Choline, Folic Acid, Vitamin D, and Fetal Brain Development in the Psychosis Spectrum

Abstract

Choline, folic acid, and Vitamin D are essential for fetal brain development that may be the first steps in the pathogenesis of the psychotic spectrum. Micronutrient deficiencies have been associated with changes in fetal brain development, manifest as early problems in childhood behavior, and cognition, and later as increased incidence of psychotic and autism spectrum disorders. Micronutrient supplements may not only prevent deficiency, but they may also positively affect brain development in the context of other maternal risk factors, including maternal infection, stress, inflammation, and substance abuse. Many genes associated with later psychotic illness are highly expressed in the fetal brain, where they are responsible for various neurodevelopmental mechanisms. Interaction of micronutrient vitamins with these genetically programmed mechanisms to prevent pathological brain development associated with later psychosis is under active investigation. In addition to their effects on brain development, micronutrient vitamins have effects on other aspects of gestation and fetal development, including the prevention of premature delivery and other developmental abnormalities. Supplemental micronutrient vitamins should be part of good prenatal care, as has already happened for folic acid and Vitamin D and is now advocated by the American Medical Association for choline. The benefits of these micronutrient supplements include protection of brain development and the possibility of decreased risk for future psychotic disorders in those children who are either genetically or environmentally vulnerable. The purpose of this review is to present the current evidence supporting the safety and effectiveness of micronutrients in gestation and to suggest areas for future research.

1. Introduction

Gestation is well-established as a critical period of pathogenesis for schizophrenia and other psychotic disorders. Decades of epidemiological studies have documented increased risk for psychotic disorders in offspring whose mothers experienced starvation, infection, stress and depression, and substance abuse during early to mid-gestation (Susser and Lin, 1993; Brown and Derkits, 2010). It is likely that the current COVID-19 epidemic will add to these findings, because SARS-CoV-2 infection induces maternal inflammation similar to other respiratory viruses (Chen et al., 2020; Centers for Disease Control, 2020). Maternal inflammatory responses to infection attack the placenta and fetus, which is one of several putative pathogenic mechanisms that alters fetal brain development and increases risk for later psychiatric and behavioral disorders (Ernst et al., 2013; Brown and Meyer, 2018; Vasistha et al., 2019). A second pathogenic element during gestation is the genetic risk for psychotic disorder. Many of the genes identified in both genome-wide association and candidate gene studies as carrying pathogenic variants for psychosis are highly expressed in fetal brain. For some of these genes, the prenatal expression is significantly greater than postnatal (Birnbaum et al., 2014).

Despite the evidence for gestation as a critical pathogenic period, little attention has been paid to therapeutic intervention possibilities. However, prenatal folic acid supplementation is a paradigmatic example of the prevention of a serious neurodevelopmental abnormality during gestation. The success of folic acid supplementation in preventing major neural-tube developmental defects has led to its universal acceptance as a prenatal preventative intervention (Viswanathan et al., 2017). Spina bifida is not solely the result of maternal folic acid deficiency. Its complex pathogenesis includes multiple genetic and environmental elements, like schizophrenia (Au, Ashley-Koch, and Northrup, 2010). However, regardless of potential causal factors for neural tube deficit in any specific fetus, folic acid supplementation is a generally effective prevention. Yet, the possibility that the neurodevelopmental risk for psychosis could be similarly diminished by folic acid or any other micronutrient has not been fully assessed.

Several problems present themselves. Neural tube developmental defects are immediately ascertained at birth, but psychosis takes decades to be expressed as a diagnosable condition. Intermediate markers of developmental problems, including attenuated or prodromal psychotic symptoms, are associated with increased risk for schizophrenia, but no symptoms or biological marker in infants, children, or even in adolescents already developing signs of illness, definitively identifies individuals destined to develop psychosis. Thus, a study would need to last for several decades, until schizophrenia or other psychoses are diagnosed, to definitively establish a preventive effect for psychoses.

A second problem is that micronutrients are already widely available as supplements and endorsed as good maternal care, because many of their benefits, including neural tube closure, are critical to the success of fetal development. Therefore, the possibilities for placebo-controlled clinical trials are ethically limited, because the placebo condition is potentially harmful to the fetus. Additionally, regardless of assignment to placebo or treatment, micronutrient vitamins are available to the mother and fetus from many other sources. Vitamins A, D, and folic acid are supplemented in many processed foods to protect fetal development even if the mother does not take supplements per se. Foods that naturally contain these nutrients, as well as choline, include vegetables, fruits, grains, eggs, and meats. Vitamin D can be synthesized in the skin by exposure to sunlight, and choline can be synthesized by the mother’s liver. Thus, a well-controlled trial comparing nutrient to no-nutrient conditions, the standard design for most medical interventions, is not possible.

Nevertheless, surveys of diet in most countries find that many mothers are deficient, with low plasma levels of Vitamin D, folic acid, and choline (Jensen et al., 2007; Brunst et al., 2014; Liu et al., 2017; Groth et al., 2017; Bailey et al., 2019; Li and Freedman, 2020). Thus, observational studies using maternal plasma measurements as a proxy summation of dietary and supplement intake to compare the outcomes of gestation with higher and lower levels of micronutrients are a possible alternative strategy. However, maternal micronutrient levels reflect confounding factors that themselves can affect fetal development. Maternal stress, alcohol and nicotine use, and socioeconomic constraints on diet are among the several factors that can affect micronutrient levels, but also impact fetal development by other mechanisms (Hunter et al., 2020).

The timing of supplementation is another difficult issue (Tables 1–3). Folic acid is most effective if begun pre-conception. Vitamin D supplementation has shown beneficial effects through the postnatal period. Choline has been supplemented in clinical trials beginning in early second trimester through late third trimester. Optimal timing is important to guide public health directives. Even with established directives, pre-conception and early gestation administration depend upon planning for pregnancy, but a substantial fraction of pregnancies are not planned, a source of maternal stress in itself.

