Chloe Weng/QW2669

NTR 338W
Final Manuscript

Abstract

Autism spectrum disorder (ASD) is characterized by challenges with social skills, limited interests, and

abnormal communication, which may be caused by the interplay between genetic and environmental factors,

including maternal nutrition, medication, and toxic products. As a possible cause of ASD, maternal nutrition

plays a critical role in modulating epigenetic events and have long-term influences on offspring. Folate is an

essential vitamin B involves in DNA methylation and amino acid homeostasis. High maternal intakes of Folate

have been hypothesis to have potential impaired neurodevelopmental consequences like autism. This review

examined the influences of high maternal folate levels on the incidence of autism(ASD). Five peered-reviewed

articles published within the past five years were identified using the PubMed database with the search term:

maternal Folate or high folic acid, and autism. Results indicated that high levels of maternal folate level at birth

(i.e., >60.3 nmol/L) were linked to 2.5 times increased risks of ASD (95% CI:1.3, 4.6). The use of FAS

dosages >1000ug/d was negatively associated with several neuropsychological outcomes in the children

including global verbal (B=-2.49; 95% CIL-4.71, -0.27), verbal memory (B=-3.59; 95% CI: -6.95, -0.23), and

cognitive function of posterior cortex (B=2.31; 95% CI: -4.45, -0.18). The baby mice from the folic acid

supplementation group (FASD) displayed short-term memory impairment (P<0.005), decreased hippocampus

size, and decreased thickness of the dentate gyrus. In summary, high maternal folate level is associated with

increased risk of ASD, impaired short-term memory, and cognitive functions in the offspring. The findings

highlight the unintended negative consequences of higher folate intake.

Keyword: Autism, Folate, pregnancy, short-term memory, cognitive development, gene expression

Introduction

Over the last two decades, the prevalence of autism spectrum disorder has increased dramatically, and

it is now estimated to be 1 to 3% of the population in high-income countries1. Autism is a broad range of

neurodevelopmental conditions characterized by challenges with social skills, limited interests, repetitive

behaviors, speech, and abnormal communication1. It can also affect other significant systems, including the

endocrine, immune, and gut micro-biotic systems 2. The onset of ASD always begins during early childhood
and remains throughout a person's life. The etiology of ASD is complex and remains unclear, which has

hindered the progress of developing the biological treatments or a cure for ASD2. The current understanding

of ASD suggests that its etiology is related to multi-factorial conditions, including genetic and environmental

factors1. Genome sequencing data have indicated hundreds of genes associated with ASD, both common

and rare, and many of the genes are involved in other neurological, psychiatric, and neurodevelopmental

conditions2. Regarding environmental risk factors, the fetal environment, nutrition, medications, vaccination,

and toxic products have been proposed to influence the development of ASD2. Moreover, maternal nutrition

during pregnancy has also been highlighted as modifiable nutritional factors that affect fetal brain development

in terms of structure and function. Thus, some scholars posit that maternal nutritional status before and during

pregnancy may have a relationship with ASD risk.

As one of the most critical maternal nutrition needs, Folate is an essential water-soluble vitamin B

required in the diet as humans cannot produce Folate. Folic acid is a synthetic form of Folate and is often used

as a supplement and fortified food3. Folate functions as a coenzyme compartmentalized between the

mitochondria and the cytosol in the body. It donates or accepts one-carbon unit in metabolism pathways, which

plays a significant role in the nucleic acid synthesis, cell division, amino acid homeostasis, epigenetic

maintenance, DNA methylation, and repair 4. After many studies in the mid-twentieth century, it has been

proved that adequate intakes of folic acid begin on the preconception period can decrease the risk of Neural

Tube Defects (NTD) in offspring. Since then, the U.S. Public Health Service had recommended that all women

of childbearing age consume at least 400ug of folic acid to prevent NTS3. Subsequently, the U.S.

implemented a mandatory fortification program in 1988, which adds 140ug of folic acid per 100 g of cereal

grain products to increase folate intake3.

ASD has been posited to be linked to abnormalities in folate metabolism. A relevant epigenetic process

crucial to neurodevelopment is DNA methylation, which depends on the availability of dietary methyl donors

such as Folate, choline, and methionine5. Changes in folate availability or other donors can affect DNA

methylation capacity, with potential impaired neurodevelopmental consequences. In addition, folate transport

through the cell is mainly facilitated by reduced folate carrier one and folate receptor (FRa) protein via

endocytosis. FRa is expressed during development in placental, choroid, and neural plate cells6. Since

Folate is involved in the transmission of the one-carbon group, and folate-dependent homocysteine re-
methylation connects Folate with the transsulfuration pathway, any alteration of folate level or folate carrier

may trigger the onset of ASD through numerous process processes in the developing brain from DNA

synthesis to neurotransmitters synthesis6.

