Study design and participants

The Norwegian Mother and Child Cohort Study (MoBa)27 includes 114 500 children born in 1999–2009. ASD cases are identified through the Autism Birth Cohort (ABC) Study,28 a case–cohort study nested within MoBa. Analyses reflect data collected through December 2014. Children from multiple births or with birth weight <2500 grams or gestational age <32 weeks (6.6%, Supplementary Figure S1) were excluded. Given questionnaire design, we focused on subjects with gestational ages ⩾32 weeks to ensure that all women could report fever for at least the first 4 weeks of the third trimester. Low birth weight is associated with a multitude of pre- and postnatal risk factors; thus, we also excluded subjects with low birth weight.29 This exclusion was also motivated by the nearly complete overlap in our study population of preterm and low birth weight status (887 subjects out of 892 with gestational age <32 weeks had low birth weight, or 99.44%).

Studies were approved by the Regional Committee for Medical and Health Research Ethics for Southeastern Norway and the Columbia University Medical Center Institutional Review Board.

Case diagnoses

ASD cases were identified through MoBa screening (3, 5 and 7 years), referrals and annual linkages to the Norwegian Patient Register (NPR).30 Through 2012, potential cases were invited for assessment by research clinicians using standardized diagnostic instruments.31, 32 Best-estimate ASD diagnoses were assigned using Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR)33 criteria. ASD cases among NPR-identified children not evaluated at the ABC Study Clinic were those assigned International Classification of Diseases, Tenth Revision (ICD-10) F84 diagnoses (Supplementary Methods).34 NPR ASD diagnoses have high validity: 58 out of 60 children with NPR ASD diagnoses met DSM-IV-TR ASD criteria at later ABC clinic assessment (positive predictive value: 96.7%, 95% confidence interval (CI), 87.5–99.4%; false positive rate: 3.3%, 95% CI, 0.6–12.5%).30

To explore the possibility that prenatal fever effects may be specific for certain ASD phenotypes, cases were classified as ASD with or ASD without mental retardation/intellectual disability (ID). Comorbidity with ID was based on intelligence quotient (IQ) data from ABC clinic assessments, as available (IQ<70)11 or NPR ICD-10 data consistent with a diagnosis of an ID. ASD cases without an ABC clinic IQ score <70 and who also had not received an NPR ID ICD-10 code were assigned to the ASD without ID group (Supplementary Methods).

Exposure data

Exposure timing

Data on prenatal fever and antipyretic use were obtained from questionnaires completed around gestational weeks 17 and 30, and 6 months postpartum. Mothers reported fever and medication use in 4-week intervals through 13+ and 29+ weeks’ gestation, respectively, on the 17- and 30-week questionnaires and from gestational week 30 through parturition on the 6-month postnatal questionnaire (Supplementary Table S1). An interval was considered positive for fever or antipyretic exposure if one or more exposures occurred during that interval. Trimesters were designated by gestational weeks as: first, 0–12 weeks; second, 13–28 weeks; and third, 29 weeks through birth. Overlap in timing across questionnaires was resolved as described in Supplementary Methods. Main analyses included women with at least one completed questionnaire containing prenatal data.

Fever and antipyretic exposures

Questionnaire items solicited data from mothers regarding the presence of specific conditions, including fever, along with their timing, as well as the names of medications used for fever, and the timing of that medication use. Response options for presence and timing of fever episodes and medication use varied across the three questionnaires (Supplementary Table S1). To enhance cross-questionnaire comparability, we collapsed data for 17-week questionnaire items—fever with rash and fever >38.5 °C—into one category. Questionnaire-specific procedures for counting fever episodes and assigning their timing to specific trimesters are detailed further in Supplementary Methods.

We assessed use of any acetaminophen- or ibuprofen-containing medications for the reported indication of fever. Methods for addressing report of multiple medications and timing of medication use are described in Supplementary Methods, along with secondary analysis procedures for examining risk modification by antipyretic use (acetaminophen or the NSAID, ibuprofen).

Supplementary Table S2 summarizes methods for resolving discrepancies in reported fever timing and in the timing of antipyretic use in relationship to fever episodes, along with the number of affected subjects. Inconsistency in reported fever timing took on three major forms. First, some mothers indicated timing of the medication taken for fever and the name of the antipyretic, but failed to report the specific timing of fever episodes for which they reported taking an antipyretic. In these instances, maternally provided medication timing defined the fever timing. Second, when timing of fever episodes was provided but timing of the selected fever-associated antipyretics (acetaminophen, ibuprofen) was not, maternally provided fever timing defined the timing of medication use. Third, missing fever timings were imputed for the high-exposure subset of mothers who endorsed the 'fever more than three times' item on the 30-week questionnaire but failed to provide complete timing for all reported fever episodes (Supplementary Methods). Supplementary Table S2 shows that the number of affected women, and thus the scope of the problem, was small.