Table 1.

Summary of major studies of prenatal folic acid effects on child development.

Study Protocol	N	Findings	Reference
U.S. case-control study of schizophrenia based on high maternal serum homocysteine (interpreted as low folic acid) in third trimester	N = 63 cases of schizophrenia with 122 controls	Odds ratio of schizophrenia increased 2.39 (1.18–4.81) in pregnancies with highest tertile of homocysteine; elevated, but non-significant effect at 3 mos (17 cases).	Brown et al., 2007
Rotterdam case-control study of maternal folic acid supplement use by gestational wk 10 children with Child Behavior Checklist toddler version at 18 mos	N = 4214	Decreased attention, social withdrawal, and aggression problems.	Roza et al., 2010
U.S. case-control study of autism spectrum disorder and folic acid intake in 1st gestational month	N = 429 cases of autism spectrum disorder with N = 278 controls	Daily folic acid intake >600μg/day 1st mo gestation associated with decreased ASD. Association stronger if mother or child had MTHFR 677 C>T variant.	Schmidt et al., 2012
Norwegian case-control study of folic acid supplements and fish oil and autism spectrum disorder	N = 85,176 with n = 270 cases	Use of folic acid 400μg per day from −4 to 8wks gestation reduced autism spectrum disorder risk. No effect of fish oil supplements in same period. No effect of folic acid at 22 wks gestation.	Surén et al., 2013
Swedish case-control study of various prenatal supplements and autism spectrum disorder	Multivitamins n = 62,840; Iron only n = 90,138; Iron and folic acid n = 25,445; Folic acid only n = 2789; None n = 91895; 5575 cases	Use of multivitamins with or without folic acid and iron decreased the prevalence of autism spectrum disorder; folic acid and iron together or alone had no significant effect.	DeVilbiss et al., 2015
U.S. case-control study of autism spectrum disorder and frequency of folic acid supplements	N = 86 cases of autism spectrum disorder with N = 1171 controls	Low (≤2 times/week) and high (>5 times/week) prenatal supplementation was associated with increased risk of autism spectrum disorder. Plasma folate at birth (≥60.3 nmol/L) had 2.5 times increased risk compared to mid 80th percentile folate levels, adjusting for MTHFR genotype. Similar findings for Vitamin B12.	Raghavan et al., 2018
Spanish study of maternal preconception and gestational folic acid supplement dose on offspring cognition at 4–5 years of age	N = 1682, Folic acid supplement ≤400μg/day 55%, 400–999μg/day 15%, ≥1000μg/day 40%	Preconception-3 mos gestation folic acid supplements ≥1000μg/day or ≤400μg/day were associated with decrease cognition.	Valera-Gran et al., 2017
Israel case-control study of folic acid and multivitamin supplementation and autism spectrum disorder	N = 572 cases of autism spectrum disorder with N = 45,300 controls	Supplementation of folic acid or multivitamins or both before but not during pregnancy as effective as during pregnancy.	Levine et al., 2018
Israel case-control study of prenatal folic acid supplementation controlling for birth order	N = 2009 cases of autism spectrum disorder with 19,886 controls	No association of amount of of folic acid purchased during gestation, if association of first birth order with both folic acid use and autism spectrum disorder was considered.	Sharman Moser et al., 2019
British randomized trial of 400μg folic acid supplement continuation past 1st trimester supplement versus placebo	N = 33 folic acid, N = 37 placebo	Significantly improved cognition at 7 yrs of age in folic acid group.	McNulty et al., 2019
Northern Sweden case-control study of maternal folic acid concentration at 14 wks gestation	N = 100 cases of autism spectrum disorder with 100 controls	Marginal increased autism spectrum risk with higher folic acid; OR per 1 SD increase over mean: 1.70, P = 0.07.	Egorova et al., 2020

Table 3.

Summary of major studies of prenatal choline or phosphatidylcholine effects on child development.

Study Protocol	N	Findings	Reference
Controlled clinical trials of Choline or Phosphatidyl-choline
600mg choline as phosphtidylcholine vs. placebo, healthy pregnancies 18wks gestation	N = 49 choline, N = 50 placebo	Mean Mullens Global Index scores 1 standard deviation above the population mean for both groups at 12mo	Cheatham et al., 2012
900mg choline as phosphatidylcholine vs. placebo, healthy pregnancies 17wks gestation	N = 46 choline, N = 47 placebo	Increased P50 auditory evoked potential inhibition 1 mo; fewer Child Behavior Checklist 1½–5 Attention and Withdrawn problems at 4y	Ross et al., 2013, 2016;
550mg choline as choline chloride vs. 100mg, healthy pregnancies 22–29wks gestation	N = 13 550mg, N = 13 100mg choline	Faster reactive saccades at 13mo; higher Child Color-location Memory Task at 7y	Caudill et al., 2018; Bahnfleth et al., 2019
2g choline as choline bitartrate vs. placebo at 24–27wks gestation vs. placebo, women continued binge drinking and drug abuse	N = 31 choline, N = 31 placebo	Higher Fagan Test of Infant Intelligence novelty preference 12mo	Jacobson et al., 2018
Other clinical studies
Serum free choline at 16–18 wks gestation and later periods, including cord blood. The study excluded over 1000 women, many with premature birth and intrauterine growth problems.	N = 404 women	No effect of maternal serum choline level on Wechsler Preschool and Primary Scale of Intelligence-Revised scales at 5.5y. Free choline was positive but non-significantly related to performance IQ.	Signore et al., 2008
Maternal plasma free choline at 16wk gestation	154 women	Positive association with Bayley Scales of Infant Development III cognitive score at 18 mos	Wu et al., 2012
Comparison of highest quartile of 2nd trimester dietary choline intake to lowest quartile.	895 women	Higher Wide Range Assessment of Memory and Learning, Design and Picture Memory subtest score 7y	Boeke et al., 2013
Maternal plasma choline at 16 wks > mean-1 standard deviation of Ross et al., 2013 supplement level vs. levels < mean-1 standard deviation. Women in this study did not receive supplements.	N = 162 women, including 25 who abused cannabis and 43 who had respiratory virus infections at 10–16 wks gestation and 25 Black women	Increased P50 auditory evoked potential inhibition 1 mo; Higher Infant Behavior Questionnaire-R Orienting/Regulation at 3 mos; fewer Child Behavior Checklist 1½–5 Attention and Withdrawn problems at 4y; Black women had lower choline levels than other groups, associated with higher rates of premature birth. [N= 7 Black women who received phosphatidylcholine in Ross et al., 2013 trial delivered at longer gestational age, near term, compared to 8 Black women who received placebo, who averaged over 2wks preterm.]	Hoffman et al., 2019; Freedman et al., 2020; Hunter et al., 2020a,b; Freedman et al., 2020
Estimate of dietary choline intake and PEMT genotype.	M = 165 women with preterm births vs. 165 with term births	Increased preterm birth with lower choline intake and PEMT rs7946 AA genotype vs. higher choline intake and PEMT rs7946 GG genotype	Zhu et al., 2020