After the U.S. fortification program's implementation, folate intake has gone up about 2.5 times through

fortified food and supplement7. As the intake increased, many scholars have wondered if there is an upper

limit to these benefits in preventing brain defects and neurological development. Furthermore, some studies

show a positive relationship between the high folate intake and lower risk of autism, while others hypothesized

the opposite. Thus, the research of the association of ASD and maternal folate level or folic acid has been

conflicting. And given the background that ASD is known to be accompanied by modified brain structure and

connectivity, the objective of this paper is to determine the effects of excessive maternal folate intake on ASD

in the offspring.

Methods

PubMed research results

A literature research was conducted using the PubMed database. The terms "(((Maternal folate [MeSH Major

Topic])) OR (high folic acid)) AND (autism [MeSH Terms])" were used with results filtered to show studies that

were published only within the last five years. The search revealed 11 results. Studies included were research

related to high folate intake during the gestational period and those published within the 5-year range. Studies

were excluded if they were not associated with maternal Folate, were not clinical trials or randomized control

trials, and were published over five years ago. Five studies papers were chosen: Barua et al. 2015, Bahous et

al. 2017, Raghavan et al. 2018, Egorova et al. 2020, and Valera-Gran et al., 2017.

Results

Of the five studies in this review, two are based on animal studies, and three are human studies. Barua et al.

analyzed 24 mice into two separate groups (high folic acid diet(20mg/kg) and control diet (2mg/kg)) throughout

mating, pregnancy, and lactation. Tissue collection, qPCR analysis of offspring RNA, western blot analyses of

total cell lysate, and total protein and DNA isolation was done on the mice to examine any alternation in gene

expression. Bahous et al. also separated the mice into two groups and fed a high folic diet and control diet.
Three-week-old mice were then evaluated for motor and cognitive function by performing behavioral testing,

including open field test, novel object recognition test, and Y-maze test. Raghavan et al. recruited 1257

mother-child pairs at birth and followed through childhood, in which maternal plasma folate and B12 of the

mothers were measured and surveyed. Egorova et al. collected serum samples from 100 women with offspring

diagnosed with autism (ASD) and 100 control women with standard developing offspring. The concentration of

sixty-two metabolic biomarkers, including vitamins, amino acids, and Folate, were assessed. In the cohort

study conducted, Valera-Gran et al. included 1682 mother-child pairs from four Spain regions. The

questionnaire was used to estimate dietary folate intake. The data collected was used to compare with the

neuropsychological development scores at age 4 to 5 using McCarthy Scales of Children's abilities.

U-shaped relationship between folate intake and risk of ASD

Raghavan et al. had a sample of 1257 mother-children pairs, and 86 of them were diagnosed with autism, and

1171 were children with neuro-typical development. In the analysis, first-trimester supplement intake greater or

equal to three times a week was proactive against ASD risk compared to those who reported taking

supplements less than three times a week. While on the other hand, maternal supplement intake greater or

equal to 5 times/ week demonstrates statically significantly increased risk for ASD (Figure 1a). The U-shaped

relationship was consistent throughout all trimesters.

Compared with non-elevated B12 levels (200-600 pmol/L), maternal plasma B12 greater than 600pmol/L levels

were associated with a higher risk of ASD in both adjusted and unadjusted models (unadjusted: 2.7, 95% CI

1.4-4.9, adjusted: 3.0 95% CI 1.6-5.7) (Raghavan et al., 2018). B12 level of less than 200 pmol/L did not have

a significant relationship with the development of ASD (Table 1).

Figure 1- Raghavan et al.: Association between maternal folate and vitamin B12 concentrations and risk of ASD in offspring in the
Boston Birth Cohort (N = 1257). The unadjusted H.R. for plasma folate (panel a) was truncated at 65 nmol/ L dues to the small sample
size beyond the specified cutoff point (unadjusted H.R. mothers whose plasma folate _65 nmol/L was 1.9, 95% CI 1.0, 3.4; n = 113).
The unadjusted H.R. for plasma vitamin B12 (panel B) was truncated at 700 pmol/L dues to declining sample sizes beyond the
specified cutoff point (unadjusted H.R. for mothers whose plasma vitamin B12 _700 pmol/L was 2.8, 95% CI 1.3, 6.0; n = 45).

