One Carbon Metabolism and Early Development: A Diet-Dependent Destiny

Abstract

One carbon (1C) metabolism is critical for early development as it provides 1C units for both biosynthesis of DNA, proteins, and lipids and epigenetic modification of the genome. Epigenetic marks established early in life can be maintained and exert lasting impacts on gene expression and functions later in life. Animal and human studies have increasingly demonstrated that prenatal 1C nutrient deficiencies impair fetal growth, neurodevelopment and cardiometabolic parameters in childhood, while sufficient maternal 1C nutrient intake is protective against these detrimental outcomes. However, recent studies also highlight the potential risk of maternal 1C nutrient excess or imbalance in disrupting early development. Further studies are needed to delineate the dose-response relationship among prenatal 1C nutrient exposure, epigenetic modifications, and developmental outcomes.

1. Importance of one carbon metabolism (OCM) for early development

The prenatal period is characterized with rapid cellular proliferation and differentiation, which makes it susceptible to maternal nutrient supply. OCM, including the folate cycle, methionine cycle and transsulfuration pathway, provides one carbon (1C) units that are used for biosynthesis of nucleic acids, proteins, and lipids required for growth and epigenetic modifications that regulate gene expression (Box 1 and Figure 1) [1]. The influence of OCM on early development and fetal programming of metabolic and cognitive health later in life is studied in various animal models and increasingly observed in human studies (Figure 2. Key Figure).

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

The Folate Cycle

In the folate cycle, tetrahydrofolate (THF) is reversibly converted to 5,10-methylenetetrahydrofolate (5,10-CH₂-THF) by the B₆-dependent serine hydroxymethyltransferase (SHMT) and provides 1C units for thymidylate synthesis. 5,10-CH₂-THF can be converted back to THF via methylenetetrahydrofolate dehydrogenases (MTHFD). Both 5,10-CH₂-THF and THF can be converted to 10-formyl-THF (10fTHF) required for purine synthesis. 5,10-CH₂-THF is also reduced to 5-methyl-THF (5mTHF) via the B₂-dependent methylenetetrahydrofolate reductase (MTHFR) which commits folate-derived methyl groups to the methionine cycle [1].

The Methionine Cycle

In the methionine cycle, methionine is converted to S-adenosylmethionine (SAM) by methionine adenosyltransferase (MAT). SAM serves as the universal methyl donor for various methyltransferases. SAM turns into SAH after transmethylation. The SAM:SAH ratio is often used as an indicator of cellular methylation potential [27]. SAH is reversibly hydrolyzed to homocysteine (Hcy) by S-adenosyl-L-homocysteine hydrolase (AHCY). Hcy can be remethylated to methionine via two independent pathways using folate or choline. The folate-dependent pathway requires 5mTHF to transfer its methyl group to Hcy by the B₁₂-dependent methionine synthase (MTR) and methionine synthase reductase (MTRR). The choline-dependent pathway requires oxidation of choline to betaine via choline dehydrogenase (CHDH). Betaine transfers its methyl group to Hcy via betaine homocysteine S-methyltransferase 1 (BHMT1). In addition to participating in the methionine cycle, choline is also acetylated to the neurotransmitter acetylcholine via choline acetyltransferase (ChAT) and converted to phosphatidylcholine (PC) via the CDP-choline pathway [44]. PC can also be made from the de novo pathway from sequential methylation of phosphatidylethanolamine (PE) by phosphatidylethanolamine N-methyltransferase (PEMT). Interestingly, PEMT is a major consumer of methyl groups provided by SAM. As such, a methyl group originated from choline for SAM synthesis can also be used for the de novo synthesis of choline [10]. This seemingly futile cycle of choline metabolism may be important for the biosynthesis of specific species of PC, since PC produced by the PEMT pathway is enriched with long-chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). Upregulation of PEMT activity may enhance PC-DHA delivery and enrichment in the fetal brain [10].

The Transsulfuration Pathway

Homocysteine leaves the methionine cycle via the transsulfuration pathway. It is first converted to cystathionine by cystathionine beta synthase (CBS) which is B₆-dependent [1]. Cystathionine is converted to cysteine by cystathionine gamma-lyase (CTH) which is also B₆-dependent. Cysteine is used to make glutathione and thus links the transsulfuration pathway with redox balance [1].

Figure 1.

