk reduction independent of folate intake or levels in both pre- and post-FA fortified
populations [14,39,40]. Polymorphisms in enzymes involved with OCM, such as MTHFR 677C>T,
MTRR 66A>G, MTHFD1 1958G>A, and BHMT 742G>A, are associated with increased risk of
NTD [14], further supporting that disturbances in OCM are detrimental to neural tube development.

Neurodevelopment
OCM has been demonstrated to affect different aspects of neurodevelopment. Both folate and
choline deficiency during late gestation reduces neurogenesis and increases apoptosis of neurons
in rodents [12,41,42]. OCM influences epigenetic modification and DNA repair of genes that are
critical for neurodevelopment [43]. OCM nutrients also affect myelination, docosahexaenoic acid
(DHA) uptake, neurotransmitter synthesis, and neuroinflammation [44].

Various behavioral studies in rodents have consistently demonstrated that high perinatal choline
intake improves offspring cognitive performance and prevents their memory decline during aging
[45]. Studies also suggest that low prenatal folate intake or Mthfr deficiency results in more anxiety
behavior and worse short-term memory in offspring [11,46]. Supplementation of 1C nutrients
reverses neural insults from other maternal exposures; prenatal choline supplementation alleviated
behavioral changes and learning and memory deficits in mice with fetal alcohol spectrum disorders
(FASD) [47] or in genetic models of autism, Down syndrome, or Alzheimer’s disease [48–50].

In humans, disturbances in OCM that lead to Hcy accumulation may impair neurodevelopment in
children (Figure 3). Maternal hyperhomocysteinemia during the periconceptional period or
early pregnancy was associated with lower psychomotor scores in infants and lower cognitive
performance and higher risk of psychological problems in childhood [51–53].

Various studies have investigated the relationship between maternal folate intake or status
and cognitive outcomes of children (Figure 3). Low maternal folate status in early pregnancy
was associated with psychological and emotional problems in two prospective cohorts
[54,55], whereas higher maternal folate status at 30 weeks of gestation was associated with
better learning, memory, and attention [56]. FA supplementation during periconception and
early pregnancy was associated with improved fetal cerebellar growth [57], improved

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Figure 3. Current human evidence regarding the time-specific associations between maternal one carbon metabolism and offspring neurocognitive
outcomes. The brackets represent the timing of exposure, and the boxes include the maternal one carbon nutrient intake or status and offspring outcomes. The
question mark represents inconsistent results across studies. Abbreviations: FA, folic acid; Hcy, homocysteine; ∝, is related to.

communication ability at 18 months [58], better verbal skills at 3–4 years [59,60], and reduced
omission error at 11 years [61] in observational studies. In two RCTs, supplementation of
400 μg of 5mTHF from gestational week 20 to delivery led to improved ability to solve conflicts
at 8.5 years (n = 136), while supplementation of 400 μg of FA from gestational week 14 to
delivery led to higher cognition scores at 3 years (n = 39), better reasoning at 7 years (n = 70),
and higher processing speed at 11 years (n = 68), respectively [62–64]. However, there are also
studies that suggest no association between folate status, intake, or supplementation and
cognitive outcomes [65,66]. Several studies have investigated the relationship between maternal
folate intake or status and autism risk in children, yet results were inconsistent, suggesting either
higher or lower risk [67–69].

Results are mixed regarding maternal B12 and cognitive development. Maternal B12 deficiency
was associated with lower cognitive performance in infants [70] and 9-year-old children [71];
yet, another two observational studies did not find an association [56,72]. An RCT (n = 178) of

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oral B12 supplementation (50 μg) from early pregnancy to 6 weeks postpartum also did not find an
effect on cognitive performance in 9-month-old infants [73].

