Background
Folate is essential in early life, with conclusive evidence that periconceptional supplementation with folic acid (FA; the synthetic vitamin form) is effective in preventing the first occurrence [
1] and recurrence [
2] of neural tube defects (NTD). This evidence has led to recommendations that are in place worldwide for women to take FA from before conceiving until the end of the first trimester. What is less clear is whether continued FA supplementation after the first trimester of pregnancy can confer longer term health benefits to the child. Emerging evidence however shows that the period of rapid growth and development of the foetal brain occurring in later pregnancy is particularly sensitive to maternal folate concentrations [
3]. Also, myelination of the brain, which is most intensive from mid-gestation to the second year of life [
4] and essential for cognitive development as it protects nerve axons and facilitates communication between neurons, may be particularly vulnerable to deficiency of folate [
5]. Several studies have investigated the association of maternal folate in pregnancy with neurocognitive development in the child [
6]. Over 40 years ago, Gross et al. showed that children born to mothers diagnosed with folate-related megaloblastic anaemia in the third trimester of pregnancy had abnormal neurodevelopment and lower intellectual abilities, compared to infants born to mothers with optimal folate status [
7]. Decades later, a longitudinal transgenerational study found that optimal maternal folate status during later pregnancy was associated with better cognitive performance in the child at 9–10 years [
8]. Another study of note used magnetic resonance imaging (MRI) of the child’s brain along with cognitive tests and found that maternal folate deficiency in later pregnancy was associated with, not only lower language and visuospatial abilities, but also reduced brain volumes in children aged 6–8 years [
9].
A major limitation in the aforementioned studies is that they are observational and thus, by design, cannot demonstrate that maternal folate nutrition is causatively linked with cognitive outcomes in the offspring [
6]. More robust evidence was however provided in our recent follow-up study of children whose mothers had participated in a randomised trial of Folic Acid Supplementation in the Second and Third Trimesters (FASSTT) [
10] and showed improved psychometrically measured cognition at 3 years and word reasoning (verbal IQ) at 7 years in the children of mothers randomised to receive FA in pregnancy [
11]. The latter study, and almost all previous research in this area, involved psychological tests to assess neurodevelopment in children, whereas direct measurements of neuronal activity in the brain have rarely been employed. Magnetoencephalography (MEG) is a non-invasive neuroimaging modality that measures the magnetic fields associated with neuronal currents generated by the brain and may thus provide a robust platform for investigating neurodevelopment in children [
12]. Therefore, in the present FASSTT Offspring trial, we aimed to evaluate the effect of FA supplementation during pregnancy on cognitive performance and MEG-assessed brain functioning in the child at 11 years. Our hypothesis was that the higher verbal IQ previously found in the 7-year-old children of mothers randomised to receive FA in pregnancy would remain evident in these children at 11 years, as measured by both cognitive testing and MEG assessment.
Discussion
We examined the effect of FA intervention in trimesters 2 and 3 of pregnancy in women taking FA as recommended in the first trimester, and provide the first randomised trial evidence that continued FA supplementation (400 μg/day) throughout pregnancy can influence cognitive performance and brain function of the child up to 11 years of age. The results not only provide a further follow-up from the FASSTT Offspring study to reinforce our previous findings of cognitive benefits in these children at 3 and 7 years but provide the first MEG brain imaging evidence that FA supplementation in pregnancy can impact brain function in the child.
Our results, reporting the third follow-up of mother-child pairs from the FASSTT trial, show that the 11-year-old children of mothers randomised to FA compared with placebo during trimesters 2 and 3 of pregnancy scored significantly higher in specified cognitive IQ domains of the WISC-IV assessment, namely, two Processing Speed tests, i.e. symbol search (by 2.9 points) and cancellation (by 11.3 points), and Verbal Comprehension (by 6.5 points) in girls. The results are entirely consistent with our earlier findings in these children at a younger age, showing higher scores in overall cognition at 3 years and in verbal IQ at 7 years in the offspring of FA-treated mothers in pregnancy [
11]. Here we show that at all time-points up to age 11 years, greater proportions of children from FA-treated mothers, compared with placebo, had cognitive scores above the median values of 10.0 in the BSITD-III test (at 3 years), 23.0 in the WPPSI-III test (at 7 years) and 102.5 in the WISC-IV test (at 11 years). The FASSTT Offspring trial thus provides more robust evidence of the role of maternal folate status in offspring cognitive function compared with previous observational studies that have reported positive associations between FA use in early pregnancy (as retrospectively reported by mothers) or maternal folate concentrations, and cognitive performance in the child [
6,
28,
29]. The role of maternal folate beyond the first trimester of pregnancy has been far less frequently investigated in previous studies, but one notable study found that higher plasma folate at the 30th gestational week was associated with better cognitive performance in over 500 children aged 9–10 years in South India [
8]. Our results are thus consistent with previous observations, but add considerably to the evidence, and indicate that FA intervention throughout pregnancy will have beneficial effects on cognitive performance in the child up to 11 years. Of note, folate intake and status of the children were considered in this study and found to be similar in both study groups, with dietary folate comparing favourably with reference values for this age group [
24] and biomarker concentrations in good agreement with those reported in children from Norway [
25], UK [
26] and USA [
27].