These problems are not fully resolved in the existing literature. The purpose of this review is not to repeat analyses that show that these micronutrients are generally beneficial, which is already well known. Rather, this review aims to identify opportunities for further research to enhance the use of these nutrients to promote fetal development and thereby possibly prevent future psychotic disorders.

2. Method

A Medline search was conducted for (schizophrenia OR psychotic) AND (prenatal OR gestation OR fetal) AND each nutrient yielded 16 articles for folic acid, 6 articles for choline, and 31 articles for Vitamin D. A search for Vitamin A largely overlapped with Vitamin D (Tables 1–3). Articles for this review were selected by the authors by two criteria: (1) latest original reports of observational, case-control, or randomized clinical trial studies, or (2) earlier studies that reported major findings.

Prenatal effects of omega-3-fatty acids, either as specific supplements or fish oil, were not considered extensively. The only case-control study found that the highest tertile of maternal docosahexaenoic acid levels was associated with an increased risk of offsprings’ schizophrenia spectrum disorders (Harper et al., 2011). A randomized trial found no additional effect of fish oil supplements beyond that afforded by folic acid on attention in 8 year children (Catena et al., 2016).

3. Results

3.1. Prenatal folic acid supplementation

Folic acid, as mentioned above, is universally accepted as a preventative measure for midline developmental defects, including spina bifida and facial clefts. The standard dose is 400 μg, but doses up to 4 mg have been safely used for women who have had previous gestations with fetal spina bifida or other neural tube defects (Wald et al., 1991). The primary reason for keeping doses generally below 1 mg is that higher doses can disguise the hematological identification of Vitamin B₁₂ deficiency and pernicious anemia (Centers for Disease Control, 1992).

Randomized trials conducted to establish the value of folic acid for the prevention of neural tube defects were halted before completion by their safety advisory committees because the effectiveness of folic acid became established early in the trial (Viswanathan et al., 2017). However, many mothers do not take folic acid supplements pre-conception or even during pregnancy itself. Case-control and observational studies have made use of differences in compliance to study the effectiveness of folic acid in prevention of mental illness, including autism spectrum disorder and schizophrenia (Table 1). The timing of the supplement in gestation and the maternal serum level or dose of supplement have been major issues.

The Generation R study in Rotterdam compared women who did or did not use folic acid supplements at various times during gestation. That study showed cognitive and behavioral benefits in early childhood in offspring of all mothers who used folic acid during pregnancy, with superior benefits if the folic acid supplement was instituted before 10 weeks gestation. Decreased problems in attention, social withdrawal, and aggression were observed (Roza et al., 2010). A Norwegian study had similar findings, with benefits if folic acid was instituted from preconception through the first 8 weeks of gestation, and no effect if folic acid was instituted at 22 weeks (Surén et al., 2013). Some countries recommend folic acid only through the first trimester. However, a British randomized, controlled trial found increased cognitive benefit in 8-year-olds whose mothers continued folic acid after the first trimester, compared to women who were switched from folic acid to placebo after the first trimester (McNulty et al., 2019).

Several studies have suggested that use of folic acid supplements five times a week or more, compared to 3 times per week, or higher maternal folate concentration might increase the incidence of autism spectrum disorder (Raghavan et al., 2018; Egorova et al., 2020). However, studies of a much larger Israeli cohort reaffirmed the benefit of supplementation early in pregnancy and suggested that the possible detrimental effect of higher doses was confounded by the proclivity of women to take higher doses in their first pregnancy (Levine et al., 2018). First birth order itself is associated with increased risk of autism spectrum disorder (Sharman et al., 2019). A Spanish study found decreased cognition in 4 to 5-year-olds whose mothers used doses higher or lower than 400–1000μg/day (Valera-Gran et al., 2017).

A confounding factor for folic acid supplementation is that most preparations include Vitamins A, B₁₂, and D. A Swedish comparison of mothers who used folic acid only found no effect on the incidence of autism spectrum disorders, compared to no supplement use. For women who used multivitamins, the addition of folic acid did not enhance effectiveness (DeVilbiss et al., 2015).

Maternal and fetal genotypes are not accounted for in most studies. MTHFR, the gene for methylenetetrahydrofolate reductase, has variants that alter its effectiveness. Schmidt et al. (2012) found that if the MTHFR 677 C>T variant, associated with reduced effectiveness, were present in either the mother or fetus, the association of higher folic acid intake with decreased risk for autism spectrum disorder was strengthened. They suggested that supplements might be particularly necessary for pregnancies with this variant.

Only one study has attempted to assess the effect of maternal folic acid on the risk for schizophrenia. Brown et al. (2007) measured maternal homocysteine levels in dried maternal blood spots in a small number of cases. Homocysteine is converted to methionine by both folic acid and Vitamin B₁₂. Higher homocysteine levels imply lower folic acid or Vitamin B₁₂ levels. The highest tertile of homocysteine levels in the third trimester, compared to the other tertiles, had double the number of offspring with schizophrenia. A similar effect in the first trimester did not reach significance, which the authors attributed to inadequate power.

