【孕前/孕期服用葉酸，有助於降低自閉症兒童】
Association Between Maternal Use of
Folic Acid Supplements and Risk of Autism Spectrum Disorders in Children
Abstract
Importance Prenatal folic acid supplements reduce the risk of neural tube
defects in children, but it has not been determined whether they protect
against other neurodevelopmental disorders.
Objective To examine the association between maternal use of prenatal
folic acid supplements and subsequent risk of autism spectrum disorders (ASDs)
(autistic disorder, Asperger syndrome, pervasive developmental disorder–not
otherwise specified [PDD-NOS]) in children.
Design, Setting, and Patients The study sample of 85 176 children was derived from the
population-based, prospective Norwegian Mother and Child Cohort Study (MoBa).
The children were born in 2002-2008; by the end of follow-up on March 31, 2012,
the age range was 3.3 through 10.2 years (mean, 6.4 years). The exposure of
primary interest was use of folic acid from 4 weeks before to 8 weeks after the
start of pregnancy, defined as the first day of the last menstrual period
before conception. Relative risks of ASDs were estimated by odds ratios (ORs)
with 95% CIs in a logistic regression analysis. Analyses were adjusted for
maternal education level, year of birth, and parity.
Main Outcome Measure Specialist-confirmed diagnosis of ASDs.
Results At the end of follow-up, 270 children in the study sample had
been diagnosed with ASDs: 114 with autistic disorder, 56 with Asperger
syndrome, and 100 with PDD-NOS. In children whose mothers took folic acid,
0.10% (64/61 042) had autistic disorder, compared with 0.21%
(50/24 134) in those unexposed to folic acid. The adjusted OR for autistic
disorder in children of folic acid users was 0.61 (95% CI, 0.41-0.90). No
association was found with Asperger syndrome or PDD-NOS, but power was limited.
Similar analyses for prenatal fish oil supplements showed no such association
with autistic disorder, even though fish oil use was associated with the same
maternal characteristics as folic acid use.
Conclusions and Relevance Use of prenatal folic acid supplements around the time of
conception was associated with a lower risk of autistic disorder in the MoBa
cohort. Although these findings cannot establish causality, they do support
prenatal folic acid supplementation.
Supplementation
with folic acid around the time of conception reduces the risk of neural tube
defects in children.1-7 This protective effect has led to
mandatory fortification of flour with folic acid in several countries,8 and it is generally recommended
that women planning to become pregnant take a daily supplement of folic acid
starting 1 month before conception.8,9
There
also is evidence that maternal folic acid supplementation during pregnancy may
be associated with reduced risk of other neurodevelopmental disorders in children.
A recent study of 38 954 children in the Norwegian Mother and Child Cohort
Study (MoBa) found that maternal intake of folic acid supplements from 4 weeks
before to 8 weeks after the start of pregnancy was associated with a lower risk
of severe language delay at age 3 years.10 A case-control study from
California of autism spectrum disorders (ASDs) showed that maternal intake of
folic acid and prenatal vitamins during the 3 months prior to pregnancy and the
first month of pregnancy was associated with a lower risk of ASDs in the
offspring, and complementary genetic analyses indicated that the association
was modified by gene variants that determine the ability to utilize available
folate.11,12
Although
ethical considerations preclude placebo-controlled randomized trials that
eliminate folic acid, observational studies of mothers who do and do not use
supplements may be informative. We used the MoBa cohort to investigate the
association between the use of maternal folic acid supplements before and in
early pregnancy and the subsequent risk of ASDs (autistic disorder, Asperger
syndrome, pervasive developmental disorder–not otherwise specified [PDD-NOS])
in the offspring.
Methods
Study Population
The MoBa
cohort13 is nationwide and includes
109 000 children born from 1999 to 2009. Mothers were recruited at
ultrasound examinations around week 18 of gestation. Cases of ASD in the cohort
are identified by a substudy of autism, the Autism Birth Cohort (ABC) study.14 The analyses in this study
reflect data collected and processed by March 31, 2012. Participation in MoBa
and the ABC study is based on written informed consent from the mother. Both
studies were approved by the regional committee of medical research ethics for
Southeastern Norway.
Measures of ASD
Cases of
ASD are identified through (1) questionnaire screening of mothers at offspring
ages 36 months, 5 years, and 7 years, (2) professional and parental referrals
of children suspected of having ASD, and (3) linkages to the Norwegian Patient
Registry. Referrals are elicited through annual newsletters to MoBa
participants and information on the Norwegian Institute of Public Health
website. The Norwegian Patient Registry collects data on diagnoses from all
hospitals and outpatient clinics in Norway beginning in the year 2008, thereby
capturing data for all children diagnosed with ASD by Norwegian health services.