Table 1-Maternal plasma folate, vitamin B12 concentrations in samples obtained 24–72 h after delivery and risk of ASD in offspring in
the Boston Birth Cohort (N = 1257)

Out of 62 biomarkers, 45 were identified in Egorova et al.'s study. Egorova et al. found that un-metabolized

folic acid was statistically insignificant in mothers in the case group compared to the control. However, similar

to Raghavan et al.'s study, high first-trimester total folate level was linked to increased risk of a child with ASD.

(OR per 1 SD increase: 1.80, 95% CI 1.22-2.37, P=0.002) (Figure 2). Other than the total folate level, the rest

of the biomarkers were found insignificantly related to the risk of ASD. Unrelated biomarkers include

kynurenine/tryptophan ratio (OR per 1 SD 0.91, 95% CI 0.66-1.26, P=0.58), CRP (OR per 1 SD increase: 1.20,

95%CI 0.89-1.61, p=0.24), neopterin (OR per 1 SD: 0.97, 95%CI 0.72-1.31, p=0.86). active smoking (serum

cotinine > 85 nmol/L) was not linked to ASD risk (OR 1.21, 95% CI 0.51-2.88, P=2.66).
Figure 2- Egorova et al.: Odds ratios (O.R.s) for ASD risk by one standard deviation (S.D.) increase in biomarker levels. O.R.s were
calculated using logistic regression adjusted for mother's age at sampling, year of sampling, sex of the child, and serum cotinine and
mTHF levels. Vitamin D3 was further adjusted for the month of sampling (light/dark months). Median serum concentration levels in
cases and controls are presented in nmol/L for biomarker kynurenic acid; 25-hydroxy vitamin D3; xanthurenic acid; trigonelline;
cystathionine; 4-pyridoxic acid; quinolinic acid; pyridoxal; neopterin; nicotinic acid; para-amino benzoylglutamate;
acetamidobenzoglutamate; hmTHF; mTHF; and in mg/l for CRP and all remaining biomarkers

Raghavan et al. also compared the lowest and highest percentile for Folate (< 14.7 and >60.3 nmol/L) and B12

(<247.0 and >536.8 pmol/L). Mothers with plasma folate levels in the top 10th percentile had significantly

increased risk of ASD compared to the middle 80th percentile. Plasma B12 in the top 10th percentile shows two

times of increased risk of ASD when compared with the middle 80th percentile. However, when Egorova et al.

assessed the combined effect of elevated maternal plasma folate and B12 on ASD, the risk was not different

between mothers with only one elevated biomarker.

Short term memory impairment on offspring of FASD mothers and reduced hippocampal area

Bahous et al. performed a behavioral test on a 3-week pup to analyze their brain function. No difference in

anxiety scores between the control group and the HMFA group. No difference was found in the Y-maze test,

meaning that there was no change in spatial memory. However, in the novel object recognition test, mice from

the HMFA group had adverse effects on short-term memory ability. In the experiment, compared with the time

spent with a familiar object, these baby mice spent significantly less time (p<0.005) as a negative

discrimination index (ratio of time spent with novel objects vs. familiar object) has resulted in this group (Figure

3A). These observations suggest that mice from HMFA mothers have short-term memory impairment. On top
of that, in measuring the hippocampus area and thickness of the dentate gyrus, both were reduced in size

compared to the control group (Figure 3) (Bahous et al.).

Figure 3- Bahous et al.: Pups born to mothers fed high folate diet had short-term memory impairment and reduced hippocampal area.
(A) Three-week-old male mice were tested using the NOR. The discrimination index is the ratio of time spent with the novel object
versus time with the familiar object; a negative discrimination index indicates short-term memory impairment. Data from two
independent experiments are shown (n 1⁄4 15–20/group). (B) The average area of the hippocampus and (C) the average thickness of
the dentate gyrus (D.G.) were reduced in FASD pups (n 1⁄4 5/group).

Choline metabolism was disturbed in the brain of HMFA pups

In past studies, altered folate metabolism is often accompanied by altered choline metabolism. Therefore,

Bahous et al. measured SAM, SAH, choline, and choline-related metabolites. Bahous et al. found no change in

Sam, SAH, and choline. However, a 30% decrease in phosphocholine (the primary storage form of choline)

was measured (Figure 4A). When measuring choline metabolites in the hippocampus area, a trend of

decreased glycerophosphocholine (GPC) suggests an increased conversion rate from GPC to choline to

replenish choline level (Figure 4B).

