Prenatal Folate and Choline Levels and Brain and Cognitive Development in Children: A Critical Narrative Review

Women’s nutritional status during pregnancy can have long-term effects on children’s brains and cognitive development. Folate and choline are methyl-donor nutrients and are important for closure of the neural tube during fetal development. They have also been associated with brain and cognitive development in children. Animal studies have observed that prenatal folate and choline supplementation is associated with better cognitive outcomes in offspring and that these nutrients may have interactive effects on brain development. Although some human studies have reported associations between maternal folate and choline levels and child cognitive outcomes, results are not consistent, and no human studies have investigated the potential interactive effects of folate and choline. This lack of consistency could be due to differences in the methods used to assess folate and choline levels, the gestational trimester at which they were measured, and lack of consideration of potential confounding variables. This narrative review discusses and critically reviews current research examining the associations between maternal levels of folate and choline during pregnancy and brain and cognitive development in children. Directions for future research that will increase our understanding of the effects of these nutrients on children’s neurodevelopment are discussed.


Introduction
A mother's nutritional status, as defined by the intake and utilization of nutrients during pregnancy has a significant effect on the development of the fetal brain and subsequent cognitive outcomes later in life [1]. To support optimal fetal development and offspring cognitive function, it is recommended that pregnant women consume adequate, but not deficient or excessive, sources of nutrients (e.g., vitamin D, folate, choline, and iron), in addition to other food components (e.g., fibre, fat, and proteins) [2]. Notably, nutrient demands increase during pregnancy as maternal body mass and metabolic demands increase is a synthetic dietary supplement present in fortified foods and vitamins [29,30]. To increase the intake of folic acid in the general population and reduce the rate of neural tube defects, fortification of grain-based food products such as flour, cereal, and pasta with folic acid has been mandated in Canada and the United States since 1998 [30][31][32][33]. Currently, over 80 countries have mandated fortification of foods with folic acid, and it is recommended that men and non-pregnant women consume 320 to 400 µg of folate per day [30,34].
Compared to folate requirements of non-pregnant women, it is recommended that pregnant women consume at least 400 to 800 µg of folate per day to ensure healthy maternal, placental, and fetal tissue growth [34][35][36][37]. The placenta extracts folic acid from maternal circulation and concentrates it into fetal circulation [35,[38][39][40]. This results in fetal levels that are two to four times higher than maternal levels [39,[41][42][43][44][45]. In the fetus, folate is critically important for cellular proliferation, neural stem cell proliferation and differentiation, decreasing apoptosis, and altering and maintaining DNA synthesis [3,9,35]. Folate deficiency during pregnancy can result in megaloblastosis (i.e., large cells with arrested nuclear maturation) and cell death, particularly in rapidly proliferating somatic cells [35]. Embryonic neural tube and neural crest cells are highly proliferative and folate deficiency during the period of neural tube closure (21 to 28 days post conception) predisposes the fetus to neural tube defects [23,46].
Given the ethical considerations regarding maternal nutrition during pregnancy, experimental research examining the effects of maternal folate deficiency on fetal brain development has been conducted in animals only. Craciunescu et al. found that maternal folate intake affected fetal forebrain progenitor cells during late gestation [47,48]. In these studies, pregnant mice were all fed the same standard diet containing 2 mg/kg of folic acid until day 11 of gestation and then folic acid was removed from the diets of the mice in the folate-deficient group [47,48]. Offspring of folate-deficient mice were found to have fewer replicating neural progenitor cells in the hippocampus, caudate, putamen, striatum, anterior and mid-posterior neocortex, and ventricular zones of the septum [47,48]. It was also found that apoptotic cells were more prevalent in the fetal septum and hippocampus of mice from mothers fed a folate-deficient diet as compared to the control mice [47,48]. Folic acid supplements before and during early pregnancy are essential for preventing neural tube defects; however, these animal studies suggest that folate availability throughout gestation is critically important for fetal brain development in both cortical and subcortical regions of the brain. Figure 1 provides a schema representing the potential mechanism of action through which folate is associated with children's brain and cognitive development. . Potential mechanism of action through which prenatal folate affects brain and cognitive outcomes in children.