3.2. Vitamins D and A

Vitamin D, like folic acid, is already part of maternal prenatal vitamins that are recommended universally. The 15μg/day or 600IU dosage is designed to reach a target serum 25-(hydroxy)Vitamin D concentration of 50 nmol/L that is considered sufficient to prevent musculoskeletal deformity (Institute of Medicine, 2010; European Food Safety Authority, 2017). These public health recommendations considered average exposure to sunlight, dietary sources including Vitamin D-supplemented dairy products, and skin color. However, skin color and exposure to sunlight vary considerably among populations. Thus, darker skin women in Nordic countries in winter have particular problems with lower levels (Mitchell et al., 2019). Low levels of Vitamin D have been found in Black American and rural Chinese populations (Brunst et al., 2014; Yu et al., 2019).

The variance in Vitamin D levels during pregnancy is important, because most studies of effects of Vitamin D on later offspring mental illness have found an optimal window of concentration. Both below and above this window, incidences of schizophrenia, autism spectrum disorder, and other developmental disorders are increased (Table 2). The boundaries of the window vary moderately between studies. It is unclear if differences between assays or differences in confounding factors, such as maternal socioeconomic status, are reasons for the variance (Table 2). Maternal gestational levels >50nmol/L and cord blood levels >25nmol/L appear to be adequate and are associated with decreased incidence of schizophrenia and autism spectrum disorder. Maternal gestational levels <25nmol/L are associated with increased incidence of schizophrenia and autism spectrum disorder (McGrath et al., 2003; McGrath et al., 2010; Vinkhuyzen et al., 2017, 2018; Eyles et al., 2018). Levels <25nmol/L are also associated with premature birth (Miliku et al., 2019). Maternal gestational levels >125nmol/L are associated with thinner, shorter infants (Hauta-Alus, 2019).

Table 2.

Summary of major studies of prenatal Vitamin D effects on child development.

Study Protocol	N	Findings	Reference
U.S. case control with third trimester serum Vitamin D	N =26 schizophrenia cases, 51 controls	No difference in Vitamin D in cases	McGrath et al. 2003
Finnish case control study of Vitamin D supplementation in first year of life	N = 79 schizophrenia cases, 9144 total	Supplementation ≥2000IU reduced risk of schizophrenia in males	McGrath et al., 2004
Danish case control study, neonatal blood	N = 424 cases schizophrenia, 424 controls	Compared to reference quintile, 40.5–50.9 nmol/L, higher and lower quintiles had greater schizophrenia risk	McGrath et al., 2010
Generation R observational study of midgestational maternal serum Vitamin D	N = 7098	Mothers who had Vitamin D in the lowest quartile <50nmol/L had an increased risk of preterm delivery and children who were small for gestational age.	Miliku et al., 2019
Generation R Case control study with midgestational serum Vitamin D	N = 4334 with 68 cases of autism spectrum disorder	Two-fold increase in cases in Vitamin D deficient <25 nmol/L versus sufficient >50 nmol/L.	Vinkhuyzen et al. 2017
Generation R observational study of mid-gestational maternal serum Vitamin D level	N = 2866 healthy women	Social Responsiveness Scale 6yrs more abnormal in Vitamin D deficient <25 nmol/L than in sufficient >50 nmol/L.	Vinkhuyzen et al. 2018
Danish case control study with neonatal cord blood.	N = 3464	Compared to the reference (fourth) quintile, those in the lowest quintile (<20.4 nmol/L) had a significantly increased risk of schizophrenia.	Eyles et al., 2018
California case-control study of Vitamin D in newborn blood spots, most within 48hrs of birth	N =563 autism spectrum disorder cases; N= 190 Intellectual Disability; N = 436 controls, 4.5–7yrs	No increase in autism spectrum disorder. Intellectual Disability prevalence in 14% deficient or 26% insufficient in Vitamin D; significant effects of maternal age	Windham et al., 2019
Finnish observational study of effect of prenatal Vitamin D on postnatal infant growth	798 healthy infants, 96% mothers Vitamin D sufficient	Mothers with prenatal Vitamin D >125 nmol/L had the shortest and thinnest infants at 6mos	Hauta-Alus et al., 2019

Vitamin A is generally supplemented with Vitamin D in both food and prenatal vitamins. Lower levels during pregnancy are also associated with increased risk for schizophrenia (Bao et al., 2012).

Vitamin D is the only micronutrient that has effectiveness after birth. Supplementation ≥2000IU in the first year of life reduced risk of schizophrenia in males, but not females (McGrath et al., 2004). The incidence of other illnesses was not changed.

3.3. Choline

Choline is a multi-functional molecule in pregnancy (Zeisel, 2006). It is tri-methylated glycine, and in starving women, it is converted to glycine. Choline is a substrate for one-carbon metabolism, which includes DNA methylation. Phosphatidyl-conjugated choline is a component of plasma membranes, including the baby’s and the placenta’s. Finally, choline is acetylated to acetylcholine and can substitute directly for acetylcholine to activate nicotinic receptors. The direct action of choline on α7-nicotinic acetylcholine receptors requires millimolar concentrations of choline that are much higher than required for methylation pathways or phosphatidylcholine synthesis (Frazier et al., 1998).

