In this study, we examined the association between acetaminophen exposure during pregnancy and the behavioral outcomes of offspring in a Brazilian birth cohort. A differential effect for sex and exposure to acetaminophen was observed. In 6-year-old boys, intrauterine exposure to acetaminophen led to increased odds of having emotional and hyperactivity/inattention problems. At the age of 11, a slight reduction in the effect size was observed. For girls, the results were nonsignificant at both follow-up times for all the considered outcomes. These findings suggest that acetaminophen exposure in the uterus may play a role in behavioral disorders in childhood, mainly in boys.

Acetaminophen is one of the most prescribed and used medications for pregnant women in Brazil [32, 33]. Comparing our results with those of previous studies, an important difference is in exposure prevalence. Although over 50% of pregnant women report using acetaminophen in the United States and Denmark [8, 34,35,36], we found that 27.5% of Brazilian mothers used this substance during pregnancy. We could not find Brazilian population-based studies reporting acetaminophen use during pregnancy.

Evidence from different prospective cohort studies has shown an association between acetaminophen use during pregnancy and neurobehavioral outcomes in childhood [6,7,8,9,10,11,12]. Three studies evaluated behavioral symptoms using the SDQ. In 7-year-old children, Liew et al. [8] observed an association between prenatal exposure to acetaminophen and higher total difficulties, conduct problems, and hyperactivity. The authors also observed increased risk when the medication was used over more than one trimester, especially near the end of the pregnancy. In addition, they found a correlation between increased frequency of use and increased risk. Stergiakouli et al. [11] demonstrated that children exposed to acetaminophen during the second and third trimesters were at higher risk of multiple behavioral difficulties, including hyperactivity and conduct problems at 7 years of age. Prenatal exposure to acetaminophen during a 32-week pregnancy was also associated with emotional symptoms. Using parent-reported SDQ scores, Thompson et al. [7] observed that acetaminophen was a risk factor for total difficulties, emotional symptoms, and conduct problems at 7 years. At age 11, however, only an association with the parent-reported emotional scale was observed.

Animal studies have supported epidemiological observational studies, providing biological plausibility for the findings. Acetaminophen is known to freely cross the placenta [37]. Recently, exposure to acetaminophen during intrauterine life was reported to affect the modulation of neurotransmission in rats [17]. Effects on neurodevelopment and behavior have also been observed in adult rats exposed to acetaminophen during intrauterine life [15, 16]. Defects in cognitive function and deficient levels of brain-derived neurotrophic factor (BDNF) were observed in rats that were administered acetaminophen during neonatal life [38]. Interference in the regulation of cellular oxidative stress and the endocrine and immune systems by acetaminophen has also been suggested as a mechanism that could lead to neurodevelopment disorders [18,19,20,21,22, 39].

Sex-related differences in symptomatology have been reported for both ADHD and emotional problems. ADHD is more commonly diagnosed in boys than in girls [40], and girls with ADHD are more likely than boys to present inattentive ADHD and separation anxiety disorder. Boys present higher rates of hyperactivity and comorbidity for disorders such as oppositional defiant disorder and conduct disorder [40, 41]. Emotional problems in early adolescence, such as anxiety and depression, are more frequent in girls than in boys [42,43,44,45]. In this study, we tested whether sex could modify the effect of acetaminophen on behavioral problems. An interaction between acetaminophen exposure and sex was observed for the hyperactivity and emotional scales, suggesting that acetaminophen may have different effects on these problems in boys and girls. Stratified analyses suggested an effect of acetaminophen on the hyperactivity and emotional scales only in boys.

A similar effect was reported for ASD symptoms in a Spanish cohort [10]. The findings in Spain revealed that exposed boys had more ASD symptoms compared to exposed girls, who showed decreased scores. Differential acetaminophen metabolization between males and females has already been reported and may play a role in this difference, as suggested by Avella-Garcia et al. [10]. Studies on animals have demonstrated that male mice show higher toxicity levels than females after acetaminophen administration [46]. Other sex differences rely on a putatively increased vulnerability to stressors [47] and to the effect of acetaminophen on testosterone regulation [39, 48], neurodevelopment, and brain masculinization [15] in male brains at the beginning of life. Thus, the hypothesis that endocrine disruption influences testicular function and the production of androgens, which further affects brain development in males, could be a plausible mechanism for the present findings.

