New Perspective on Impact of Folic Acid Supplementation during Pregnancy on Neurodevelopment/Autism in the Offspring Children – A Systematic Review

It has been conclusively established that folic acid supplementation prior to and during early pregnancy (up to 12 weeks of gestation) can prevent neural tube defects (NTDs). We hypothesized that folate effects may extend from neuro-structural defects to alterations in neuro-behavioural and emotional skills including autism spectrum disorders (ASDs) and other developmental disorders. The objective of this review was to comprehensively evaluate evidence on the impact of folic acid on neurodevelopment other than NTDs. We conducted an online search of relevant literature compiled by the National Library of Medicine from Medline and EMBASE (searched on Dec 31, 2014: http://www.ncbi.nlm.nih.gov/entrez/query/fcgi and http://www.elsevier.com/online-tools/embase). We first created 3 files (search restricted to English literature) using the following key words: 1) folate or folic acid (171322 papers identified by this search); 2) maternal or pregnancy or pregnant or gestation or gestational or prenatal or antenatal or periconception or periconceptional (1349219 papers identified by this search); and 3) autism or autism spectrum disorders or developmental delay or development or neurodevelopment or mental or cognitive or language or personal-social or gross motor or fine motor or behaviour or intellectual or intelligence or Bayley Scale (8268145 papers identified by this search). We then merged the 3 files and reviewed the papers that addressed these three issues simultaneously. A total of 22 original papers that examined the association between folic acid supplementation in human pregnancy and neurodevelopment/autism were identified after the screening, with 15 studies showing a beneficial effect of folic acid supplementation on neurodevelopment/autism, 6 studies showed no statistically significant difference, while one study showed a harmful effect in > 5 mg folic acid supplementation/day during pregnancy. Folic acid supplementation in pregnancy may have beneficial effects on the neurodevelopment of children beyond its proven effect on NTDs.


Introduction
There are major emotional, societal, and economic implications of impaired neurodevelopment and/or autism in children [1,2]. These children will often require specialized schooling and other community resources. Although the survival/life-span of these infants may not be seriously affected, many of them may need treatments throughout their lifetime, and the cost to the public health care system could be huge. When they reach adulthood, productivity is often lower than those with normal development, indirectly increasing the societal burden.
It has been established that supplementation with folic acid around the time of conception reduces the risk of neural tube defects (NTDs) in the offspring [3,4,5,6]. However, whether folic acid has a similar effect on impaired neurodevelopment and/or autism remains elusive. This article therefore focuses on assessing the role of folic acid supplementation during pregnancy and folate metabolism on neurodevelopmental outcomes including autism spectrum disorders (ASDs), other than NTDs.

Search strategy
We conducted an online search of relevant literature compiled by the National Library of Medicine from Medline and EMBASE (searched on December 31, 2014 of the site: http:// www.ncbi.nlm.nih.gov/entrez/query/fcgi and http://www.elsevier.com/online-tools/embase), with restriction to human studies. We first created 3 files (restricting our search to English literature) using the following key words: 1) folate or folic acid (171322 papers identified by this search); 2) maternal or pregnancy or pregnant or gestation or gestational or prenatal or antenatal or periconception or periconceptional (1349219 papers identified by this search); and 3) autism or autism spectrum disorders or developmental delay or development or neurodevelopment or mental or cognitive or language or personal-social or gross motor or fine motor or behaviour or intellectual or intelligence or Bayley Scale (8268145 papers identified by this search). We then merged the 3 files. All abstracts of the papers identified by merging the 3 files were screened by two independent reviewers in our group to exclude irrelevant studies (such as those on NTDs); because the causation between folic acid supplementation in pregnancy and NTDs has been established, our review is interested in outcomes other than NTDs.

Study selection
We included randomized controlled trials (RCTs), cohort studies, and case control studies that examined the association between folic acid supplementation during pregnancy and neurodevelopment/autism in the offspring children. Data extraction was conducted independently and screened all records at the title level by two reviewers (Chao Sheng and Ri-hua Xie). To enhance sensitivity, records were only removed if both reviewers excluded at the title level.
The second level of review was at the abstract level followed by another round of review at the full-text level. Two independent reviewers abstracted data using a standardized form. When there was a disagreement it was resolved by discussion with a third reviewer (Yunfei Gao). Corresponding authors were contacted via e-mail at least three times to obtain data if the outcome of the neurodevelopment/autism in the offspring children could not be readily abstracted from the publication.

