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We evaluated the relationship between prenatal folic acid supplementation and autism spectrum disorder (ASD) in 3-year-old offspring. We used data from the Japan Environment and Children’s Study, a nationwide prospective birth cohort study. We analyzed the data to determine the association between folic acid supplement use and the incidence of ASD in offspring, and classified participants into three groups based on the time of initiation of folic acid supplementation, as follows: (1) preconception users of folic acid supplements and (2) post-conception users, and (3) non-users. The dietary folate intake of study participants was also classified into three groups (&lt;200 µg, 200 µg to &lt;400 µg, ≥400 µg). Overall, 361 offspring of 96,931 participants with single pregnancies were diagnosed with ASD (0.37%). A total of 7,046 participants (7.3%) used folic acid supplements before conception, 29,984 (30.9%) took them after detection of pregnancy, and 59,901 (61.8%) never received them. Multivariate logistic regression analyses demonstrated no association between prenatal folic acid supplementation and ASD in offspring (preconception use: adjusted odds ratio [AOR], 1.189; 95% confidence interval [CI], 0.819–1.727 and post-conception use: AOR, 1.072; 95% CI, 0.840–1.368); additionally, no association was observed with the use of folic acid supplements and/or multivitamin supplements (preconception use: AOR, 1.273; 95% CI, 0.921–1.760 and post-conception use: AOR, 1.132; 95% CI, 0.885–1.449). Moreover, no significant association was observed in participants with combined prenatal supplement use and dietary folate intake. Maternal use of folic acid supplements from the pre- or post-conception period was not significantly associated with ASD in 3-year-old offspring in Japan. Evaluation of the dietary folate intake from preconception also showed no significant association.

Prenatal folic acid supplementation and autism spectrum disorder in 3-year-old offspring: the Japan environment and children’s study