The derivation of the study sample is described in . 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 <32 weeks at birth, children with birth weight <2,500 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, and children whose mothers reported supplement use but had not specified the type and duration. In total, 12,003 children were excluded, for one or more reasons. The final study sample included 85,176 children. At the end of follow-up, the age range was 3.3–10.2 years and the mean age 6.4 years.
A total of 270 children (0.32%) in the study sample have been diagnosed with ASD: 114 (0.13%) with autistic disorder, 56 (0.07%) with Asperger’s syndrome, and 100 (0.12%) with PDD-NOS. The distribution of ASD cases by year of birth is shown in eTable 1 of the online supplement
. 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 have 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%). PPV estimates are lower for the individual ASD subtype diagnoses: 80% (12/15) for autistic disorder (95% CI, 52–96%), 38% (5/13) for Asperger’s syndrome (95% CI, 14–68%), and 73% (8/11) for PDD-NOS (95% CI, 39–94%). PPV estimates for the subtype diagnoses are preliminary, as the number of cases in each group is still low.
The proportions of mothers reporting folic acid use are shown in . 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 week 9–12 and then reverted to 45.8% in week 13–17. The distribution of folic acid use across categories of parent and child characteristics is shown in . 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 non-smokers, to have pre-pregnancy BMI 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.
Folic Acid Supplement Use by Pregnancy Interval
Parent and Child Characteristics by Maternal Folic Acid Use a
Women who took folic acid in early pregnancy had a higher response rate to the screening questionnaire completed when the children were 36 months. For the study sample overall, the response rate was 62% in folic acid users and 55% in non-users. For children born in 2005–08, i.e., the youngest children, the difference was somewhat larger, with a response rate of 61% in folic acid users and 50% in non-users. Consequently, ASD children 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 displayed in . There was an inverse association between folic acid use and the subsequent risk of autistic disorder. In children whose mothers took folic acid, 0.10% (64/61,042) had autistic disorder, 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.
Risk of Autistic Disorder According to Maternal Folic Acid Use
The use of fish oil supplements followed similar patterns as folic acid use in the study sample: it was associated with the same parental characteristics (eTable 2
), it increased throughout the period of recruitment to the cohort (eTable 2
), and it increased from before pregnancy through the first trimester (eFigure 1
). Despite these similarities, there was no association between fish oil supplement use and autistic disorder risk, as shown in . 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 those taking ≥400 μg 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.
For Asperger’s 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): 2002–04 for Asperger’s syndrome (n=30,117 including 48 cases) and 2002–06 for PDD-NOS (n=59,152 including 91 cases). Our power to detect an OR of similar magnitude to that found for autistic disorder (OR=0.61) was limited: 36% for Asperger’s syndrome, and 61% for PDD-NOS. For Asperger’s 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 non-users, 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 non-users, generating an adjusted OR of 1.04 (95% CI, 0.66–1.63).
Results of exploratory analyses are displayed in . They should be cautiously interpreted, as 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 (, analysis #1). The use of other vitamins and minerals in addition to folic acid did not appear to affect autistic disorder risk (, analysis #2). 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 (, analysis #3). The analysis in which cases were stratified according to language level suggested that the inverse association may be strong in those with severe language delay and weak in those with moderate or no delay (, analysis #4). The analysis stratified by year of birth suggested that the inverse association may be stronger in the older children (born in 2002–04) than in the younger children (born in 2005–08) (, analysis #5).