Gestational Choline and Fetal Brain Development
Choline is also an essential nutrient but was only officially recognized as such in 1998 [49]. There are currently no mandates regarding choline supplementation of food in Canada or the United States [49][50][51]. Although choline is produced endogenously in the human liver, the amount of choline naturally produced is insufficient to meet the needs of the human body and thus adequate levels of choline can only be obtained through diet [52]. Animal food products such as beef, eggs, chicken, fish, and pork are major dietary sources of choline containing more than 60 mg of choline per 100 g [49,[52][53][54]. Plant foods such as nuts, legumes, and cruciferous vegetables are also good dietary sources of choline containing at least 25 mg of choline per 100 g [53,54]. Choline can also be obtained through dietary supplements containing choline only, choline in combination with B-complex vitamins, or in some multivitamin products [54]. Choline dietary supplements typically contain between 10 mg and 250 mg of choline per dose and often provide choline in the form of choline bitartrate, phosphatidylcholine, or lecithin [54].
It is recommended that choline intake should increase from 425 mg/day in nonpregnant women to 450 mg/day in pregnant women [54]. Large amounts of choline from maternal diet are transferred across the placenta from the mother to the fetus [53,[55][56][57]. Choline concentrations in the amniotic fluid are 10 times greater than in maternal blood, and plasma choline concentrations are 3 and 6 to 7 times greater in the umbilical cord and fetus, respectively, than they are in the maternal blood [44,45]. It is believed that choline plays a similar role to folate in brain development, including acting as a methyl donor in DNA methylation [9,51]. Like folate, choline is important for the prevention of neural tube defects as it is required for closure of the neural tube [9,22,58,59]. Choline also plays a significant role in hippocampal development and has been associated with the development of neural pathways and the expression of genes involved in memory processes [50,51].
In human diets, the most common dietary form of choline is phosphatidylcholine (PC); however, it can also be found as free choline, phosphocholine, sphingomyelin, glycerophosphocholine, and lysophosphatidylcholine [53]. Choline is a component of sphingomyelin, which is a constituent of the myelin sheath of nerve axons and facilitates efficient transmission of nerve signals [51,53]. As a methyl donor in DNA methylation, choline is thought to regulate the expression of genes involved in regulating synaptic plasticity, learning, and memory [50,51]. As with folate, choline also plays a role in the conversion of homocysteine to methionine and helps regulate homocysteine concentrations in the body [55]. Further, it acts as a major constituent of phospholipids in cell membranes and signaling lipids in cells and is involved in the biosynthesis of lipoproteins [9,51,53,55]. It is also a constituent of the neurotransmitter acetylcholine (ACh), which acts in both the peripheral and central nervous systems [9,53,55,60]. ACh influences processes in the developing brain including progenitor cell proliferation and differentiation, neurogenesis, gliogenesis, cell survival, morphology and migration, and synaptic plasticity, and supports the development of the hippocampus [53]. ACh released from cholinergic neurons that extend into the hippocampus and cerebral cortex from the basal forebrain also regulates multiple processes including attention, learning and memory [61].
Like experimental studies examining the effects of folate deficiency on brain development in mice, animal models have also shown that gestational choline levels have significant effects on fetal brain development. Studies have found that maternal choline deficiency resulted in decreased progenitor cell proliferation and neurogenesis, and increased apoptosis in the fetal hippocampus [56,62,63]. Further, maternal choline supplementation stimulated hippocampal cell division and enhanced hippocampal neurogenesis [56,64]. Other studies have found that modification of choline intake levels in pregnant rats resulted in changes in the timing of migration and differentiation of neuronal progenitor cells in fetal hippocampal regions [62,65]. Thus, to support healthy brain differentiation and development, particularly in regions important for learning and memory such as the hippocampus, research suggests that it is important for a fetus to have access to adequate amounts of choline throughout gestation. Figure 2 provides a schema representing the potential mechanism of action through which choline is associated with brain and cognitive development in children. Choline *Hippocampus* Figure 2. Potential mechanism of action through which prenatal choline affects brain and cognitive outcomes in children.