As the precursor of acetylcholine, choline plays a significant role in memory. Bahous et al. measured

acetylcholinesterase's mRNA levels (AchE), which cleaves acetylcholine at the synaptic cleft in the

hippocampus and cortex area. As a result, AchE mRNA was found significantly decreased in the offspring from

the HMFA group in the cortex but not in the hippocampus (Figure 4C).
Figure 4- Bahous et al.: Pups born to mothers fed high folate diet had disturbances in choline and acetylcholine metabolism. (A) The
phosphocholine concentration (PCho), the choline storage form, was significantly decreased in the liver (n 1⁄4 6/group). (B) In the
hippocampus, there was a trend towards reduced glycerophosphocholine (GPC) in FASD pups (n 1⁄4 7/group, #P 1⁄4 0.06). (C) mRNA
expression of acetylcholinesterase (AchE) was reduced in the cortex of FASD pups (n 1⁄4 7/group). (D) mRNA levels of the nicotinic
receptor of acetylcholine, Chrna7, and AchE in the cortex were significantly positively correlated (n 1⁄4 7/group; r 1⁄4 0.894, P < 0.05).
ChAT staining to measure (E) staining intensity and (F) numbers of cholinergic neurons in the Substantia Innominata (S.I.). There was
a trend towards reduced intensity.

The negative association of neuropsychological development and high Folate in 4-5 years old

Valera-Gran et al. used the McCarthy Scales of Children's Abilities (MSCA) test to analyze children's

neuropsychological development. And multiple linear regression models were used to link any associations

between intake of Folate and FAS and MSCA scales. The results (Table 1) show that when compared with

children from mother with FAS dosages intake of 400-999 ug/d, the FAS dosages intake of >1000 ug/d was

linked to significant variation of -2.49 points in global verbal scores and of -3.59 (P=0.074) in verbal memory,

-2.31(P=0.007) in cognitive function of posterior cortex, and -3.26 (P=0.234) points in cognitive function of lest

posterior cortex. Children with mothers with FAS intake < 400ug/d showed significantly lower scores proving

the "U-shaped relationship" (verbal memory: -2.93, P=0.007).

When repeating the analysis from the 4th to 7th month of pregnancy, Valera-Gran et al. found a tendency for a

positive association between higher folate intake and verbal memory score (B=0.98 95% CI, -0.02, 1.99) and a

negative association between higher folate intake and visual executive function (B=-0.93, 95%CI: -1.89, 0.03).

However, no relevant association was found when the folate intake is greater than 1000ug/d (Valera-Gran et

al.).

Table 1 Valera-Gran et al.: A pooled analysis of the linear association between FAS and dietary folate intake in the periconceptions
period and neuropsychological development in children aged 4-5y, INMA cohort study.

To further enhance the reliability of the findings, Valera-Gran et al. used forest plots and meta-analyses to

estimate the associations between high FAS dosage (>1000ug/d) during the preconception period and global

verbal, verbal memory, and CFLPC scores compared with the reference group (FAS dosages of 400-999ug/d).

Valera-Gran et al. observed a negative association (Figure 5) for global verbal, verbal memory, and CFLPC

scores among the children with mothers of folate intake greater than 1000ug/d.

Figure 5- Valera-Gran et al.: pooled estimate association between folic acid supplements (>1000 compared with 400–999 mg/d) in the
periconception period and child neuropsychological development in children aged 4–5 y. Overall estimates from the models were
2 1 2
obtained from fixed-effects pooled analysis (I # 50%). Overall, four cohort estimates were obtained from random-effects models (I .
50%). CFLPC, cognitive function of left posterior cortex; CFPC, cognitive function of posterior cortex.
Maternal F.A. alters expression of candidate autism susceptible genes and transcriptional factors in offspring's
brain

Transcription factors play a significant role in gene expression regulation and neuronal differentiation. Out of

the 17 genes analyzed by Barua et al., the expression of transcriptional factors Nfix, Runxl, and Vgll2 exhibited

alterations where HMFA resulted in the downregulation in male pups and upregulations in female mice. To

investigate whether HMFA diets result in alternation in the imprinted genes, Barua et al. compared the levels of

transcript genes in the brain. In analyzing the candidate autism susceptible gene Auts2, the male mice showed

a downregulation, whereas the female mice showed an upregulation. In addition, the result showed that in

male mice from the HMFA diet, the expression of the imprinted gene Dio3 was upregulated while the

expression of H19, Snrpn, Xist, Gabl, and Slc22as did not show significant changes. In the female mice, the

imprinted genes H19 and Xist were upregulated, while the expression of Dio3, Snrpn, Gabl, and Slc22a3 did

not show significant changes (Figure 6).