One Carbon Metabolism Metabolic Pathways. Folate cycle metabolites and enzymes (green boxes): 5,10-CH₂-THF, 5,10-methylenetetrahydrofolate; 5,10-CH=THF, 5,10-methenyl-tetrahydrofolate; 10fTHF, 10-formyl-tetrahydrofolate; 5mTHF, 5-methyl-tetrahydrofolate; DHF, dihydrofolate; Gly, glycine; Ser, serine; dTMP, thymidine monophosphate; dUMP, deoxyuridine monophosphate; DHFR, dihydrofolate reductase; MTHFD1/2, methylenetetrahydrofolate dehydrogenase; MTHFR, 5,10-methylenetetrahydrofolate reductase; SHMT1, serine hydroxymethyltransferase 1; TYMS, thymidylate synthase. Methionine cycle metabolites and enzymes (red boxes): DMG, dimethylglycine; Hcy, homocysteine; Met, methionine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine, Sar, sarcosine; AHCY; S-adenosyl-L-homocysteine hydrolase; BHMT, betaine-homocysteine S-methyltransferase; CHDH, choline dehydrogenase; GNMT, glycine N-methyltransferase; MATI/III, methionine adenosyltransferase; MTR, methionine synthase; MTRR, methionine synthase reductase. Transsulfuration pathway metabolites and enzymes (orange boxes): Cth, cystathionine; Cys, cysteine; Hse, homoserine; CBS, cystathionine β-synthase; CTH, cystathionine γ-lyase. Key methyltransferase enzymes (blue box): DNMTs, de novo and maintenance DNA methyltransferases; HMT, histone methyltransferase; PRMT, protein arginine methyltransferase; PEMT, phosphatidylethanolamine N-methyltransferase. Choline metabolites and enzymes (purple boxes): CDP-choline, cytidine diphosphate-choline; PC, phosphatidylcholine; pCholine, phosphocholine; PE, phosphatidylethanolamine; CCT, CTP:phosphocholine cytidylyltransferase; ChAT, choline acetyltransferase; CK, choline kinase; CPT, cholinephosphotransferase; PLD, phospholipase D. Coenzymes (grey circles): vitamin B₂, B₆ and B₁₂. *enzymes mainly expressed in the liver; ^# BHMT1 is also expressed in the kidney.

Figure 2.

Potential mechanisms by which one carbon metabolism influences early development. Maternal intake of one carbon (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 the 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 eventually affect fetal growth and metabolism. Taken together, maternal one carbon 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. CH₃: methyl group; CRH, corticotropin-releasing hormone; FATP, fatty acid transport protein; GLUT, glucose transporter; IGF2, insulin-like growth factor 2; mTOR, the mechanistic target of rapamycin; SAM, S-adenosylmethionine; SNAT, sodium-coupled neutral amino acid transporter

Nutrients including methionine, folate (see Glossary), choline and its oxidized metabolite betaine, vitamin B₁₂, B₂, and 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 mechanisms by which OCM impacts early development and recent findings in the time-specific and dose-dependent influence of maternal OCM on offspring developmental outcomes.

2. Factors that influence OCM in the prenatal period

OCM in the prenatal period is influenced by the supply of substrates and activity of enzymes. Increasing maternal intakes of folate and vitamin B₁₂ during pregnancy increases maternal and cord blood status of these nutrients [8, 9]. Increasing maternal choline intake leads to higher plasma choline levels and enhanced oxidation of choline to betaine [10]. Since these nutrients are interrelated in OCM, disturbances in one nutrient influence the status of others. Pups from folate-deficient mouse dams had lower betaine levels in the brain [11] , while supplementing choline to these dams partially preserved neurogenesis [12]. Since the enzyme methionine synthase (MTR) that mediates methyl group donation from 5-methyl-tetrahydrofolate (5mTHF) to homocysteine (Hey) requires B₁₂ as a cofactor, a folate-excess and B₁₂- deficient condition leads to the so-called “methyl folate trap” that impedes OCM [1].

Various single nucleotide polymorphisms (SNPs) have been discovered in genes encoding 1C metabolic enzymes such as MTHFR, MTHFD1, MTR, PEMT, and BHMT1, influencing 1C metabolite status in the maternal-fetal dyad and the risk of adverse pregnancy and neonatal outcomes such as preeclampsia, preterm birth, and neural tube defects (NTD) [13, 14]. One carbon metabolic enzymes are also regulated by hormonal changes during pregnancy. For example, PEMT, the enzyme that mediates de novo choline synthesis, contains three estrogen response elements in the promoter, and its expression is increased by 17-ß estradiol [15] and thus has an increase in activity during pregnancy [10].