Potential Interactions between Gestational Folate and Choline
Few studies have examined the interactive effects of folate and choline on fetal brain development. Craciunescu et al. found choline supplementation may modify the effects that dietary folate availability has on neural progenitor cells in the fetal forebrain during late gestation [48]. Specifically, they reported that the rate of mitosis in folate-deficient cholinesupplemented (FDCS) mice (i.e., 4.95 g choline chloride/kg diet, 0.0 mg folic acid/kg diet) was less than that of control mice who were supplemented with standard amounts of folate and choline (i.e., 1.1 g choline chloride/kg diet, 2 mg folic acid/kg diet) but greater than that of folate-deficient mice (i.e., 1.1 g choline chloride/kg diet, 0.0 mg folic acid/kg diet) [48]. Further, hippocampal apoptosis rates in FDCS mice were found to be significantly lower than those of folate-deficient mice, but the same as those in control mice [48]. As both folate and choline are methyl-donor nutrients that influence neurogenesis and apoptosis, it is possible that choline supplementation might mitigate the effects of folate deficiency on brain development, and this may be mediated by epigenetic events (i.e., methylation of DNA and histones important for epigenetic control of gene expression) [66,67]. Investigations of the interactive effects of folate and choline is important because they could provide a better understanding of how these nutrients work together to support brain and cognitive development. Future research may also provide further evidence that maternal gestational supplementation of these two nutrients is critical for optimal fetal brain development and subsequent cognitive outcomes in children.

The Current State of Knowledge on the Association between Maternal Folate and Offspring Cognitive Outcomes
Animal studies (see Table 1) suggest a relationship between maternal folate intake during pregnancy and offspring brain development; however, a limited number of studies have examined the effects of maternal folate levels on cognition. Jadavji et al. found that maternal folate deficiency in mice (i.e., 0.3 mg folic acid/kg diet) was associated with short-term memory impairment in offspring based on their poorer performance on the novel object recognition task and the Y-maze test compared to offspring of mice fed folate sufficient diets (i.e., 2 mg folic acid/kg diet) [68]. Additionally, Ferguson et al. found that maternal folate deficiency (e.g., 400 nmol of folic acid/kg diet) in mice produced offspring who exhibited more anxiety-related behavior on the elevated plus maze test [69]. These animal studies suggest that maternal gestational folate deficiency is associated with poorer cognitive and behavioural outcomes in offspring and that gestational folate availability is important for offspring cognitive and behavioural development.
Human studies (see Table 1) have also examined the association between maternal folate levels during pregnancy and child cognitive outcomes; however, various methods have been used to measure maternal folate levels. Several studies considered maternal use of folic acid supplements only during pregnancy. Julvez et al. found that in four-year-old children, verbal, motor-executive function, and verbal-executive function scores on the McCarthy Scales of Children's Abilities, and social competence and inattention symptom scores on the California Preschool Social Competence Scale were positively associated with maternal use of folic acid supplements during the first trimester of pregnancy [70]. Similarly, Wehby and Murray assessed the effect of using folic acid supplements at conception and/or during the first trimester of pregnancy on cognitive development at three years of age using 16 items from the Denver Developmental Screening Test and found that prenatal folic acid supplementation had a positive effect on children's overall cognitive and gross motor development [71]. A study conducted in the United Kingdom reported that seven-year-old children of mothers who received 400 µg folic acid per day during the second and third trimester of pregnancy had higher scores on the Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III) as compared to the children of mothers who were given a placebo [72]. Further, compared to a nationally representative sample of children seven years of age, the children of the folate-supplemented mothers had higher verbal, performance, general language, and Full-Scale IQ scores on the WPPSI-III [72]. It is notable that the folic acid supplement given to the women in this study was at the low end of the recommended range for folic acid intake for pregnant women (e.g., 400-800 µg/day) [34,36,37,72,73]. In another study, Chatzi et al. examined neurodevelopmental outcomes on the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) in 