Choline is a naturally occurring nutrient found in the human body and in food. Most choline is present in food as phosphatidylcholine in the cell membranes of the animal or plant. The FDA recommendation for minimum daily dietary intake in pregnancy, currently 550 mg in pregnancy, is not based on consideration of the effect on fetal brain development (Food and Drug Administration, 2016). The minimum daily intake was initially set at 500 to 550 mg per day in men because lower levels are associated with increased risk of liver disease. The level for women was then set at 425 mg, based on their smaller body size. In pregnancy, however, placenta choline transporters concentrate choline in the fetal circulation. The fetus and its surrounding amniotic fluid have concentrations of choline three to five times higher than the concentration in the mother’s blood (Zeisel, 2006). The concern was that the mother’s loss of choline to the fetus might result in injury to the maternal liver, and therefore the recommended daily amount for pregnant women was raised (Institute of Medicine, 1998). The daily upper limit is 3500 mg choline, half the amount that produces hypotension. Within these limits, the FDA classifies choline and phosphatidylcholine, also called lecithin, as Generally Recognized as Safe, which means that choline and phosphatidylcholine can be added to foods or marketed as supplements without prescription.

Choline in the blood comes not only from the diet, but also from the mother’s own synthesis of choline. Choline has a reservoir in liver cells in the form of phosphatidylethanolamine. The liver stabilizes choline blood levels, when dietary intake is inadequate, by converting some of its phosphatidylethanolamine stores in the cell membrane into phosphatidylcholine, which then loses the phosphatidyl portion to become choline. A key regulator in this conversion is the enzyme phosphatidylethanolamine N-methyltransferase. However, its gene PEMT has genetic variants that cause it to be less efficient and increase the risk of liver disease and premature birth (Zeisel, 2006; Zhu et al., 2020). A second complication is that dietary intakes of folic acid and methionine interact with choline and, in turn, choline interacts with their levels. Deficiency in one of these can increase the need for more choline. For example, many people who abuse alcohol become folic acid deficient and therefore require more choline (Jacobson et al., 2018). Dietary surveys in the United States, Canada, and China find that most pregnant women consume less than 500 mg of choline daily, below the range in the FDA’s recommendations (Wu, 2012; Brunst et al., 2014; Groth et al., 2017, Li and Freedman, 2020).

The case for choline as a necessary prenatal supplement is now emerging, and therefore studies are reported here in more detail (Table 3). Based on animal experimentation literature, several research groups, including ours, received permission from the FDA and other governmental agencies to study the effects of maternal choline concentration on early offspring brain development and for randomized clinical trials of phosphatidylcholine or choline supplements compared to placebo in pregnant women. Choline citrate or choline chloride is a pure form, used in two of the clinical trials, but the choline is not easily absorbed from the intestine into the maternal bloodstream (Cho et al., 2020). Choline that is not absorbed from the small intestine may reach the large intestine, where some bacteria can metabolize it to trimethylurea, a fishy-smelling molecule, or to trimethylamine N-oxide, which is toxic to the heart. If choline is conjugated into phosphatidylcholine, the form normally found in foods, it is better absorbed and less likely to be metabolized by bacteria. Levels of trimethylamine-N-oxide are three times higher in women who receive choline supplements than women who receive phosphatidylcholine. Two clinical trials, including ours, chose phosphatidylcholine to optimize absorption and to minimize side effects. Two others chose choline. Higher maternal plasma choline levels were reported in the trials that used phosphatidylcholine, consistent with its better absorption. None of the four trials described below reported serious adverse effects for either the mother or baby of phosphatidylcholine or choline. Mild effects, including dizziness, nausea, headache, bloating, constipation and diarrhea, flatulence, and body odor, occurred as frequently in the placebo groups as in the supplemented groups (Ross et al., 2013).

3.3.1. Results of randomized controlled clinical trials of prenatal choline or phosphatidylcholine supplementation

In our clinical trial (Ross et al., 2013), phosphatidylcholine supplements, 6300 mg daily, were instituted at 17 weeks gestation and continuing until delivery. Maternal gestational plasma choline concentrations obtained with the phosphatidylcholine supplement were 15.21 ± 8.14μM, compared to 7.85 ± 2.54μM for placebo. As soon as the newborns regained their birth weight, they received 100 mg phosphatidylcholine drops daily for the first 3 months of life. Forty-seven women and their babies received these supplements, and 46 received placebos, by random, double-blinded assignment. Compared to placebo, phosphatidylcholine supplementation improved the inhibition of cerebral P50 auditory responses, recorded electrophysiologically when the offspring were 1 month old. This physiological feature of schizophrenia, some forms of bipolar disorder, autism spectrum disorder, and attention deficit disorder, can already be measured in newborns (Hunter 2011). Deficiency in P50 inhibition, loss of the normal ability to inhibit the cerebral evoked response to repeated stimuli, is associated with patients’ neuropsychological problems with distractibility (Hamilton et al., 2018). Poor sensory filtering is a basic clinical problem for many patients with schizophrenia (Javitt and Freedman, 2015). Deficiencies in P50 inhibition are also heritable and co-segregate with risk for schizophrenia in families (Greenwood et al., 2016).

P50 inhibition was normal in 76% of newborns of mothers who received phosphatidylcholine supplements, compared to 43% of newborns of mothers who received placebo. A pharmacogenomic analysis was conducted for a variant in the newborn’s CHRNA7, the gene for the α7-nicotinic acetylcholine receptor that is the target of the choline intervention. This variant, which is also associated with schizophrenia in adults, increased the likelihood that a newborn would have poor P50 inhibition in the placebo group. In the phosphatidylcholine-treated group, the newborn’s CHRNA7 variants did not affect P50 inhibition.

Half the children in the trial were assessed at 40 months of age using the Child Behavior Checklist, 1½−5 year version (Ross et al., 2016). Mean rating percentiles on the Attention Problems scale were significantly lower for those children of both sexes in the phosphatidylcholine supplement group compared to those in the placebo group. Ratings on the Withdrawn Problems scale showed effects of both the phosphatidylcholine supplement and sex. For male children in the supplement group, mean rating percentiles were significantly lower than ratings for those who received placebo; the difference was not significant for females. The children’s ratings of Attention and Withdrawn problems, as well as the Total Problems scale, at 40 months of age were significantly related to their P50 inhibition as newborns. As observed for P50 inhibition, there was a significant genotypic effect of the CHRNA7 variant associated with schizophrenia for Withdrawn Problems in the placebo-treated group. This genetic effect was absent in the phosphatidylcholine-treated group, who had fewer Withdrawn problems regardless of CHRNA7 genotype.