When a
child with ASD or potential ASD is detected through any of the mechanisms
described above, he or she is invited to participate in a clinical assessment
that includes the research-standard instruments for diagnosis of ASD, the
Autism Diagnostic Interview–Revised15 and the Autism Diagnostic
Observation Schedule,16 which have proven high
reliability and validity in making diagnoses of ASD in children. Assessments
are conducted without knowledge of previous questionnaire responses. Diagnostic
conclusions are best-estimate clinical diagnoses derived from test and
interview results and from information collected from parents and teachers.
Diagnoses are based on Diagnostic and Statistical Manual
of Mental Disorders (Fourth Edition) (DSM-IV) criteria, and
the case definition includes codes 299.00 (Autistic Disorder), 299.80 (Asperger
Syndrome), and 299.80 (Pervasive Developmental Disorder–Not Otherwise
Specified).
The
registry contains International Statistical Classification of Diseases,
10th Revision codes determined by Norwegian specialist health
services, and the ASD case definition of the ABC study includes codes F84.0
(Childhood Autism), F84.1 (Atypical Autism), F84.5 (Asperger Syndrome), F84.8
(Other Pervasive Developmental Disorder), and F84.9 (Pervasive Developmental
Disorder, Unspecified). In this article, we have used the terms autistic
disorder for code F84.0 and PDD-NOSfor codes
F84.1, F84.8, and F84.9.
Measures of Folic Acid
Use and Dietary Folate Intake
Since
1998, the Norwegian Directorate of Health has recommended that all women
attempting to become pregnant should take one 400-μg folic acid supplement per
day from 1 month before conception through the first trimester. Folic acid
supplements are available over the counter in Norway. Multivitamin supplements
containing folic acid are also available, but at the time participants were
recruited to MoBa, all such supplements contained less than 400 μg of folic
acid.
In MoBa,
detailed information about the mothers' supplement intake before conception and
in early pregnancy was obtained through questionnaire report at week 18 of
gestation. No foods were fortified with folic acid at the time when
participants were recruited; synthetic supplements thus represented the only
source of folate apart from the ordinary diet for the pregnant women. The women
were asked to record their intake of vitamins, minerals, and other supplements
according to the ingredient lists on the supplement containers, within 4-week
intervals from before the start of pregnancy. They were not asked to specify
the exact amounts, so if folic acid was only taken as part of a multivitamin
supplement, the daily dose would be lower than 400 μg.
Additional
information about supplement use and dietary intake in mid pregnancy was
obtained through a food frequency questionnaire completed in week 22. In this
questionnaire, women were asked to write the name of supplements they were
currently taking (in week 22), and exact amounts of vitamins and minerals were
calculated on the basis of this information. The food frequency questionnaire
has been described in a previous article17 and validated through blood
samples and 4-day food records from a subsample of the cohort.18
Measures of Timing
For our
primary analyses, we examined an interval from 4 weeks before to 8 weeks after
the start of pregnancy. The start of pregnancy was defined as the first day of
the last menstrual period before conception, in keeping with the standard
definition used in the follow-up of pregnant women in Norway. Children of
mothers who used folic acid supplements during the entire or parts of the
exposure interval were compared with children whose mothers did not use folic
acid supplements during the interval. The exposure interval was chosen on the
basis of an a priori hypothesis that the effect of folic acid on the
development of the central nervous system is most prominent in this period, and
it also corresponds to the interval used in the previous study of language
delay.10 The interval covers or precedes
events of critical importance to the fetal brain, such as the closure of the
neural tube 28 days after conception (gestational week 6) and the embryonic
period that includes development of the basic brain structures 15 to 56 days
after conception (gestational weeks 5-10).19
Potential Confounders
We
explored a number of factors that might influence a potential association
between supplement use and ASD risk: parental education, parental age, whether
the pregnancy was planned, maternal smoking during pregnancy, maternal body
mass index (calculated as weight in kilograms divided by height in meters
squared), weight gain at weeks 18 and 30, parity, and year of birth.
Statistical Analyses
Analyses
were performed using SPSS version 19.0 (SPSS Inc). Odds ratios (ORs) with 95%
CIs for the association between folic acid use and risk of each ASD were
estimated from logistic regression models. The adjusted models included
adjustment for year of birth, maternal education level, and parity, because
these were the only covariates that had any influence on the OR estimates.