The current sex-specific findings, showing a positive effect of prenatal FA on verbal IQ in 11-year-old girls (by 6.5 points), but not in boys, are not entirely unexpected given existing evidence of sex differences in brain activity patterns associated with cognition and behaviour and in functional brain development in infancy and early childhood [
4]. Also, between 3 and 60 months, females are reported to exhibit a higher rate of myelination than do males in the genu of the corpus callosum, in left frontal and left temporal white matter and in the right optic radiation [
30]. Furthermore, in previous epigenetic analysis of cord samples from these children at birth, we investigated candidate genes related to brain development or function and reported significant DNA methylation effects in
IGF2 in girls, but not in boys, arising from maternal FA intervention [
31], perhaps offering a biological basis for the sex-differences found here in the relationship of prenatal FA with cognitive outcomes in childhood. Sex differences in brain development, however, likely reflect a dynamic interplay of many biological (e.g. prenatal and neonatal hormone production and direct sex chromosome effects) and other mechanisms [
4]. In any case, this aspect requires further investigation.
In addition to using standardised IQ tests to measure child cognition, we applied magnetoencephalographic brain imaging for the first time in a study of this kind. In broad support of the cognitive outcomes, the neuronal activity assessments using MEG indicated more efficient semantic processing of language in the 11-year-old child as a result of prenatal intervention with FA. Specifically, the increased power at Beta and High Gamma bands in response to the language task in children from the FA group suggest more active engagement of local neuronal networks. Previously, increased power at the High Gamma band using MEG was reported as a result of intensive computer-based training [
32]. The fact that we observed similar band power patterns in response to the language task in children of FA-supplemented mothers to those reported in children with enhanced cognition as a consequence of intensive training suggests that the current MEG findings are clinically relevant.
The finding of FA-related effects in language processing as assessed by MEG is broadly in line with the higher verbal comprehension scores in the current WISC-IV assessment at 11 years and our earlier findings of higher verbal IQ in these children at 7 years in response to prenatal FA. Likewise, a recent study reported greater cerebral cortical thickness in children aged 8–18 years who were exposed prenatally to population-wide FA fortification compared to those born before the introduction of this mandatory policy in the USA [
33], whilst in the Generation R Study, maternal folate deficiency diagnosed after the first trimester was associated with reduced brain volume (measured using magnetic resonance imaging) in 6–8 year old Dutch children [
9]. Thus, our randomised trial results, together with the available observational evidence, are consistent in indicating that prenatal maternal FA supplementation affects neurocognitive development and may have a specific role in semantic processing of language up to 11 years. In addition, there may be clinical implications of our findings for later life in that the achievement of full cognitive potential of the child is considered paramount for future academic attainment, and higher intelligence in childhood is associated with better cognitive reserves in adulthood that could potentially delay cognitive decline in later life [
34,
35]. Future MEG studies might investigate the functional neural connectivity patterns within emerging neural networks, both in resting-state and under task conditions, that support developing language and cognition.
Although the precise mechanism explaining the effect of FA during pregnancy on neurodevelopment of the child is unknown, it must in some way involve the essential role of folate in one-carbon metabolism, encompassing a complex network of interdependent pathways required for a number of biological processes that could impact neurodevelopment, including myelination, neurotransmitter synthesis and methylation [
36]. It is also possible that folate-related epigenetic changes via DNA methylation are involved [
3]. In previous epigenetic analysis of cord blood samples in these children as newborns, using a candidate gene approach we showed that prenatal FA throughout pregnancy resulted in significant effects in DNA methylation in specific genes linked with brain development or function (
IGF2,
BDNF and the widely dispersed retrotransposon,
LINE-1) [
31], and in epigenome-wide screening of the same samples, we identified a novel mechanism for folate-dependent regulation of the
ZFP57 gene [
37].
The main strength of this study is the research design, involving a follow-up study in children of mothers who had participated in a randomised trial in pregnancy, which enabled the demonstration of a causative link between prenatal FA and subsequent neurocognitive development in the child. The use in a sub-set of children of the neuroimaging technique, MEG, objectively investigated the effects of FA intervention in pregnancy on brain function in childhood. Furthermore, this is the third follow-up of the FASSTT Offspring cohort (with previous investigations at age 3 and 7 years), providing the opportunity to track cognitive development in childhood, and the broad agreement in results at all time-points provides some degree of internal validation to the findings. In addition, a range of factors previously reported to be associated with cognitive performance, and thus potential confounders in the relationship between prenatal FA and offspring intelligence, were considered [
23,
38]. However, this study also has limitations, the most notable of which is the relatively small sample size. Although every effort was made to maximise the participation rate from the original FASSTT trial (
n = 119), the final sample of 68 mother-child pairs may have lacked sufficient statistical power to detect small differences in some components of the WISC-IV cognitive test. Also paternal factors, recently emerging as potentially important for child neurocognitive development, were not considered.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.