Emerging research suggests a positive relationship between maternal choline intake

or status and cognitive outcomes (Figure 3). Maternal plasma choline and betaine at
16 weeks of gestation were associated with better cognitive scores at 18 months [65].
Higher maternal choline intake was also related to better visual memory at 7 years in
the Project Viva study [66]. A controlled feeding RCT (n = 24) demonstrated that infants
born to mothers consuming 930 versus 480 mg choline/day during their third trimester
of pregnancy had faster processing speed [74]. In another RCT (n =100), choline supple-
mentation from the second trimester of pregnancy to infancy improved cerebral inhibition
at 5 weeks [75] and reduced attentional problems at 40 weeks of age [76]. Supplementing
women who were heavy drinkers with 2 g of choline during pregnancy in an RCT (n = 69)
improved visual recognition memory of infants at 12 months, providing pilot human evidence
that choline prevents the growing fetus from neural insults by alcohol [77]. A recent prospec-
tive cohort study showed that higher maternal plasma choline (concentrations ≥7.07 μM at
16 weeks of gestation) was also associated with less attention problems and withdrawn
syndrome in children exposed to maternal cannabis use or infection [78]. However, there
are also studies that found no association between maternal choline intake and cognitive
scores [60,79].

Overall, despite promising findings between maternal 1C nutrients and offspring cognitive
outcomes in observational cohorts and small trials, large RCTs that validate the causative rela-
tionship between the two are still limited. Interpreting the relationship between maternal OCM
and offspring cognitive development is complicated by the different timing of exposure, supple-
mentation dosage, SNPs, and the different aspects of cognitive outcomes measured at varied
time points of childhood in studies. The null findings in some prospective studies may also be
related to the challenge in sensitively estimating 1C nutrient intake in free-living populations. It
should also be noted that the influence of 1C nutrients may depend on the dosage levels. The
excess of 1C nutrients, especially folate, has also been related to adverse neurodevelopmental
outcomes, which is discussed in a later section.

Early growth
OCM affects cellular biosynthesis, methylation of genes in growth-related pathways (e.g., IGF),
and placental macronutrient delivery, thereby influencing growth of the fetus (Figure 2)
[20–22,25]. Maternal supplementation of multiple 1C nutrients or betaine alone increases
fetal weight and postnatal growth performance in normal or growth-restricted prenatal
environments [80–82]. By contrast, both choline and betaine supplementation prevented fetal
overgrowth in high-fat-fed obese mouse dams [25,83].

Low maternal folate status was related to increased risk of fetal growth retardation, small for
gestational age (SGA), and lower birth weight [84]. Meta-analyses of cohort studies [85] and
RCTs [86] suggest that FA supplementation both before and after conception was associated
with lower risk of SGA. Krikke et al. showed that maternal folate levels in early pregnancy were
associated with a slight increase in body mass index (BMI) in children at 5–6 years of age,
suggesting that the positive relationship between maternal folate and growth extends beyond
the fetal period [87].

However, high supplemental folate intake along with low vitamin B12 status in the second trimes-
ter of pregnancy increases the risk of SGA in a region with high prevalence of B12 deficiency in

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India [88], suggesting that excess folate or imbalance of 1C nutrients may negatively affect fetal
growth. In a more B12-replete population, maternal B12 status was not associated with birth
weight [89]. Interestingly, the highest quartile of maternal B12 status was associated with lower
weight gain between birth and 3 years of age [90].

Studies regarding other 1C nutrients and early growth in humans are limited. A recent study
demonstrated that a ‘varied and balanced’ 1C nutrient intake pattern at periconception was
associated with higher birth length and weight [91]. Two studies associated maternal betaine
status with lower birth weight [92,93]. Maternal choline status was positively related to weight
and fat mass at birth but not in early childhood [94]. Overall, while deficiencies in 1C nutrients
during pregnancy seem to restrict fetal growth, whether supplementing these nutrients in case
of an obesogenic environment may protect against offspring obesity is a new area of exploration.

Cardiometabolic outcomes
Cardiometabolic diseases, such as atherosclerosis and type 2 diabetes, are present with
altered OCM and aberrant epigenetic modifications in multiple tissues or organs (e.g., artery,
liver, and pancreas) during pathogenesis [95,96]. High-fat feeding in dams was demonstrated
to disrupt 1C metabolites and enzymes and resulted in genome-wide DNA hypermethylation in
offspring mice [97]. Gestational diabetes mellitus (GDM) in human pregnancies was associated
with higher folate but lower betaine status in the cord blood [98] and altered DNA methylation
of crucial metabolic genes, such as LEP, in newborns [99]. Therefore, under conditions of
OCM derangement secondary to maternal cardiometabolic disturbances, maternal 1C nutrient
intake may be critical to overcome the epigenetic programming that predisposes offspring to
cardiometabolic diseases later in life.