Study quality assessment
The quality of included cohort and case control studies was assessed with the Newcastle Ottawa Scale [7]. Using this checklist, Yunfei Gao evaluated each of the included articles, with additional inputs from Chao Sheng and Ri-hua Xie. The details are shown in Table 1. Divergent views were resolved by consulting a third reviewer.

Data extraction and synthesis
Data extracted from each study included the first author's last name, publication year, main outcome, sample size, study design, age of children, effect of folic acid, and comments on the study. Because of major heterogeneity in original studies in terms of study design and outcome

Literature search
A total of 3,348 papers were identified. Sixty five full-text articles were assessed for eligibility after screening. Most frequently the removed papers were animal studies or reviews or commentary/discussion in the interpretation of the study findings on other pregnancy outcomes (e.g., NTDs) or studies in humans but the effects of folic acid supplementation during pregnancy on neurodevelopment/autism in the offspring children was not examined. See details of selection in Fig 1. A total of 22 original papers that looked at the association between folic acid supplementation in pregnancy and neurodevelopment/autism were identified after the screening in Table 2.
Because of major heterogeneity in study design, exposure measurement, and outcome measurement, no attempt was made for quantitative synthesis of effect by meta-analysis. The 43 full-text excluded articles with reasons of exclusion were listed as supplement information in S1 Appendix.

Study characteristics
The 22 eligible studies include 2 RCTs, 18 cohort studies, and 2 case control studies. The baseline characteristics and further summarized information are outlined in Table 2. The main outcomes include ASDs, autism, developmental delay, cognition, attention function, neurodevelopment, emotional problems, and behavioural problems. The children range in age from 12 months to 11 years. Among the 21studies, 7 studies included more than 1000 children.

Outcomes
Fifteen studies showed a beneficial effect of folic acid supplementation on neurodevelopment/ autism, 6 studies found no statistically significant result, while one study found a harmful effect at high dose of folic acid supplementation (see Table 2). There were 3 studies that had ASD as the main outcome measure. The first was a cohort study of 85,176 children aged 3.3 to 10.2 years [8]. The rate of ASD in children whose mothers took folic acid was 0.10%, whereas the rate for mothers who did not take folic acid was 0.21%, with adjusted odds ratio (OR) of folic acid users 0.61 (95% confidence interval (CI), 0.41-0.90). Another study was the Childhood Autism Risks from Genetics and Environment, a case-control study in the United States [9]. In the 837 mother-child pairs, the mean folic acid intake in the first month of pregnancy was significantly greater for mothers of normally developing children than for mothers of children with a confirmed diagnosis of ASD. A mean daily folic acid intake of ! 600ug during the first pregnant month was associated with reduced ASD risk (adjusted OR: 0.62; 95% CI: 0.42-0.92; P = 0.02). This finding was consistent with another case-control study by the same author [10], which showed that mean folic acid intake in early pregnancy was significantly higher for mothers of normally developing children than for mothers of children with ASD.
Several studies found similar beneficial effects of folic acid supplementation on other areas of neurodevelopment. For example, a study in Massachusetts [11] showed that for each 600 ug/day increment in total folate intake during the first trimester, Peabody Picture Vocabulary Test-III score at age 3 years was 1.6 points (95% CI 0.1-3.1; p = 0.04) higher. Forns et al found that omission errors (defined as the number of targets to which the individual did not respond) were lower in those whose mothers took dietary supplementation with folic acid and vitamins during pregnancy [12]. In a cohort study [13] involving 553 mother-child pairs in Greece, neurodevelopment at 18 months was assessed using the Bayley Scales of Infant and Toddler Development (3rd edition). Compared with non-users, daily intake of 5 mg supplemental folic acid was associated with a 5-unit increase on the scale of receptive communication and a 3.5-unit increase on the scale of expressive communication. Roth et al assessed severe language delay (defined as minimal expressive language: only 1-word or unintelligible utterances at the age of 3 years) in a cohort of 38,954 children, and found that adjusted ORs for 3  *Results were considered beneficial if they consistently showed better outcomes in the group with folic acid supplementation in pregnancy (or higher maternal folate level) than the comparison group. If study results consistently showed poorer outcomes in the group with folic acid supplementation in pregnancy (or higher maternal folate level) than in the comparison group these results were defined as harmful. Where there was no difference between (or among) comparison groups, or conflicting findings on different outcome measures (i.e. protective on one outcome but harmful on another, study results were defined as no association. doi:10.1371/journal.pone.0165626.t002 patterns of exposure to maternal dietary supplements (no supplement as the reference) were 1.04 (95% CI, 0.62-1.74) for other supplements but no folic acid; 0.55 (95% CI, 0.35-0.86) for folic acid only; and 0.55 (95% CI, 0.39-0.78) for folic acid in combination with other supplements, demonstrating a clear protective effect of folic acid supplementation during pregnancy [14]. A study by Steenweg-de et al found a higher risk of emotional problems in 3 year old children using the Child Behavior Checklist (CBCL) whose mothers did not use supplements or started folic acid supplements late in pregnancy (OR: 1.45; 95% CI: 1.14, 1.84) compared to children whose mothers started folic acid supplement in early pregnancy [15]. Similarly, in a prospective cohort study, Roza et al examined the association between folic acid supplement use during the first trimester and behavioural and emotional problems identified by the CBCL in 4,214 toddlers at the age of 18 months. This study found that folic acid supplement use protected both from internalizing (OR of no use 1.