A previous study from another group asked parents to use the Child Behavior Checklist to rate their adult children retrospectively as they were in early childhood. Half had adult children with schizophrenia. For Attention problems, the effect size for children who later developed schizophrenia was Cohen’s d’ = 0.45, and for Withdrawn, d’ = 1.73 (Rossi et al., 2000). In the prenatal phosphatidylcholine study, d’ for Attention was −0.59, and for Withdrawn it was −0.79. Thus the effect size of the change observed with phosphatidylcholine is similar in magnitude, but opposite in direction, to the difference between children who later developed schizophrenia and those who did not.

In a separate group of 27 mothers with schizophrenia enrolled over 5 years, 17 had newborns who completed the 1-month recording. Among those treated with placebo, 38% had normal P50 inhibition, compared with 69% of those treated with phosphatidylcholine (Ross et al., 2016).

Other clinical studies of prenatal choline effects on outcomes in early childhood include three double-blind, placebo-controlled trials and two observational studies. One double-blind trial reported decreased reactive saccadic latency from 4–13 months of age in 8 children of healthy mothers who received 550mg choline supplementation beginning at 27–29 weeks gestation, compared to 8 children who received 100mg choline. Both groups received prepared meals containing 380 mg choline so that one group received a total of 930 mg daily versus the other group who received 480 mg. The 550 mg supplemented group has plasma choline 7.4 ± 2.0 μM, compared to 6.5 ± 1.1 μM for the 100 mg group, a non-significant difference, perhaps because of choline’s poor absorption. The authors interpret the increased reaction time as indicating increased processing speed (Caudill et al., 2018). The magnitude of the effect depended highly on the number of days the mother consumed the choline supplement, which suggests that earlier in the third trimester is better than later. A follow-up of the children at 7 years of age found improved performance in the 550 mg supplemented group on a test of visual memory (Bahnfleth et al., 2019). The mothers and infants did not receive postnatal supplementation, nor is breastfeeding noted as a factor.

The Caudill et al. (2018) trial found that choline supplementation increases methylation of CRH gene in the placenta at birth. In the cord blood, leukocyte CRH and NR3C1 were hypomethylated, and cortisol levels were 30% decreased. Cortisol is highly pathogenic for fetal neuronal development in early gestation, and thus this decrease is a potentially beneficial (Freedman et al., 2021).

A third trial of mid-pregnancy choline supplementation was conducted in women who have heavy alcohol use during pregnancy, common in the Capetown Coloured community in which these women live. This South African community is a historic mixture of Indonesian, European, and Black African heritages, which is considered distinct from other ethnic groups. The mothers were recruited in mid-pregnancy through the 23^rd gestational week. During the study, they drank an average of 1.3 times per week, consuming drinks containing >4 oz. of alcohol. Supplemented mothers received 1.3 grams of choline bitartrate, which is 1 gram of choline, twice per day. Four weeks after entering the trial, approximately 24–27 weeks gestation, the supplemented group’s plasma choline concentration was 10.7 ± 4.2 μM, compared to 8.3 ± 1.9 μM for the placebo group, a significant difference. One-third of the infants had full fetal alcohol syndrome or a sub-syndromic form. Choline supplementation did not alter this outcome. Infants of the mothers who received the supplement had increased novelty preference scores at 12 months of age on the Fagan Test of Infant Intelligence, which the authors interpreted as better visual recognition memory. (Jacobson et al., 2018).

The earliest clinical trial to be reported was conducted in women selected to be healthy with uncomplicated pregnancies. Women who used tobacco, drugs, and alcohol were excluded. In addition, they had to intend to breastfeed for at least 90 days. In the trial, 5400 mg phosphatidylcholine supplementation in healthy women beginning at 18 weeks gestation versus placebo raised plasma choline concentration to 13.6 ± 0.6 μM, compared to 7.7 ± 0.3 μM for the placebo group. The supplement was continued as long as the mother breastfed. Milk of supplemented mothers has choline concentration 106 ± 10 (standard error) nmol/mL, compared to 83 ± 8 nmol/mL for the placebo group. Both differences were significant. The trial reported no effect of the supplements on the Visuospatial Memory Delayed Response Task, the Mac-Arthur-Bates Short Form Vocabulary Checklist: Level I, or the Mullen Scales of Early Learning Global Development Index at 10–12 months of age versus placebo. Mean Mullen Global Index scores were one standard deviation above the normal range for offspring of both placebo and supplemented mothers (Cheatham et al., 2012). In contrast, the mean Mullen Global Index scores were below average in the Ross et al. (2013) trial. The mothers in this study were mostly college educated. The mothers in the placebo group reported an average annual income of $68,000, and the supplemented group reported $62,000. The authors interpreted their findings as indicating that the mothers were already consuming adequate amounts of choline in their diets and that the supplement was therefore not necessary. They intended the Visuospatial Memory Delayed Response Task to specifically assess hippocampus development. In the placebo group but not in the supplement group, the range of performance included individuals more than 2 standard deviations below the mean. It is possible that the phosphatidylcholine supplementation, but not the placebo, may have improved performance for a more impaired subgroup of the infants.

3.3.2. Observational studies of the effects of prenatal maternal choline levels

We conducted a second study of choline in gestation, without using supplements to change the woman’s plasma choline level, which meant that FDA restrictions on the randomized trials in the U.S. to healthy women did not affect recruitment. In this second study, choline levels at 16 weeks gestation greater than 7.07μM, which are within 1 standard deviation of the mean level obtained with phosphatidylcholine supplements in the randomized trial, were considered to be higher levels, comparable to the level achieved supplement. Levels below 7.07μM were considered to be lower levels. The proportion of women with higher choline concentrations higher than 7.07μM was 31% (Hunter et al., 2021).