18-month-old children of mothers who reported taking daily supplements of 5000 µg of folic acid or more throughout pregnancy compared to children of mothers who did not use a folic acid supplement throughout pregnancy [74]. They found that children of mothers who reported taking a daily supplement had a 5 unit increase on receptive communication and a 3.5 unit increase in expressive communication on the Bayley-III scales [74]. The maternal folic acid intake of the women in this study was well above the recommended intake for pregnant women (e.g., 400-800 ug/day) [34,36,37,73,74]. Finally, a recent meta-analysis conducted by Chen et al., which investigated the effect of maternal folic acid supplementation on children's neurodevelopment, concluded that appropriate maternal folic acid supplementation may have positive effects on children's intelligence and development and reduce the risk of language problems, ADHD, autism traits, and behavioral problems [75]. Cumulatively, the research evidence suggests that maternal folic acid supplementation, even at the lower end of the recommended range throughout pregnancy, is an important predictor of children's cognitive outcomes. However, in the majority of these studies, women's actual folate levels were not measured and folate intake from their diets was not considered.
In addition to the above supplementation studies, other studies have used food frequency questionnaires (FFQs) to estimate levels of maternal prenatal folic acid and examine associations with cognitive outcomes. For example, Villamor et al. found that for each 600 µg per day increase in total folate intake during the first trimester of pregnancy assessed using FFQs there was a 1.6-point increase in scores on the Peabody Picture Vocabulary Test, Third Edition (PPVT-III) in three-year-old children [76]. The women in this study had a mean estimated folate intake of 949 ± 390 µg/day, which was well above the recommended daily intake for pregnant women [76]. In contrast, Boeke et al. did not find an association between increased folate intake assessed using FFQs during the first and second trimesters of pregnancy and higher scores on the Wide Range Assessment of Memory and Learning, Second Edition (WRAML2) or the Kaufman Brief Intelligence Test, Second Edition (KBIT-2) in children at age seven [77]. The women in this study had a mean daily estimated folate intake of 972 ± 392 µg/day and 1268 ± 381 µg/day in the first and second trimesters of pregnancy, respectively, which were well above the recommended intake levels for pregnant women [77]. The results of these studies suggest that maternal folic acid intake may be an important predictor of some areas of cognitive development (e.g., language) but not of other areas (e.g., memory and intelligence). The use of FFQs to quantify maternal folic acid intake accounts for most dietary sources of folic acid and thus provides an accurate estimation of women's actual folate intakes; however, there can be variability among FFQs in the dietary sources of folic acid considered and participants' recall can limit the accuracy of this method [78,79].
Studies have also measured maternal folate concentrations (i.e., plasma/serum folate, red blood cell (RBC) folate) directly from blood samples collected during pregnancy. Although plasma/serum and RBC folate concentrations are both associated with folic acid intake [20,21], they result from different biologic processes and are not interchangeable [80,81]. Plasma/serum folate concentrations reflect very recent intake, whereas RBC folate concentrations reflect the long-term average of intake over the life span of red blood cells and folate stores in the liver [82]. As noted above, it is recommended that pregnant women should consume at least 400 to 800 µg of folate per day; however, a corresponding plasma/serum folate level has not been established [34][35][36][37]83,84]. Therefore, the results of studies that have examined the associations between plasma/serum and RBC folate levels in maternal blood and child outcomes may not be comparable. A study in India that measured maternal plasma/serum folate concentrations at 28 to 32 weeks' gestation (i.e., third trimester) reported a positive association with children's performance on a test of cognitive function (i.e., Kaufman Assessment Battery for Children) [85]. The women in this study had mean plasma/serum folate concentrations of 34.7 ± 19.2 nmol/L, which were within the normal range (i.e., 7-46 nmol/L) for non-pregnant women [34,[85][86][87]. Ars et al. found that low plasma/serum folate concentrations below 8 nmol/L during the first trimester of pregnancy were associated with smaller total brain volume and poorer language and visuospatial skills on the NEPSY-II-NL in children at six years of age [88]. In contrast, maternal plasma/serum folate concentrations