Discussion
A total of 22 original papers that looked at the association between folic acid supplementation in pregnancy and neurodevelopment/autism were identified after the screening, with 15 studies showing a beneficial effect of folic acid supplementation on neurodevelopment/autism, 6 studies found no statistically significant effect, while one study found a harmful effect at high dose of folic acid supplementation [18]. Two papers that suggested an adverse effect of folic acid on ASDs were not included in our review because no data on individual subjects were available in these two studies [19,20]. Both papers used ecological data to support their hypothesis: prenatal folic acid supplementation and autism diagnoses in the United States since the 1980s in King's study, and published autism incidence rates and prescriptions for folic acid in Rochester, Minnesota from 1976 to 1997 in Beard's study. Beard's study found a correlation coefficient of 0.87 (95% CI 0.19-0.99) between autism rates and the prescription prenatal vitamins containing folic acid and a correlation coefficient of 0.62 (95% CI 0.38-0.95) between autism rates and pediatric folic acid. However, during the same period, major changes in other factors such as diagnostic criteria, public awareness, disease surveillance, and screening efforts have all played an important role in the increased rates of diagnosed ASDs, so ecological data may not be suitable to analyze the association between folate and ASDs. Data from recent ASDs surveillance in the United States revealed a major increase in ASDs prevalence during a period with no change in policies regarding prenatal folic acid supplementation or folic acid food fortification (2002 to 2008), suggesting that ecological analyses are seriously flawed [21]. On the other hand, in a small sample of children (77) born to mothers used folic acid supplementation >5 mg/day during pregnancy had a statistically significantly lower mean psychomotor scale score (difference, -4.35 points; 95% CI, -8.34 to -0.36) than children whose mothers used a recommended dosage of folic acid supplements (0.4-1.0 mg/day) [18]. The finding from a single study with small sample needs to be replicated. Castro et al conducted a systematic review of studies involving on relationship between folic acid and ASD. All 11 papers included in Castro's review met the inclusion criteria in our review. It concluded that although lower folate levels were associated with increased risk of ASD, the effects of folateenhancing interventions on the clinical symptoms of ASD have yet to be confirmed [22]. Folic acid, or folate (vitamin B9) is an essential nutrient that is required for DNA replication and as a substrate for a range of enzymatic reactions involved in amino acid synthesis and vitamin metabolism. Demands for folate increase during pregnancy because of increased demands from the fetus. It has been conclusively established that folic acid deficiency prior to and during early pregnancy (up to 12 weeks of gestation) causes increased risk of NTDs, and periconceptional supplementation of folic acid can dramatically lower this risk (as much as 70%) [3,4,5,6]. If folic acid deficiency prior to and during early pregnancy can cause NTDs, it may also cause milder forms of fetal brain damage that could be expressed as impaired neurodevelopment/autism in early childhood, and this effect may not be restricted to pre-conception and early gestation (<12 weeks of gestation, as the neural tube closes at that time so no NTDs after that). Laboratory investigations in animals and humans have shown that folate plays an important role in early brain development. In humans, there are active placental transports of folate and fetal brain folate levels are higher than adult levels [6]. In rats, the concentrations of many folate-dependent enzymes were substantially higher during early development than adult levels [23]. Dams and developing pups fed with diets eliminating folic acid 1 week prior to birth were less viable and had lower brain weights, lower activity level, and lower level of S-adenosyl-L-methionine concentrations in brain tissue of surviving offspring than animals reared on normal diets [24]. Ferguson et al examined whether gestational dietary folate deficiency not producing severe NTDs could lead to other functional impairments in mice [25]. They found that prenatal folate deficiency in mice led to an increase in anxiety-related behaviours. Worthy of our attention is that Padmanabhan et al found a hypomorphic mutation of the mouse MTRR gene, which results in developmental delay, as well as congenital malformations, including neural tube, heart, and placental defects, showing that folate metabolism has distinct transgenerational epigenetic functions that are responsible for specific developmental processes [26]. Even with normal dietary folate, the hypomorphic mutations in the MTRR gene associated with reduced expression may still lead to congenital abnormalities.
A few clinical studies have compared metabolites or cofactors of the folate-methionine pathway in children with autism [27][28][29][30][31][32][33][34][35][36][37]. While results from these studies have not been consistent a dysfunctional folate-methionine pathway has been identified that may have an impact on developmental delays including autism [38]. This pathway is crucial for DNA synthesis, DNA methylation, and cellular redox balance.
Clinical case series have also linked folic acid deficiency to other types of fetal brain damage such as intracranial calcification [39]. Del Campo et al reported several cases of patients who, in addition to the structural anomalies typical of maternal methotrexate exposure, have significant developmental delay, and suggested that prenatal exposure to folic acid antagonists increases the risk of mental retardation [40]. Arakawa et al observed that the EEG maturation was delayed in children born to mothers with low serum folate [41]. A recent study evaluated the nutritional intake in 111 Chinese children with autism (aged 2 to 9 years) and compared with the national Dietary Reference Intakes (DRI) [42]. They found that the children with autism had adequate or exceeded intakes in energy, protein, vitamins B1, B2, E, niacin, magnesium, and iron, but had inadequate intakes in folic acid, vitamins A, B6, C, calcium, and zinc, nutrients known to be important for brain development and function [42].