3. OCM, placental function, and biosynthesis during early development

The folate cycle provides 1C units for nucleotide synthesis in the growing fetus. Moreover, OCM is interrelated with macronutrient biosynthesis and transport. PC is an essential component of very-low-density lipoprotein (VLDL) that mediates endogenous lipid transport [16]. Both choline and folate deficiencies lead to steatosis by impacting PC availability [17]. The 1C nutrients also modify DNA methylation and expression of metabolic genes, which may then exert lasting influence on macronutrient metabolism of offspring [18, 19].

The critical role of 1C nutrients on placental functions, which regulates macronutrient transport to the growing fetus, is recently revealed (Figure 2). Higher maternal choline intake during pregnancy suppressed the anti-angiogenic gene soluble fms-like tyrosine kinase 1 (sFLTI) expression in the placenta in humans and promoted placental angiogenesis in mice [20, 21]. Better placental angiogenesis facilitates macronutrient transport and growth of the fetus [22]. In 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 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.

4. 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.

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 with de novo methylation that establishes sex-specific imprints during gametogenesis [119]. Histone methylation, especially tri-methylation at histone 3 lysine 4 (H3K4me3) helps to maintain proliferation and pluripotency of embryonic stem cells [120].

Adequate intake of 1C nutrients influences gametogenesis, 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 (⩾ 20mmol/L) in follicular fluid [1]. Oocyte maturation and zygotic implantation is negatively impacted by hyperhomocysteinaemia. Human embryonic stem cells require sufficient 1C nutrients to maintain H3K4me3 to remain undifferentiated [120].

Studies have investigated whether the maternal SAM:SAH ratio is related to DNA methylation in offspring. A study in Gambia demonstrated that women who conceived in the rainy season (vs. dry season) had higher 1C nutrient status and serum SAM:SAH ratio in early pregnancy [27]. Accordingly, there was higher total methylation 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 third trimester pregnancy women, the higher choline intake group had higher global DNA methylation in the placenta than the lower intake group, although the placental SAM:SAH ratio did not differ [29].

The influences of maternal 1C nutrient intake on the offspring epigenome and phenotypes are clearly demonstrated in studies of the 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, LEP), and stress response (e.g., NR3C1, 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 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) of 400μg/day FA supplementation through the second and third trimesters that IGF2 methylation was lower in the supplemented group only in the female offspring while BDNF methylation was lower only in the male offspring [31]. Although 1C nutrients provide the methyl group for DNA methylation, maternal 1C nutrient 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 the 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 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 vs. 480 mg/d) in the third trimester increased NR3C1 and CRH DNA methylation (encoding glucocorticoid receptor and corticotropin-releasing hormone, respectively) and thus lowered 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 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.

5. Maternal OCM and offspring functional outcomes

5.1. 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 supplementation 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.

5.2. 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, 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, 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 which lead to homocysteine 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].

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. FA, folic acid; Hcy, homocysteine

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 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, supplementing 400 μg of 5-mTHF from gestational week 20 until delivery led to improved ability to solve conflicts at 8.5 years (n = 136), while supplementing 400 μg FA supplementation from gestational week 14 until delivery led to higher cognition scores at 3 years (n = 39) and 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 oral B₁₂ supplementation (50 μg) from early pregnancy to 6 weeks postpartum also did not find an effect on cognitive performance in 9 months 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 supplementation 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 2g 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 prospective cohort study showed that higher maternal plasma choline (concentrations ⩾7.07 μM at 16 weeks of gestation) was also associated with less attention problem 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 relationship between the two are still limited. Interpreting the relationship between maternal OCM and offspring cognitive development is complicated by the different timing of exposure, supplementation 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 nutrient 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.

5.3. 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]. In 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. shows that maternal folate levels in early pregnancy were associated with a slight increase in BMI in children at 5-6 years of age, suggesting 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 trimester of pregnancy increases the risk of SGA in a region with high prevalence of B₁₂ deficiency in 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 [90].

Studies regarding other 1C nutrients and early growth in humans are limited. A recent study demonstrates 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.