at 16 and 36 weeks gestation (i.e., second and third trimesters) were not associated with scores on the Bayley-III at 18 months of age [89]. The women in this study had mean plasma/serum folate concentrations of 36.4 ± 8.08 nmol/L at 16 weeks gestation, which was in the normal range for non-pregnant women [34,86,87,89]. Tamura et al. examined the association between RBC folate status during the second and third trimesters of pregnancy and children's neurodevelopment at age five years and found no associations [90]. Neurodevelopment was assessed using the Differential Ability Scales, Visual and Auditory Sequential Memory Tests, Knox Cube Test, Gross Motor Scale, and Grooved Pegboard Test [90]. In this study, the women's mean RBC folate concentrations were 873 nmol/L, 1070 nmol/L, and 1096 nmol/L at 19, 26, and 37 weeks gestation, respectively, which were slightly above or slightly below the recommended RBC folate concentration of 906 nmol/L for pregnant women [34,37,90]. Like studies that used FFQs, which estimate recent folate levels, there was some support that maternal plasma/serum folate levels were associated with children's cognitive outcomes. However, both FFQs and plasma/serum folate assess a woman's folate levels at a given time point. They are not predictive of folate levels across pregnancy as levels may vary depending on when the blood sample was taken [91][92][93]. In contrast, the Tamura et al. study, which investigated the association between RBC folate (which is reflective of longer-term intake), and child outcomes did not report any associations, suggesting that the inconsistencies in the findings of human studies may be due to differences in the measure used to assess folate levels.

The Current State of Knowledge on the Associations between Maternal Choline and Offspring Cognitive Outcomes
In rat and mice models (see Table 2), high maternal choline intake during pregnancy has been found to enhance the cognitive abilities of offspring; however, the optimal amount is not known [50]. Pregnant rats whose diet was supplemented with choline chloride were found to produce offspring with enhanced visuospatial memory skills as compared to non-choline-supplemented rats [94]. Choline supplementation in pregnant rats has also been linked to increased ability to retain a larger number of items in working memory, increased memory precision due to less proactive interference, an increased tendency for object exploration, and increased thresholds for implementing chunking strategies in offspring [94][95][96][97]. Further, choline supplementation in pregnant rats facilitates temporal memory and accelerates the maturation of relational cue processing in offspring [51,98]. These findings suggest that choline supplementation during pregnancy may be predictive of better cognitive function in offspring, particularly offspring memory.
Enhanced cognitive function associated with increased maternal intake of choline during gestation has also been noted in human studies (see Table 2). The methods used to investigate choline levels in women were like those used in studies that examined maternal folate levels (i.e., supplementation, FFQs, measurements from blood samples). One study reported that infants born to women who were supplemented with 930 mg of choline chloride per day, compared to infants of women who were receiving 480 mg of choline per day, displayed improved information processing speed measured using a visual attention task [99]. It is of note that the choline levels in both groups were above adequate intake levels for pregnant women [34,54,99]. Another study used a FFQ to estimate choline intake during the first and second trimesters and found that prenatal choline intake was positively associated with memory scores on the WRAML2, but not intelligence scores on the KBIT-2 in seven-year-old children [77]. The women in this study had a mean estimated daily choline intake of 328 ± 63 mg/day, which was below the recommended adequate intake level for pregnant women (i.e., 450 mg/day) [34,54,77]. A study that directly measured maternal plasma-free choline at 16 weeks gestation reported positive associations with infant cognitive development at 18 months of age on the Bayley-III [89]. The mean plasma-free choline concentration of the women was 7.07 ± 1.78 umol/L and the mean estimated daily choline intake was 383 ± 98.6 mg/day at 16 weeks gestation, which was below the recommended adequate intake level for pregnant women [34,54,89]. However, in a study conducted by Signore et al., maternal gestational free and total serum choline concentrations measured at multiple timepoints throughout pregnancy (i.e., at 16 to 18 weeks, 24 to 26 weeks, 30 to 32 weeks, and 36 to 38 weeks gestation) were not associated with Wechsler Preschool and Primary Scales of Intelligence-Revised (WPPSI-R) Full-Scale