Strengths
To our knowledge, this is the first systematic review examining the impact of folic acid supplementation during pregnancy on neurodevelopment/autism in the offspring children. We did an extensive search of relevant literature and selected studies strictly. After merging and analyzing the selected studies, we provided a preliminary conclusion.

Limitations
Our study has some limitations. First, the magnitude of the protective effect of folic acid supplementation observed in most of the included studies was quite small as compared with the known effect of folic acid supplementation on NTDs. We speculate that compared with NTDs, the diagnosis of neurodevelopmental disorders is more subtle and requires a much longer observation period. As a result, the potential effect of folic acid supplementation may have been offset by measurement errors or loss of follow ups. Second, most of the included studies were observational. However, one RCT [43] showed beneficial effects of folic acid supplementation, consistent with a majority of the observational studies, which adds weight to the evidence. Third, because of major heterogeneity in original studies in terms of study design and outcome and exposure measurements, no attempt to summarize the effect by meta-analysis was made. Finally, there may be studies with negative results that were not published in the searchable databases because of potential publication bias. Although the major heterogeneity of the included studies prevented us from a formal assessment of publication bias, global inspection of all included studies did not find any systematic trends in terms of positive/negative findings.

Implications for research
The limited data identified suggests that folic acid supplementation in pregnancy protects against impaired neurodevelopment including ASDs in children, and may improve cognitive function and intellectual and motor function. However, it is hard to draw a conclusion due to the limitations of the identified studies. Large scale RCTs with validated diagnosis and high follow up rate are needed in order to produce robust evidence regarding the effects of folic acid supplementation in pregnancy on fetal neurodevelopment.

Conclusion
In summary, our review of the literature suggests that folic acid supplementation in pregnancy may protect against impaired neurodevelopment including ASDs in children, and may improve cognitive function, intellectual, and motor function.