The results presented in this study suggest that the magnitude of the effect on emotional and hyperactivity/inattention problems slightly decreased from age 6 to 11. Our sensitivity analysis using EPDS showed that the absence of an association in the adjusted analysis at the age of 11 may be due to a lack of power in the analyses for emotional problems. Our results at different ages are in line with the results reported by Thompson et al. [7], which showed differences between both ages for the parent-reported symptoms. Studies evaluating other age groups, including adolescence, are necessary to better understand the relationship between the exposure and the evaluated outcomes.

The results should be interpreted considering certain limitations. The first limitation relates to the retrospective gathering of data on medication use during pregnancy, which may have resulted in difficulty recalling acetaminophen use. Thus, the low prevalence of acetaminophen use observed in this study may be a result of underreporting. We compared our data with that collected in the same population, in 2015, by another birth cohort study (2015 Pelotas Birth Cohort). In this birth cohort, acetaminophen use information was assessed prospectively during pregnancy, as well as in the perinatal interview (retrospectively). The prevalence for use in the perinatal interview was 51% (unpublished data), which agrees with most of the data from published studies [8, 32,33,34]. Considering that underreporting use of acetaminophen may be a limitation for the exposure definition in our study, we expect that effect size observed in the association analyses is underestimated. Therefore, false negative results are more likely to be observed than false positives regarding this limitation. Second, both the dose-response and timing of exposure associations (higher risks for the third trimester of pregnancy exposure) had been reported previously [8,9,10,11, 13]. However, because of possible memory issues, our data did not allow for the evaluation of exposure according to the trimester of pregnancy.

Furthermore, we cannot rule out the effect of using other medications during pregnancy on behavioral outcomes. However, other types of nonsteroidal analgesics were not associated with worse outcomes. Nonsteroidal analgesic use was also included as a covariable in the models. Thus, it is unlikely that co-medication with other analgesics influenced the findings. These results are consistent with the specificity of acetaminophen observed by Thompson et al. [7] and with the results from previous studies that used the same adjustment approach with other analgesics [8]. It has also been shown that children whose mothers have externalizing disorders (e.g., conduct disorder or drug/alcohol dependence) are at an increased risk for developing behavior problems [49]. Although we do not have information regarding externalizing problems in mothers, we included smoking and drinking behavior during pregnancy, which could be considered as proxies. Other psychological variables from mothers would also improve the robustness of our findings. Fever and inflammation during pregnancy are known risk factors for the development of neurological diseases [50]. Werenberg-Dreier et al. [51] showed that exposure to fever and infection over specific timeframes during pregnancy was associated with the occurrence of ADHD. Although the women in the present study reported using the medication, their reasons for doing so were not available. This information would also be important to improve the robustness of our findings. However, this information was not collected in 2004.

Another critical limitation, which has been greatly discussed in the literature, was the presence of residual confounding due to socioeconomic variables. Additional file 1: Table S1 shows socioeconomic variables, including educational level, economic index, skin color, and parity, which are variables that behave as proxies for socioeconomic position (SEP) in Brazil. These variables were all associated with the use of acetaminophen during pregnancy and with most of the outcomes at different ages, especially hyperactivity/inattention problems. While other variables were not statistically associated with our main exposure, we included some of them in the adjusted models given recent evidence suggesting them as possible confounders of this association. However, these variables might not be as important as SEP variables in our models. It is unlikely that residual confounding due to a lack of control over other SEP measurements, such as income and work, may have biased our results. Models were adjusted for variables associated with exposure. Further, the results considered most of the effects of SEP. In addition, crude and adjusted measures of the association between acetaminophen exposure and emotional problems at 11 years of age are very similar, reinforcing the idea that the association was, probably, not confounded by these variables.