IQ, visuospatial processing or memory in children at five years of age [100]. There are no recommendations regarding serum choline concentrations in maternal blood during pregnancy; therefore, no conclusions can be drawn as to whether these women were below, within, or above adequate recommended serum choline levels for pregnant women. The results from these studies suggest that maternal choline may be predictive of children's memory, attention, and processing speed, even at low levels; however, future research is needed that examines these relationships when choline intake during pregnancy is at or above recommended intake levels. Additionally, the form of choline measured in studies should also be considered as one study found that plasma free choline concentrations were not predictive of dietary choline intake during pregnancy [101]. Considering the evidence from animal models supporting the importance of maternal choline for brain development and particularly hippocampal function, and that choline supplementation might mitigate the negative effects of folate deficiency on brain development, future research that examines folate and choline together and considers possible interactions between these two nutrients is needed.

Gaps in the Current State of Knowledge and Directions for Future Research
Animal studies have established that gestational availability of folate and choline influences numerous processes that are critical for healthy development of the fetal brain. The current literature also suggests that maternal folate and choline levels during pregnancy may be important predictors of children's cognitive outcomes; however, there are inconsistencies in the findings that have yet to be resolved. These inconsistencies may be a result of several methodological constraints and confounding factors that have not been accounted for in the current literature.
One such methodological constraint is that the methods used to assess folate and choline levels in the current literature are not consistent across studies and some have inherent limitations that limit validity of the findings. For example, several studies have simply assessed whether women did or did not take folic acid or choline supplements during pregnancy and did not account for dietary sources of folate or choline [70][71][72]74,99]. FFQs were also used frequently to estimate women's folate and choline intake; however, studies have shown that there can be significant variability between the dietary sources of these nutrients that are considered, and levels of these nutrients in the dietary sources [78,79]. This variability could influence the consistency of the results across studies that use this method. Other studies have assessed plasma/serum levels of folate or choline concentrations; however, there are currently no recommendations regarding their adequate or recommended levels in pregnant women [85,89,100]. Thus, classifying pregnant women as below, within, or above recommended ranges for these nutrients during pregnancy based on their concentrations in serum/plasma blood samples is not currently possible. The use of various methods to assess women's folate and choline levels during pregnancy limit the possibility of comparing the results of the research conducted to date. Further, the lack of consistency in the findings reported in the human literature could be due to the different methods used to measure folate and choline levels. Future research is needed that investigates the biological relevance of folate and choline intake and plasma/serum levels and RBC folate levels on human brain development and their association with children's cognitive outcomes.
Another potential methodological constraint is that the gestational trimester in which folate and choline levels are measured could be differentially associated with children's cognitive outcomes [70]. Changes in concentrations of folate and choline throughout pregnancy and the impact of varying concentrations of these nutrients on children's neurodevelopmental outcomes are areas of research in need of further investigation. Lewis et al. and Signore et al. both found that maternal free and total choline concentrations remain relatively constant throughout gestation [100,102]. However, Fayyaz et al. found that folate concentrations tend to increase throughout pregnancy [86]. Interestingly, Villamor et al. found that maternal folate intake during the first trimester, but not the second trimester of pregnancy, was associated with children's scores on the PPVT-III at three years of age, suggesting that the timing of folate supplementation during pregnancy could influence children's neurodevelopment [76]. However, the precise timing and the ideal concentrations of these nutrients at different time points during pregnancy to ensure optimal neurodevelopment are currently unknown.