Maternal plasma choline levels are influenced not only by the mother’s diet but also by her own biological synthesis of choline and by her stress, which causes choline to be retained in the maternal liver (Hunter et al., 2021). Choline levels were not associated with maternal body mass index (BMI). Prenatal vitamin and folic acid use exceeded 90% in both mothers with high and low choline. Genetic variants in PEMT also did not differ between women with high or low choline levels. Average choline levels for the entire group of mothers in the current study rose at 28 weeks gestation, consistent with other reports of increasing choline concentrations after mid-pregnancy. The mother’s increasing estrogen activates her PEMT gene to increase her choline synthesis, and the placenta increases its transport of choline to support the accelerating growth of the fetus (Zeisel, 2006). Maternal plasma choline concentrations at 28 weeks were not associated with the outcomes in childhood that we measured because these outcomes depend upon fetal brain formation that occurs earlier in pregnancy (Freedman et al., 2019).

Similar to the findings of the clinical trial of phosphatidylcholine supplementation, higher maternal choline concentrations at 16 weeks gestation were associated with increased inhibition of the P50 cerebral auditory evoked potential. P50 inhibition is a neurobiological mechanism associated with better ability to maintain attention (Hamilton et al., 2018). Both male and female children of mothers with higher 16-week gestational choline levels had fewer Attention problems on the Child Behavior Checklist at 4 years of age than children of mothers with lower choline levels. The Attention score at 4 years of age was correlated with P50 inhibition at 1 month of age. The male children were less likely to be withdrawn in social situations than male children of mothers with lower plasma choline concentrations (Hunter et al., 2021).

Because this observational study was not under FDA regulation, the effects of prenatal choline levels were observed on a wider range of maternal co-morbidities, most that have been associated with later offspring mental illnesses, including psychotic disorders (Brown and Derkits, 2010; Hunter et al., 2011). Maternal infections, including respiratory virus infections, were associated with increased maternal C-reactive protein levels and deficiencies in newborn P50 inhibition and childhood attention and social behavior; these associations were decreased in offspring of mothers with higher choline levels (Freedman et al., 2019, 2020; Hunter et al., 2019). Offspring of mothers who used cannabis through the 10^th week of gestation also had poorer P50 inhibition and childhood attention and social behavior that were mitigated if mothers had higher choline levels (Hoffman et al., 2019).

Black American women recruited from low-income neighborhoods of Denver had the lowest choline levels of any ethnic subgroup within the observational cohort (Hunter et al., 2020). There was no evidence that differences in the allele frequencies of single nucleotide variants in PEMT or MTHFR contributed to lower maternal choline levels. White women living in the same low-income neighborhoods did not have similarly low choline levels, and neither did women in Uganda. Black American women’s low choline levels were related to their high stress, as monitored in their hair cortisol.

Low choline levels in these 25 Black American women were associated with preterm birth, averaging over 2 weeks before term. A relationship of low choline intake to pre-term birth has also been found in China (Zhu et al., 2020). Analysis of gestational age at birth in 15 Black American women in the Ross et al. (2013) phosphatidylcholine trial found that supplementation significantly decreased pre-term Black birth compared to placebo. Although the sample size is small, it is the largest group of Black American women studied in a choline clinical trial. These women, despite the stress from institutional racism that affects their lives, sought timely prenatal care, complied with all study requirements, and were less likely to be obese or abuse drugs than White women in the study. Nonetheless, their hair cortisol levels were higher and their plasma choline levels were lower than the White women’s. These differences were associated with adverse effects on the development of their children, as measured by poorer ability to pay attention and relate to caregivers after birth. The mothers themselves rated these problems in their children on the Infant Behavioral Questionnaire-R (SF).

Whether the children of these Black American women will be at increased future risk for schizophrenia is unknown, and results will be complicated by the historic high rate of diagnosis of schizophrenia in Black Americans (Minsky et al. 2003). It should be noted that offspring of women of other ethnic backgrounds in highly stressed urban environments, e.g. Surinamese, Turkish, and Moroccan immigrant women in Amsterdam, are reported to have increased risk for schizophrenia (Veling et al., 2008). Thus, stress is the likely pathogenic factor, with low gestational choline from chronic hypercortisolemia being one possible mechanism that is potentially remediable by nutrient supplementation.

An observational study from Canada reported mean maternal plasma choline concentration 7.07 ± 1.87 μM at 16 weeks gestation. The distribution was skewed, with a median 6.70μM. An assessment of self-reported dietary intake found most mothers below current recommended minimum daily intake, but correlation with diet reported accounted for only 4% of the variance in plasma concentration. The authors estimated 7μM as consistent with an adequate intake; 56% of women were below this level. Higher maternal blood plasma choline concentrations at 16 weeks gestation were associated with increased Cognition scores at 18 months of age on the Bayley Scales of Infant Development (Wu et al., 2012).

An earlier study of 404 women in Alabama, of whom 72% were Black and 70% below the poverty level, found mean 16-week serum choline concentrations that were higher than plasma concentrations reported in other studies, 9.34μM (Signore et al., 2008). Autotaxin released from platelets during clotting increases choline levels in serum after phlebotomy by liberating choline from phosphatidylcholine in the platelets, whereas chelation to prevent clotting in plasma samples inhibits autotaxin (Ohkawa et al., 2018). Therefore, the samples in this study may not accurately reflect maternal gestational choline levels. A positive, but non-significant effect, was found on the WPPSI-R Performance Scale for 16–18 week gestation serum choline concentrations.

The longest duration study, based on assessment of choline in the maternal diet in 895 women in Massachusetts, found a relationship of higher dietary intake to increased performance in the first and second trimester on the Wide Range Assessment of Memory and Learning, Design and Picture Memory subtests, in the 7-year-old offspring (Boeke et al., 2013). Dietary intake was similar to that observed in other U.S. and Canadian studies. Similar to the findings of the Colorado observational study, positive effects were observed for only the highest quartile of choline intake.