Figure 6- Barua et al.: qPCR showing relative expression of the gene transcripts in the C.H. of a male and b female offspring from the
HMFA group (20 mg/kg diet). The results were normalized to Hprt and Rn45s transcript expression and expressed as relative values
compared to corresponding transcripts from the CD group (2 mg/kg diet). Data are expressed as the mean ± SEM; asterisks denote
statistically significant change (*P < 0.05, **P < 0.01, ***P < 0.001).

Effects of Maternal F.A. on DNA Methyltransferases and methylation

When comparing the 5-mC levels of genomic DNA from both sex, no significant link was found in overall

methylation (male brain F.A.: P<0.011, female brain F.A. P<0.001). The result shows that the offspring from

HMFA mothers had no impact on global DNA methylation levels of the offspring epigenome. However, analysis

of the gene encoding DNA methyltransferases revealed an upregulation in Dnmt3b in the male and female

offspring (Barua et al.).

Discussion

The purpose of this review is to find the relationship between high maternal folate intakes during

pregnancy and the risk of autism in offspring. Raghavan et al. found that adequate maternal folate intake is

linked to a decreased risk of ASD in the offspring, whereas infrequent intakes and frequent folate intake are

linked to increased risk of ASD in the offspring4. In addition, Raghavan et al. observed the highest ASD risk

in children of mothers with serum folate and B12 elevated in the 90th percentile4. This result confirmed the "U-

shape" relationship between maternal serum folate level and the risk of ASD3. Thus, while folate deficiency is

detrimental, excessive intake is also problematic. Bahous et al. found that high serum maternal were

associated with impaired short-term memory and a 30% decrease in phosphocholine, a neurotransmitter that

plays a role in memory processing 1. They also found decreased size in the hippocampus and the thickness

of the dentate gyrus1. When analyzing the neuropsychological development in the children 4-5 years old in

Valera-Gran D et al.'s study, the dosage intake > 1000ug/d in gestational stage women is associated with

significant variation in global verbal scores, the left posterior cortex's cognitive function, and verbal memory in

children5. In a mice study, Baura et al. found the expression of the transcriptional factors gene Nfix (essential

role in neural stem cell), Runxl (a regulator in neuronal differentiation coordination), and Vgll2 (affects the

ventromedial hypothalamus) were related to altered regulation in the offspring pups2. In addition, the study

found downregulation of the candidate autism susceptible gene Auts2 in male offspring and up-regulation in

female offspring5.

In the study of Valera-Gran D et al., the FAS dosages >1000 ug/day consumed by mothers during the

periconception have a negative association with brain activities linked to verbal ability in children. However,

Chatzi et al. found that the high dosage of 5000ug FAS per day was associated with an enhancement in the

offspring's receptive and expressive communication skills and verbal comprehension6. This finding

contradicts Valera-Gran D et al.'s results. However, this could be due to the different ways each study

categorized the FAS dosage (adjusted and unadjusted), the period of the pregnancy evaluated, and the

various FAS assessments used in the studies.

Even though the three human studies included in this paper found a positive relationship between folate

levels and maternal, these studies could not directly attribute the sources of the high maternal serum folate
level. It may be possible that adverse outcomes (NTDs, stillbirths, and developmental disabilities) due to

inadequate intakes of Folate in the previous birth delivery prompted some mothers to consume higher folate

amounts through food or supplement1. Since the fortification program in the 1990s, many people, including

pregnant women, increased their folate intake through fortified foods, which possibly creates an accumulation

of folic acid during the gestation period. According to a recent NHANES study, women at the gestation stage

who consumed more folic acid supplements were more likely to consume fortified foods 7). Even though

excessive intakes of Folate do not necessarily correlate with serum folate level, the combination of the dietary

FAS and Folate through fortified food may potentially affect maternal serum folate level. Reduced folate carrier

(RFC), a folate-specific transporter, plays an essential role in delivering Folate to cells for systemic

circulation8. A transport process processes the absorption of the monoglutamate (a hydrolyzed form of

Folate) with an acidic pH optimum via the proton-coupled folate transporter, PCFT, and SLC46Al18. It is also

possible that genetic variation and alternation in these transporters may affect increased folate absorption and

metabolism. In addition, women at the gestational period are found to have advanced absorption of Folate and

other biomarkers and excrete less Folate in urine during the third trimester than non-pregnant people9. All of

which can lead to elevated serum folate levels. Considering the fetal brain is vulnerable to nutrition absorptions

during the third trimester in addition to the elevated maternal folate level, it is possible that the offspring may

accumulate high levels of folate level that could lead to ASD.