We
estimated the power of these analyses for autistic disorder, which was the ASD
subtype with the highest number of diagnosed cases in the study sample. The
power calculations were based on the observed distributions of the outcome and
the exposure, ie, an overall prevalence of 0.13% for autistic disorder and a proportion
of 68% of the study sample exposed to folic acid within the exposure interval.
The type I error probability was set at α = .05 (2-sided). Under
these conditions we had a power of 93% to detect an OR of 0.50, 73% to detect
an OR of 0.60, 45% to detect an OR of 0.70, and 18% to detect an OR of 0.80.
To assess
the possibility of residual confounding, we explored whether maternal illness
and medication use during pregnancy had any effect on association. Information
about maternal illness and medication use was obtained from the questionnaire
completed in week 18 and from the Medical Birth Registry. We adjusted the
logistic regression models for the presence of anxiety, depression, epilepsy,
preeclampsia, and diabetes during pregnancy (separately for each disorder). We
also made separate adjustments for use of medications for anxiety, depression,
and epilepsy and for the use of hormone treatment and in vitro fertilization to
become pregnant.
We
conducted a secondary analysis of the association between maternal use of fish
oil supplements and the risk of ASD, to investigate whether the associations
were specific to folic acid or similar across different types of supplements.
If they were similar, the associations would more likely be attributable to
health-conscious maternal behaviors in general and not the supplements per se.20 We also examined the association
between folic acid use in week 22 of pregnancy and subsequent risk of ASD, to
evaluate whether any associations, if present, were similar in early pregnancy
and mid pregnancy.
The study
did not have sufficient power for subgroup analyses, but we performed some
exploratory analyses for the autistic disorder subtype to look for clues to
interactions that could be tested in future studies. We explored (1) the timing
of initiation of folic acid supplementation; (2) maternal use of other
vitamins, minerals, or both from 4 weeks before to 8 weeks after the start of
pregnancy; (3) maternal total daily intake of folate in week 22 (diet and
supplements combined, adjusted for dietary folate equivalents); (4)
stratification of autistic disorder cases by language level at 36 months; and
(5) stratification of the study sample by year of birth (2002-2004 vs
2005-2008).
Results
The
derivation of the study sample is shown in Figure 1. A total of
97 179 cohort participants were eligible for the analyses. To isolate
folic acid exposure from other exposure reported to increase the risk of ASD,
we excluded children with gestational age less than 32 weeks at birth, children
with birth weight less than 2500 g, and multiple births. We also excluded
children for whom we did not have data on maternal supplement use before
conception and in early pregnancy as well as children whose mothers reported
supplement use but had not specified the type and duration. In total, 12 003
children were excluded, for 1 or more reasons. The final study sample included
85 176 children. At the end of follow-up, the age range was 3.3 through
10.2 years (mean, 6.4 years).
A total
of 270 children (0.32%) in the study sample have been diagnosed with ASDs: 114
(0.13%) with autistic disorder, 56 (0.07%) with Asperger syndrome, and 100
(0.12%) with PDD-NOS. The distribution of ASD cases by year of birth is shown
in eTable 1. Of the ASD cases,
135 (50.0%) had been clinically assessed through the ABC study. The remaining
135 had specialist-confirmed diagnoses of ASD recorded in the Norwegian Patient
Registry. Registry diagnoses had a high validity for ASD as a whole: of the 39
children assessed in the ABC study after being detected through the registry,
38 were found to meet DSM-IV criteria
for ASD, generating a positive predictive value (PPV) of 97% (95% CI,
87%-100%). Estimates of PPV are lower for the individual ASD subtype diagnoses:
80% (12/15 [95% CI, 52%-96%]) for autistic disorder, 38% (5/13 [95% CI,
14%-68%]) for Asperger syndrome, and 73% (8/11 [95% CI, 39%-94%]) for PDD-NOS.
Estimates of PPV for the subtype diagnoses are preliminary, because the number
of cases in each group is still low.
The
proportions of mothers reporting folic acid use are shown in Figure 2. In the first
interval (weeks 4 to 1 before the start of pregnancy), 32.9% of mothers took
folic acid. The proportion increased to 70.7% in weeks 9 through 12 and then
reverted to 45.8% in weeks 13 through 16. The distribution of folic acid use
across categories of parent and child characteristics is shown in Table 1. Women who used
folic acid within the exposure interval (4 weeks before to 8 weeks after the
start of pregnancy) were more likely to have college- or university-level
education, to have planned the pregnancy, to be nonsmokers, to have
prepregnancy body mass index below 25, and to be first-time mothers. Folic acid
use increased substantially by year of birth, from 43.2% in 2002 to 83.7% in
2008.