Another issue of concern is that the research to date has examined a limited number of potential confounders. Demographic factors such as family socioeconomic status and maternal and paternal level of educational attainment have been associated with maternal nutrient use and children's cognitive development [103,104]. Sample characteristics such as maternal pre-pregnancy body mass index (BMI) and child sex have also been found to be confounding factors that can influence children's cognitive development [105,106]. Finally, other important micronutrients that are often supplemented during pregnancy such as vitamins B-12 and B-6, omega-3 long-chain polyunsaturated fatty acids, and iron have also been found to be associated with children's cognitive outcomes [107][108][109][110][111][112][113][114][115]. These variables need to be considered as potential confounding factors when assessing the effects of folate and choline on children's neurodevelopment.
In addition to the already identified methodological and analytical issues, it is important to note that there is an imbalance in the number of studies that considered the effects of maternal folate compared to maternal choline. More human studies have examined the association between maternal folate levels and children's cognitive outcomes and no studies have investigated their interactive effects. The evidence from animal models suggesting that gestational choline supplementation may reduce the adverse effects of folate deficiency on fetal brain development, supports the conduct of human studies that investigate how these two nutrients interact to influence cognitive outcomes in children.
Further, limited research has investigated possible biological mechanisms (e.g., DNA methylation alterations) that may underlie these associations. It has been noted that folate and choline likely play similar roles in the development of the fetal brain and central nervous system and that their interaction may influence fetal brain development [9,48]. However, to date, no human studies have examined potential synergistic effects of maternal folate and choline levels during pregnancy and potential underlying biological mechanisms (i.e., DNA methylation) on children's cognitive outcomes One last point that needs to be considered in future research is the level of supplementation. Several studies have reported that supplementing with large amounts of folate or choline during pregnancy may result in adverse child health outcomes, including low birth weight and increased risk for developing autism spectrum disorder (ASD) or colitis [116][117][118][119][120][121]. However, there is little research on the association between very high maternal choline or folate supplementation and adverse child neurodevelopmental outcomes. Further, the precise levels above the daily recommended levels for folate and/or choline that would be harmful to children's neurodevelopment have yet to be determined.
In summary, the findings from this narrative review suggest that future nutritional research examining the associations between maternal folate and choline levels and children's cognitive outcomes should develop and use consistent and accurate methods to measure women's folate and choline levels across the different trimesters of pregnancy and consider a wider range of potential confounding variables (e.g., demographic factors, levels of other nutrients) in order to reduce inconsistencies in results that could be attributable to discrepancies in methodology. Additionally, future research is needed that addresses the following issues: (a) What are the optimal intake levels/status of folate and choline needed during pregnancy to promote optimal child neurodevelopment? (b) Do the optimal levels/status vary across the different trimesters of pregnancy? and (c) Is there a risk to children's neurodevelopment if maternal intake/status is above or below the optimal levels? Additionally, potential biological pathways (e.g., DNA methylation) should be examined to further understand the mechanistic basis for both folate's and choline's role in children's brain and cognitive development and how these nutrients might interact within the context of early development to further support children's neurodevelopment. It is important that future research address these methodological limitations and mechanistic questions to better understand how maternal folate, choline, and their interaction during pregnancy, influence brain and cognitive development in children.

Conclusions
Folate and choline are important methyl-donor nutrients that play critical roles in the development of the fetal brain. Animal studies indicate that gestational folate and/or choline levels are associated with structural and cellular alterations in various brain regions such as the cerebral cortex and hippocampus. The current human literature also suggests that maternal folate and choline concentrations during pregnancy may play a role in children's cognitive development; however, there are some inconsistencies in published research. To address these inconsistencies, future research needs to use consistent methods to assess women's folate and choline levels during pregnancy, and consider confounding factors (e.g., parental socioeconomic status, maternal pre-pregnancy BMI, infant birth weight, maternal levels of other micronutrients during pregnancy, gestational trimester), relative maternal folate and choline concentrations (e.g., below, at, or above recommended levels) and the potential synergistic effects that maternal folate and choline levels may have on fetal brain development and in turn children's cognitive development.