Potential confounders

We evaluated variables that might influence associations between primary maternal exposures (fever in all three trimesters) and ASD risk: maternal age; smoking and parity; parental education; birth month; and birth year (Supplementary Methods and Supplementary Tables S3A and S3B). Potential confounders demonstrating a significant association (P<0.05) with both the primary exposure (fever in any of the three trimesters) and the outcome (ASD) were retained in our analyses.

Statistical analysis

Main analyses

Crude and adjusted odds ratios (ORs and aORs, respectively) of ASD risk in association with maternal fever in each trimester, and for any time during pregnancy, and their associated 95% CIs were estimated by logistic regression. We first examined associations between fever and risk by fitting separate models for each time period, using all subjects responding to at least one questionnaire containing prenatal data. Analyses examining ASD risk in association with fever at any time during pregnancy included subjects responding to all questionnaires.

Subsequent analyses restricted the study sample to mothers who responded to all questionnaires addressing prenatal fever. We used three adjusted models: (1) Adjusted Model 1 (aOR1), adjusting for fever exposure in either/both of the other trimesters; (2) Adjusted Model 2 (aOR2), adjusting for all other selected confounders; and (3) Adjusted Model 3 (aOR3), adjusting for both fever in other trimesters and all confounders. This same population of respondents was used for stratified analyses comparing ASD risk in association with prenatal fever within the subgroup of ASD cases with comorbid ID and within the ASD subset without such comorbidity.

Dose–response analyses

The cumulative effect of maternal fever was estimated by counting numbers of individual time intervals wherein mothers reported fever episodes. We categorized fever exposure into three levels (0 (referent), 1–2, ⩾3 episodes), with exposure periods of any time during pregnancy or >12 weeks of gestation. Using logistic regression, we estimated ORs and aORs for both exposure periods.

Fever-associated risk stratified on use of antipyretics

In exploratory analyses, we examined whether the risk effect of fever exposure was modified by acetaminophen use. Mothers reporting fever in each trimester were divided into two subgroups based on reported use or nonuse of acetaminophen specifically for fever. Crude and adjusted ORs (aOR1, aOR2 and aOR3, as above) of ASD for each subgroup (compared with the no-fever referent) were estimated by logistic regression. We performed similar secondary analyses substituting ibuprofen for acetaminophen to further examine antipyretic modification of fever-associated ASD risk. Although both agents reduce fever, as an NSAID, ibuprofen has anti-inflammatory potential that acetaminophen does not; if, as prior studies have suggested, fever-related activation of maternal immunity mediates neurodevelopmental consequences among offspring, use of ibuprofen as an antipyretic might be anticipated to be associated with greater reduction in fever-associated ASD risk than acetaminophen (Supplementary Methods).

Supplementary and sensitivity analyses

Influences of confounders on ORs of ASD were examined individually and sequentially (Supplementary Methods and Supplementary Tables S4A and S4B).

To address possible influences relating to trends across birth years in ASD prevalence (incomplete ascertainment of cases in later birth years, given follow-up in some subjects only through age 5) and maternal fever report, we performed an analysis of fever effects stratified by early (1999–2004) and late (2005–2009) birth year periods.

We also examined fetal sex effects. Trophoblasts from male placentae are reported to respond to infection-related signals with proinflammatory cytokine production,35, 36 consistent with studies suggesting increased risk of preterm birth in males, particularly in the context of infection.36, 37, 38 Because of low numbers of girls with ASD, we pursued potential influences of sex through sensitivity analyses restricted to boys (Supplementary Methods).

Other sensitivity analyses addressed potential biases relating to gestational age at birth and fever timing imputation. Preterm births (32–36 weeks) might have obscured the ability to assess the impact of fever and antipyretic events in later pregnancy; thus, we restricted to term births (that is, gestational age >36 weeks) in sensitivity analyses. In an additional sensitivity analysis, we excluded mothers who reported high levels of fever exposure on the 30-week questionnaire (>3 fever episodes) without specifying the timing of those episodes (Supplementary Methods).