Women who
took folic acid in early pregnancy had a higher response rate to the screening
questionnaire completed when the children were aged 36 months. For the study
sample overall, the response rate was 62% in folic acid users and 55% in
nonusers. For children born in 2005-2008, ie, the youngest children, the
difference was somewhat larger, with a response rate of 61% in folic acid users
and 50% in nonusers. Consequently, children with ASDs born to women who used
folic acid may have had a higher probability of being diagnosed at an early
age.
Results
of the logistic regression analysis for autistic disorder are reported in Table 2. There was an
inverse association between folic acid use and subsequent risk of autistic
disorder. Autistic disorder was present in 0.10% (64/61 042) of children
whose mothers took folic acid, compared with 0.21% (50/24 134) in children
whose mothers did not take folic acid. The adjusted OR of autistic disorder was
0.61 (95% CI, 0.41-0.90) in children of folic acid users. Adjustment for
maternal illness and medication use did not affect the OR (eTable 2).
The use
of fish oil supplements followed patterns similar to those for folic acid use
in the study sample: it was associated with the same parental characteristics (eTable 3), it increased
throughout the period of recruitment to the cohort (eTable 3), and it increased
from before pregnancy through the first trimester (eFigure). Despite these
similarities, there was no association between use of fish oil supplements and
risk of autistic disorder, as shown in Table 3. The adjusted OR of
autistic disorder was 1.29 (95% CI, 0.88-1.89) in children of mothers who used
fish oil supplements.
The
inverse association found for folic acid use in early pregnancy was absent for
folic acid use in mid pregnancy: the adjusted OR for autistic disorder was 0.96
(95% CI, 0.60-1.55) for mothers taking 400 μg or more per day in week 22 and
1.02 (95% CI, 0.62-1.67) for those taking less than 400 μg per day at that time
(Table 3).
For
Asperger syndrome and PDD-NOS, we restricted the analyses to birth years with a
cumulative incidence of 0.08% or higher (higher than the lowest level observed
for autistic disorder): 2002-2004 for Asperger syndrome
(n = 30 117, including 48 cases) and 2002-2006 for PDD-NOS
(n = 59 152, including 91 cases). Our power to detect an OR of
magnitude similar to that found for autistic disorder (OR, 0.61) was limited:
36% for Asperger syndrome and 61% for PDD-NOS. For Asperger syndrome, the
proportion of diagnosed cases was 0.12% (21/17 218) in children of folic
acid users and 0.21% (27/12 899) in children of nonusers, generating an
adjusted OR of 0.65 (95% CI, 0.36-1.16). For PDD-NOS, the proportion was 0.15%
(58/39 543) in children of folic acid users and 0.17% (33/19 649) in
children of nonusers, generating an adjusted OR of 1.04 (95% CI, 0.66-1.63).
Results
of exploratory analyses are reported in Table 4. These results
should be cautiously interpreted, because none of the exploratory analyses had
a statistical power of more than 50% to detect an OR of 0.60. There did not
seem to be a strong gradient in risk by timing of initiation of folic acid use
within the primary exposure interval. The use of other vitamins and minerals in
addition to folic acid did not appear to affect risk of autistic disorder. The
analyses based on the food frequency questionnaire data from week 22 did not
reveal any apparent association between maternal total daily folate intake in
week 22 (diet and supplements combined) and subsequent risk of autistic
disorder in children. The analysis in which cases were stratified according to
language level suggested that the inverse association may be strong in children
with severe language delay and weak in those with moderate or no delay. The
analysis stratified by year of birth suggested that the inverse association may
be stronger in the older children (born in 2002-2004) than in the younger
children (born in 2005-2008).
Comment
This study found that
maternal use of supplemental folic acid from 4 weeks before to 8 weeks after
the start of pregnancy was associated with a lower risk of autistic
disorder—the most severe form of ASD—in children.
Use of
folic acid supplements was associated with higher socioeconomic status and more
health-conscious maternal behavior patterns in the study sample. We cannot
exclude the possibility that some portion of the inverse association represents
residual, unmeasured confounding. However, if residual confounding was
substantial, we would have expected to find a lowering of risk associated with
fish oil supplement use as well, because the use of fish oil was associated
with the same parental characteristics in the study sample. No such lowering of
risk was observed. We also would have expected the inverse association between
folic acid use and autistic disorder risk to persist in mid pregnancy (week
22), which it did not. There was no evidence of residual confounding in
analyses adjusting for maternal illness and medication, which might reflect the
fact that the pregnant women in the cohort were generally healthy and had low
proportions of medication use during pregnancy. We did not have data on more
rare psychiatric disorders, but we believe that such disorders are unlikely to
have had any significant influence.