In rodent studies, maternal 1C supplementation in high-fat-fed obese dams alleviated increases

in whole body adiposity and blood glucose levels in offspring [100]. Supplementing 1C nutrients
to high-fat-fed dams attenuated higher fat preference and expression of the mu-opioid receptor
(MOR), which codes the rewarding properties of food in offspring [101]. Supplementation of the
1C nutrient choline to high-fat-fed dams led to similar relief in excess adiposity and liver fat content
by reducing the expression of lipogenic genes in the fetus, and male offspring also had better
glucose tolerance when fed a high-fat diet after weaning [102,103]. Interestingly, prenatal choline
supplementation alleviated weight, adiposity, and insulin resistance in offspring rats faced with
postnatal high-fat feeding, yet increased weight gain and leptin levels in offspring receiving a
normal-fat control diet [104]. By contrast, maternal folate depletion led to higher circulating triglyc-
eride levels in mouse offspring when fed a high-fat diet from weaning [105]. The balance between
1C nutrients also seems to matter, as a recent study demonstrated that a low or normal-choline/
high-folate, but not a high-choline/high-folate, gestational diet increased weight gain and plasma
insulin and leptin in offspring [106].

Human evidence regarding maternal OCM and cardiometabolic outcomes is relatively limited.
Low maternal serum folate levels at early pregnancy were associated with higher BMI, whereas
low maternal B12 was associated with higher heart rates in children at 5–6 years [87]. By contrast,
higher maternal folate intake at 32 weeks of gestation was associated with greater lean body
mass in offspring at 9 years [107]. Wang et al. demonstrated 40% lower odds of elevated systolic
blood pressure in children whose mothers had folate levels above (versus below) the median
during pregnancy [108]. However, a cohort in India with higher prevalence of B12 deficiency
suggested that high maternal erythrocyte folate along with low B12 predicted the highest levels
of insulin resistance in children [109]. Consistently, higher maternal folate concentrations were
associated with higher insulin resistance in children in another birth cohort in India [110]. An

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RCT (n = 3524) in Nepal suggested that FA supplementation was protective for metabolic Outstanding questions
syndrome [111], whereas in a subsample of 1132 participants, B12 deficiency in mothers was What are the time- and dose-specific
associated with a 27% increase in insulin resistance [112]. As for other 1C nutrients, a effects of prenatal 1C nutrient expo-
sures on fetal epigenetic changes in
controlled-feeding RCT demonstrated that higher choline intake in the third trimester of
humans? Do these changes have last-
pregnancy led to lower cord blood cortisol levels [29]. Since chronic cortisol elevation impairs ing effects on health outcomes?
blood glucose and blood pressure, increasing maternal choline intake may provide a protective
mechanism against fetal programming of cardiometabolic diseases. Is there a bell-shaped curve regarding
the relationship between maternal 1C
nutrient intake and benefits on early
Potential risk of 1C nutrient excess development?
While ensuring sufficient maternal supply of 1C nutrients is critical for early development, emerging
Can maternal OCM be modified to
research suggests the potential risk of excess 1C nutrients, especially excess FA consumption.
avert the negative influence of other
There is no upper tolerable intake level (UL) for natural folate, but a UL of 1000 μg was established maternal exposures (e.g., obesity) on
for FA, the synthetic form of folate used in fortified foods and supplements, with concerns that high offspring health?
FA intakes may mask B12 deficiency, increase unmetabolized FA in the body, and inhibit key
What is the right balance of 1C nutrients
1C enzymes, such as dihydrofolate reductase (DHFR) and MTHFR [113]. Several studies in for a pregnant woman? Should 1C
North American populations demonstrate that a considerable number (e.g., 26% in one nutrient intake be personalized with the
study) of pregnant women exceed the UL from supplements and fortified products consideration of genetic polymorphisms
[114,115]. The high folate intake leads to supranutritional folate status [e.g., a serum level to optimize OCM for early development?