4. Discussion

Significant opportunities exist for meaningful research into the potential effects of folic acid, Vitamin D, and choline for enhancing fetal brain development and thereby lowering the risk for psychotic disorders and autism spectrum disorders. This review focused on these three nutrients because of the current research indicating their possible value. However, it is likely that deficiencies in other nutrients may be pathogenic and, conversely, that supplementation of other nutrients may have helpful effects.

For folic acid, studies that could be presumably accomplished with existing cohorts include the effect of maternal folic acid use, with or without multivitamins, on the risk for psychotic disorders. The only study of effects on schizophrenia used homocysteine as an indirect measure. The positive versus negative effects of higher doses at each trimester requires resolution. Future studies could investigate folic acid serum levels, rather than utilizing mothers’ self-report. If folic acid does not have adverse effects at higher levels, then doses as high as 4 mg, the dose used for pregnancies with known risk of neural tube defect, would be safe if maternal Vitamin B12 levels were also measured. If all mothers received a baseline dose of 400 μg, the current standard of care, the effect of higher doses could be ascertained in a randomized, placebo-controlled design. Measurements of maternal folic acid levels should also control for potential effects of genetic differences in MTHFR.

Maternal Vitamin D plasma levels could be routinely verified at mid-gestation and the fetal level in cord blood at birth. Unlike folic acid and choline, Vitamin D has been found to be effective postnatally at decreasing risk of later schizophrenia. Therefore, a trial of supplementation for the newborn whose mother had a low mid-gestational Vitamin D level or who had low cord blood Vitamin D level would seem warranted.

The randomized and observational studies of choline converge on several points: (1) choline or phosphatidylcholine supplements are needed by most women to reach optimal levels to effectively support fetal brain development, especially in pregnancies with stress, infection, and substance abuse; (2) supplements should be initiated as soon in the second trimester as possible or perhaps preconception to support fetal brain development, and (3) adverse effects of effective doses are minimal. Based on the currently available data, an optimal dosage beginning pre-conception or as soon as possible during gestation, is phosphatidylcholine 4200 mg per day (four 600mg capsules in the morning and three capsules at dinner). This form produces the highest plasma levels of choline and lowest levels of trimethylamine oxide. Alternatively, if they prefer, most women can safely take choline citrate in liquid form 1300 mg 0.5 teaspoon (400 mg choline) twice per day in 8 oz. of fluid.

The scientific evidence lacking is large-scale clinical trials, similar to those that support folic acid and Vitamin D as interventions. Paradoxically, the large-scale studies for folic acid and Vitamin D are retrospective studies of pregnancies with compliant and non-compliant mothers in populations that had already adopted these interventions as standard good prenatal care (Wilcox et al., 2007). Blood spots and serum usually collected in archival populations would be unsuitable for choline levels because of contamination from platelet phosphatidylcholine (Okhawa et al., 2018). Suitable plasma sampling would be needed. Until that time, smaller, single-site trials, some ongoing (Hoffman, 2017), will be needed to support the initial adoption of choline into prenatal care. The American Medical Association has endorsed choline supplements for all pregnancies, but it has not reached widespread acceptance as standard of care (American Medical Association, 2017).

Ongoing mechanistic studies for all three nutrients may help identify individuals likely to benefit. Folic acid’s mechanistic roles in DNA synthesis, one-carbon metabolism, and the homocysteine-methionine transition via methylene tetrahydrofolate reductase, whose gene is MTHFR, are well known. MTHFR polymorphisms in the mother or fetus predict who benefits most from folic acid supplementation. Choline’s activation of α7-nicotinic acetylcholine receptors encoded by CHRNA7 facilitates the transition of excitatory and inhibitory neurotransmission to their mature forms (Liu, Neff, and Berg, 2006; Lozada et al., 2012). These transitions are not complete in schizophrenia, as assessed in post mortem samples (Hyde et al., 2011; Kerwin, Patel, and Meldrim, 1990). Fetuses with CHRNA7 variants associated with schizophrenia benefit especially from phosphatidylcholine supplements (Ross et al., 2013, 2016). A broader consideration of choline’s molecular effects through CHRNA7 or other genes is needed. Vitamin D activates the Vitamin D Receptor, a nuclear zinc-finger protein, but how it affects brain development or can be used to identify vulnerable pregnancies is still under investigation (Cui et al., 2017).

A problem for all prospective micronutrient studies is to identify an outcome that can be measured early in childhood in a relatively circumscribed number of subjects, because a prospective randomized trial for psychotic disorders themselves would require thousands of subjects followed for decades. The Generation R study used the Child Behavior Checklist to identify effects of folic acid on attention, social withdrawal, and aggression. Ross et al. (2106) also used the Child Behavior Checklist to assess effects of phosphatidylcholine. These same problems are identified retrospectively in the childhood of individuals who developed schizophrenia as adults (Rossi et al., 2000). The Child Behavior Checklist is a widely-used instrument completed by the parents that might thus provide a suitable intermediate variable for early assessment of potential effects of micronutrient interventions (Achenbach and Rescorla, 2000).

In addition to opportunities for biomedical research, there is a pressing need for better dissemination of all three nutrients into prenatal care. The effectiveness of these nutrients for early childhood temperament and behavior as measured by instruments like the Child Behavior Checklist and for early childhood cognition is a more practical outcome than the prevention of psychosis because the time duration from gestation to outcome is more manageable, and effects can be ascertained with fewer children than the thousands likely needed to assess differences in the incidence of psychosis. This strategy receives support from a recent study that found the risk for psychosis from maternal inflammation in pregnancy is mediated by the adverse effect of the inflammation on academic performance (Ramsay et al., 2020). Positive outcomes in early childhood related to early school achievement are also more likely to be a compelling rationale for the recommendation of nutrients, including choline, to women than the prevention of psychosis, a rare, stigmatized illness whose risk is remote. Improvement in childhood development achieved now from prenatal micronutrients could then be analyzed for preventive effects for psychosis by the next generation of psychiatric researchers.