Our
findings indicate that the inverse association may be largely driven by the
children with autistic disorder and severe language delay at 36 months, who
were presumably the more severely affected children. It is also worth noting
that the OR estimate for those with missing data on language level
(nonresponders to the screening questionnaire) was similar to that for those
with severe language delay. This suggests that mothers with severely affected
children may have had lower response rates and that an ascertainment bias may
have been present. The possibility of such bias, combined with the small
numbers in each stratum, warrants caution in the interpretation of these
findings.
The
participation rate among women invited to participate in MoBa was 38.5%, and
the cohort is not fully representative of the Norwegian population. Comparisons
with nationwide registry data have demonstrated that the mothers in the cohort
were more likely to be first-time mothers, less likely to be single mothers,
and had higher levels of education, higher mean age, and lower levels of
smoking than other pregnant women.21
We tested
the generalizability of our findings by replicating the analyses in a
nationwide data file containing data from the Medical Birth Registry of Norway,
the Norwegian Patient Registry, and Statistics Norway. We included children
born in 1999-2007 and applied the same inclusion criteria as for the MoBa-based
analyses. The replication sample included 473 095 children, of whom 822
(0.17%) had diagnoses of autistic disorder recorded by Norwegian specialist
health services. Folic acid use is substantially underreported in the Medical
Birth Registry; our comparisons with the MoBa questionnaire data found that
half of the mothers reported in the registry to be nonusers of folic acid
actually had reported folic acid use in the questionnaires. This underreporting
is a major limitation and will bias association measures toward the null.
Despite this, we did find a significant inverse association in the nationwide
sample: mothers with reported folic acid use had an adjusted OR of 0.83 (95%
CI, 0.71-0.97) for autistic disorder in their children. When the MoBa
participants were analyzed using the registry-based folic acid variable, the
adjusted OR was 0.75 (95% CI, 0.46-0.96). The similarity between MoBa
participants and nonparticipants suggest that our MoBa-based analyses have not
been significantly affected by selection bias.
The
strengths of the study were the cohort design, large sample size, and
prospective data collection as well as the combination of screening, referrals,
and registry linkage for detection of ASD cases. The richness of the exposure
data allowed for differentiations between different supplements and between the
various stages of pregnancy. Our ability to compare the study sample with a
nationwide sample was also an advantage.
The main
limitation was the incomplete ascertainment of ASD cases in the cohort. The
prevalence of diagnosed ASD was lower than what has been reported in the United
Kingdom and the United States,22,23although that discrepancy is not
merely attributable to underascertainment, because the nationwide ASD prevalence
is also lower in Norway.24 For the country as a whole, the
prevalence is estimated to be 0.8% in 12-year-olds,24 which is not very different from
the 0.66% prevalence observed for children born in 2002 and 2003 in the MoBa
cohort (eTable 1).
Underascertainment was less of a problem for autistic disorder than for the
other ASD subtypes, but it was still reassuring that the inverse association
for autistic disorder was stronger in the older children (born in 2002-2004),
for whom case ascertainment was closer to completion. The relative weakness of
the inverse association among the younger children (born in 2005-2008) may have
resulted from the fact that mothers who took prenatal folic acid supplements
had higher questionnaire response rates, causing the ASD cases among their
children to be identified earlier. As mentioned previously, there also was the
possibility of ascertainment bias arising from lower response rates among
parents of the children more severely affected with autistic disorder. If such
bias were present, the OR estimate for the younger children would be biased
toward the null.
Another
limitation of the study was the reliance on subtype diagnoses of ASD. These
have not been found to have high reliability across assessment sites in studies
in the United States and may be removed altogether from the upcoming DSM-V classification
system.25 Our own validation of registry
diagnoses indicated that subtype diagnoses were less reliable than for ASD as a
whole, but there was still a high level of agreement (PPV, 80%) for autistic
disorder diagnoses, which was the outcome of primary interest.
Our
main finding was that maternal use of folic acid supplements around the time of
conception was associated with a lower risk of autistic disorder. This finding
does not establish a causal relation between folic acid use and autistic
disorder but provides a rationale for replicating the analyses in other study
samples and further investigating genetic factors and other biological
mechanisms that may explain the inverse association.
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