Bahous et al. found altered acetylcholine(Ach) metabolism is a result of high maternal folate levels.

Glycerophosphochole (GPC) was decreased in the pup hippocampus area; this could result from increased

conversion from GPD to choline (precursor of Ach) to maintain the amount of choline10. Bahous et al. also

found a trend of decreased ChAT in the cholinergic neurons and reduced mRNA levels of AchE in the cortex.

Since AchE plays a vital role in Ach metabolism, which leaves Ach at the synaptic cleft and breaks down Ach,

a decreased AchE may suggest alterations in Ach metabolism, contributing to memory impairment in the

offspring. The hippocampus and the cortexes are critical regions for memory and the ability to learn. The

reduced thickness of the dentate gyrus and size of the hippocampus area found in the FASD offspring group is

consistent with the impaired short-term memory impairment finding11.

Molecular pathways control the process of brain development through complex transcription gene

interactions. Barua et al. found that transcriptional factors Nfix, Runxl, and Vgll2 were significantly altered in the
mice offspring due to HMFA5. The study had shown that there are differences between male and female

mammalian brains at the level of cellular composition as well as gene expression 12. In a recent study, the

transcriptional factor Nifx was found to play an essential role in neural stem cell(NSCs) quiescence by

regulating the hippocampus area's microenvironment. The results show Nfix's effect in controlling NSC's

quiescence13. Barua et al. also found that HMFA resulted in significant downregulation in male offspring and

upregulation in female offspring5. Such alteration in expression may affect brain development, resulting in

possible neural defects. In addition, from the HMFA groups, the expression of transcriptional factor Runxl was

found upregulated in the female pup and downregulated in male offspring5. Runxl, a critical regulator in

neuronal differentiation coordination, was expressed during the first two postnatal weeks in the forebrain14.

Other studies have indicated that the altered expression of Runxl results in impaired development in post-

mitotic motor and sensory neurons13. Similarly, this study found the HMFA diet altered the expression of

Vgll2 5. In a recent study, Vgll2 is found to be a critical neonatal marker that affects the ventromedial

hypothalamus (nucleus involved in feeding, fear, sexual activity, and thermoregulation) on neonatal (postnatal

day 0) 15. These findings from the study suggest the HMFA may affect the expression in transcriptional

genes 15.

Limitations and strengths

While each of the five reviews has suggested a similar result, there are some limitations within each

study. In the study of Raghavan et al., the case and neurotypical development classification was based on

EMR, instead of using adjudication based on the research-reliable gold standard diagnostic assessments such

as Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Observation Schedule1. The result of

this method could misclassify children with other development or behavioral issue as ASD. Since the maternal

plasma folate was measured 24-72 hours postpartum, the data may only reflect the third trimester, excluding

the folate level's effects in the previous trimesters1. Also, estimates from the self-reporting diet and

supplement of the human studies could cause sampling bias as it can rise from social desirability, recall period,

sampling approach, and selective recall 5. Egorova et al. limited the sample size due to restrictions of sample

volume of 200uL, and only samples volume more than 700uL were eligible for the study2. As a result, the

study may have limited statistical power and an inflated false discovery rate. Valera-Gran et al.'s analysis

accounted for a wide range of adjusted potential confounding factors, affecting the study results. However, the
study includes detailed data of FAS dosages during different pregnancy periods, which may strongly support

the relationship between maternal folate level and risk of ASD in children 5. Barua et al. utilized a small

sample size in the animal study; however, the findings were similar to previous studies 3. Bahous et al.'s

study exposure time was short (5 minutes for the novel object recognition test and familiar test), requiring more

exposure time to further verify the result 4.

Currently, the studies cannot conclude firmly about the potential effect of excessive folate intake. The

results only confirmed the beneficial effects of adequate folate intake. Still, since the etiology of ASD is

complicated, these studies can only provide a new perspective on the impact of excessive folate intake. The

discovery provided a pathway for further future studies with a larger cohort to elucidate the potential effect of

high maternal folate level on ASD and other related brain development. Although confirmation is still needed,

the usage of FAS dosage >1000ug/day during pregnancy should be monitored unless medically prescribed.

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