5.4. 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 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 dams led to similar relief in excess adiposity and liver fat content by reducing expression of lipogenic genes in 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]. On the contrary, maternal folate depletion led to higher circulating triglyceride 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 demonstrates 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 rate of children at 5-6 years [87]. In 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 (vs. below) the median during pregnancy [108]. However, a cohort in India with higher prevalence of B₁₂ deficiency suggests 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 RCT (n=3524) in Nepal suggests that FA supplementation was protective for metabolic syndrome [111] whereas in a subsample of 1132 participants B₁₂ deficiency in mothers was associated with 27% increase in insulin resistance [112]. As for other 1C nutrients, a controlled feeding RCT demonstrated that higher choline intake in the third trimester of pregnancy led to lower cord blood cortisol levels [29]. Since chronic cortisol elevation impairs blood glucose and blood pressure, increasing maternal choline intake may provide a protective mechanism against fetal programming of cardiometabolic diseases.

6. Potential risk of 1C nutrient excess

While ensuring sufficient maternal supply of 1C nutrients is critical for early development, emerging research suggests the potential risk of excess in 1C nutrients, especially excess FA consumption. There is no upper tolerable intake level (UL) for natural folate, but a UL of 1000 μg was established for FA, the synthetic form of folate used in fortified foods and supplements, with concerns that high FA intakes may mask B₁₂ deficiency, increase unmetabolized FA in the body, and inhibit key 1C enzymes such as DHFR and MTHFR [113]. Several studies in North American populations demonstrate that a considerable number (e.g., 26% in one study) of pregnant women exceed the UL from supplement and fortified products [114, 115]. The high folate intake leads to supranutritional folate status (e.g., a serum level over 45.3 nmol/L suggested by the WHO [116]) that suggests metabolic capacity has been exceeded. Several studies supplementing FA in rodent diets at 4-20mg/kg body weight versus control (2mg/kg) suggest that high maternal FA intake increased anxiety behavior and impaired learning and short-term memory of offspring with altered DNA methylation, neurotrophic factor expression and DHA concentration in the brain [117]. The Infancia y Medio Ambiente (INMA) Project which enrolled over 2000 Spanish children suggests that those with maternal FA intake exceeding UL during pregnancy were associated with lower psychomotor and neurocognitive scores [3, 4]. Maternal plasma folate exceeding the 45.3 nmol/L cutoff was associated with greater risk of autism in the Boston Birth Cohort [69].

A recent study by De Crescenzo et al. identified an interesting phenomenon that high maternal FA intake mirrored the detrimental effect of folate deficiency on cortical neurogenesis and behavioral development of offspring, and that both FA excess and deficiency suppressed folate’s participation 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 (LGA) 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).

Figure 4.

Proposed relationship between prenatal one carbon exposure and developmental outcomes. 1C, one carbon; CVD, cardiovascular disease.

7. 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 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 macronutrient 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 adverse growth and developmental outcomes.

The challenges of research in this area include that: (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 intakes, in varied forms and combinations, with early development.

Outstanding Questions.

• What are the time- and dose-specific effects of prenatal one carbon (1C) nutrient exposures on fetal epigenetic changes in humans? Do these changes have lasting effects on health outcomes?

• Is there a bell-shaped curve regarding the relationship between maternal 1C nutrient intakes and benefits on early development?

• Can maternal one carbon metabolism (OCM) be modified to avert the negative influence of other maternal exposures (e.gobesity) on offspring health?

• What is the right balance of 1C nutrients for a pregnant woman? Should 1C nutrient intakes be personalized with the consideration of genetic polymorphisms to optimize OCM for early development?

• How can prenatal vitamin supplements be better designed to complement dietary intake of 1C nutrients in different populations?

• How does paternal OCM during the periconceptional period affect early development?

• Does 1C nutrient exposure affect early development in a sexually dimorphic manner? How do hormonal treatments such as in cases of in vitro fertilization and transgender pregnancies influence OCM during early development in both sexes?

Figure I.

(in Box 2). 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 methyltransferases, thereby influencing DNA and histone methylation.

Trends:

• One carbon metabolism (OCM) exerts its impacts on offspring development by participation in biosynthesis, epigenetic modification, and remodeling of placental function.

• Dysregulation in maternal OCM impairs early development, leading to abnormalities in growth, body composition, and cardiometabolic and neurocognitive functions in the offspring.

• Growing evidence from animal and human studies suggests that sufficient maternal supply of one carbon (1C) nutrients may overcome the adverse influence of other intrauterine exposures, such as maternal obesity and alcohol intake, on early development.

• Recent research suggests the potential risk of maternal 1C nutrient excess and imbalance on growth and neurodevelopment. Further studies to delineate the dose-response relationship between maternal 1C nutrient intakes and offspring outcomes are urgently needed.