over 45.3 nmol/l suggested by the World Health Organization (WHO) [116]], which suggests How can prenatal vitamin supplements
metabolic capacity has been exceeded. Several studies supplementing FA in rodent diets at be better designed to complement
4–20 mg/kg body weight versus control (2 mg/kg) suggest that high maternal FA intake dietary intake of 1C nutrients in
different populations?
increased anxiety behavior and impaired learning and short-term memory of offspring with
altered DNA methylation, neurotrophic factor expression, and DHA concentration in the How does paternal OCM during the
brain [117]. The Infancia y Medio Ambiente (INMA) Project, which enrolled over 2000 Spanish periconceptional period affect early
children, suggests that those with maternal FA intake exceeding UL during pregnancy were development?

associated with lower psychomotor and neurocognitive scores [3,4]. Maternal plasma folate Does 1C nutrient exposure affect early
exceeding the 45.3 nmol/L cutoff was associated with greater risk of autism in the Boston development in a sexually dimorphic
Birth Cohort [69]. manner? How do hormonal treatments,
such as in cases of in vitro fertilization
and transgender pregnancies, influence
A recent study by De Crescenzo et al. identified an interesting phenomenon that high maternal FA OCM during early development in both
intake mirrored the detrimental effect of folate deficiency on cortical neurogenesis and behavioral sexes?
development of offspring, and that both FA excess and deficiency suppressed folate’s participa-
tion in the methionine cycle [2]. These results are consistent with a study by Bahous et al. that high
dietary folate in pregnant mice led to pseudo-MTHFR deficiency, thereby decreasing the use of
folate as a methyl donor [5].

The potential harmful effect of FA excess is also noted for fetal growth and cardiometabolic
functions. Excessive gestational FA intake impaired insulin secretion in mouse offspring [6].
FA intake beyond the first trimester of pregnancy was associated with increased risk of large
for gestational age in a birth cohort in China [7]. High FA and low B12 intake during pregnancy
was associated with insulin resistance of children in the PUNE cohort in India [109]. Excess in
overall 1C nutrient intake is also related to changes in IGF pathway regulation and leptin
secretion [19,118], suggesting that a bell-shaped curve of benefit versus risk may exist for
1C nutrients in general (Figure 4).

Concluding remarks and future perspectives

Altered 1C metabolism due to maternal 1C nutrient intake or genetic deficiencies can result in
inappropriate growth at birth and metabolic and cognitive impairments later in life, while increasing
maternal 1C nutrient supply may overcome developmental disturbances resulting from other
adverse exposures in early life, as demonstrated in various animal studies. Emerging observational

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Figure 4. Proposed relationship

between prenatal one carbon
(1C) exposure and developmental
outcomes. Abbreviation: CVD,
cardiovascular disease.

Trends in Endocrinology & Metabolism

and interventional studies in humans provide evidence that resonates with findings in animals.
Underlying mechanisms of action include direct impacts of OCM on fetal biosynthesis and macro-
nutrient metabolism and epigenetic programming of the fetal genome as well as modification of the
placental epigenome and metabolism, which subsequently affects fetal nutrient acquisition and
metabolism. However, there are also concerns related to excess maternal 1C nutrient supply
and imbalance between 1C nutrients that could also result in OCM derangement, leading to ad-
verse growth and developmental outcomes.

The challenges of research in this area include the following: (i) the influence of OCM may be time
specific, demonstrated by the differential DNA methylation changes and growth and cognitive
outcomes, with 1C nutrient exposures at different time points of gestation; (ii) the complexity of
1C metabolism regulation by dietary intakes, interactions between 1C pathways and nutrients,
and genetic polymorphisms, which may partly explain the inconsistent results in epigenetic and
functional outcomes in studies; (iii) the difficulty in conducting human studies that pinpoint the
causative relationship among 1C nutrient exposure, epigenomic and cellular changes, and
long-term functional outcomes (see Outstanding questions). Further research is needed to
delineate the dose–response relationship of 1C nutrient intake, in varied forms and combinations,
with early development.

Acknowledgments
The authors give special thanks to Dr Marie Caudill (Cornell University) for her insights into conceptualizing and editing this
manuscript. This research is funded by the National Institute of General Medical Sciences (NIGMS) of the National Institutes
of Health (NIH) (grant number 5SC3GM132010).

Declaration of interests
The authors declare no conflict of interest.

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