Maternal smoking status before and during pregnancy and bronchial asthma at 3 years of age: a prospective cohort study from the Japan Environment and Children's Study (JECS)

Maternal smoking exposure during pregnancy is an established risk factor for childhood asthma, but the association between maternal pre-pregnancy smoking status and asthma risk is not well understood. This study examined the association between maternal smoking status before and during pregnancy and bronchial asthma at 3 years of age. The data of 75,411 mother-child pairs, excluding the missing data of exposure and outcomes from the Japan Environment and Children's Study (JECS) were used. The association between prenatal maternal smoking status and the risk of bronchial asthma at 3 years of age was determined using multivariate logistic regression analysis.


Introduction
Asthma is one of the most common illnesses in children. The trend of the prevalence of asthma varies with country and region. The global incidence of asthma is increasing in low-income countries but is at or decreasing in some developed countries [1].
Maternal second-hand smoke (SHS) exposure during pregnancy is also associated with an increased risk of asthma in children [10][11][12]. Regarding postnatal SHS exposure, ve European birth cohort studies from the Mechanisms of the Development of Allergy reported that SHS exposure in infants was not statistically signi cant but was associated with an increased risk of asthma up to 14-16 years of age [13].
There is controversy about the health effects on children from mothers who quit smoking before or early in pregnancy. A large-scale population-based retrospective cohort study using the United States National Vital Statistics System data from 2011-2018 found that mothers quitting smoking during the rst or second trimester of pregnancy are associated with an increased risk of preterm birth [14]. In contrast, it has been reported that maternal smoking cessation before and early in pregnancy does not increase the risk of a small-for-gestational-age infant and childhood overweight at 3 years [15]. Regarding asthma in children, a retrospective hospital-based birth cohort study in Finland found that paternal smoking cessation during pregnancy was associated with a reduced risk of asthma in children, while the effect of maternal smoking cessation during pregnancy and the risk of asthma in children were unclear [16].
Limitations of previous studies include recall bias in retrospective studies, differences in the de nition of smoking environment, and the inability to adjust for essential confounding factors. Therefore, there is a need for veri cation using a large-scale prospective birth cohort. The purpose of this study was to clarify the association between the prenatal maternal smoking status and the risk of bronchial asthma at 3 years of age by adjusting for covariates before and after birth using a large cohort Japan Environment and Children's Study (JECS).

Study design and population
This study used data from JECS, a nationwide birth cohort study. JECS is a project aimed at investigating the effects of environmental factors on child health and development. The detailed protocols have been published elsewhere [17,18]. Fifteen Regional Centres across Japan participate in JECS, and women in early pregnancy and their partners who lived in the area around the Regional Centre were recruited and followed up by the Centre. Participants were recruited between January 2011 and March 2014.
Participating children will be tracked until they reach the age of 13 years. The JECS protocol was reviewed and approved by the Ministry of the Environment's Institutional Review Board on Epidemiological Studies and the Ethics Committees of all participating institutions (Ethical Number: No.100910001). The JECS is conducted in accordance with the Declaration of Helsinki and with written informed consent from all participants.
We used the dataset 'Jecs-ta-20190930-qsn' which was released in October 2019. Of the 100,304 live births, we excluded those with missing data on maternal smoking status before and after pregnancy and bronchial asthma at 3 years of age. Finally, 75,411 mother-child pairs were analysed in the present study ( Figure 1).

Exposure and covariates
Prenatal parental smoking history and exposure to SHS were collected by a self-administered questionnaire during the second or third trimester of pregnancy. A mother was instructed to choose among whether 'Never'; 'Previously did, but quit before recognizing current pregnancy'; 'Previously did, but quit after nding out current pregnancy'; or 'Yes, I still smoke respective to herself and her partner'. For second-hand smoking, a mother answered how often she had a chance to inhale tobacco smoke at any indoor places at mid/late pregnancy by choosing frequency from either almost never or never, once a week, 2-3 times a week, 4-6 times a week, or every day. We have recategorised it into three categories: seldom, 1-3 times a week, and 4-7 times a week.
Regarding the second-hand smoking of children after birth, a mother was asked in the questionnaire at the age of one and a half years. The mothers answered the question of whether the child is exposed to someone's cigarette smoke by choosing the options of rarely, sometimes, and often. We have recategorised into two, with (sometimes and often) or without (rarely) smokers near children.
There are potential confounding factors for the association between prenatal tobacco smoke exposure and asthma [2][3][4]. We adjusted the analyses for child sex, mother's history of bronchial asthma, maternal age at birth, pre-pregnancy body mass index, maternal educational level (≤12 or >12 years), older siblings, gestational age at birth (<37 or ≥37 weeks), and mode of delivery (vaginal or caesarean). Additionally, we adjusted for potential confounding factors after birth with the following: attends a childcare facility at 1 year of age, breastfeeding at 1 year of age, and RS virus infection at 1 year of age.

Outcome de nitions
Information of outcomes was collected using a self-administered questionnaire when the children were 3 years of age. The parents answered the question if your child was diagnosed with bronchial asthma by a doctor after age 2 years. The outcomes of this study were de ned using this questionnaire (yes or no).

Statistical analyses
The association between smoking exposure and bronchial asthma at 3 years of age was subjected to logistic regression analysis to estimate the crude odds ratio (cOR) and adjusted odds ratio (aOR), adding all the covariates and 95% con dence interval (CI). To elucidate the association between prenatal maternal smoking exposure and bronchial asthma at 3 years of age, we extracted a population without second-hand smoking in children after birth (n=56,212). The association between prenatal maternal smoking exposure and bronchial asthma at 3 years of age was examined using a logistic regression analysis adjusted with covariates excluding second-hand smoking of children at the age of one and a half years. To elucidate the involvement of second-hand smoking in children after birth and bronchial asthma at 3 years of age, logistic regression analysis was performed by stratifying prenatal maternal smoking status. All statistical analyses were performed using SPSS version 27. A p-value of 0.05 (twosided) was considered statistically signi cant.

Results
The characteristics of the study participants are shown in Table 1. Among the 75,411 mother-child pairs with complete data, 5430 (7.2%) children had bronchial asthma at 3 years of age. Regarding the maternal smoking status before childbirth, 45,248 (60.0%) were counted as 'Never', 18,160 (24.1%) as 'Previously did, but quit before recognizing current pregnancy', 9,301 (12.3%) as 'Previously did, but quit after nding out current pregnancy', and 2,702 (3.6%) as 'Yes, I still smoke'.  were also signi cantly associated with a higher risk of bronchial asthma at 3 years of age compared to never smoking. Mothers who were frequently exposed to SHS at home, workplace, or any other indoor places during pregnancy were signi cantly associated with a higher risk of bronchial asthma at 3 years age compared to mothers who were seldom exposed to SHS  Adjusted for maternal and partner's smoking status before birth, frequency of second-hand smoke during pregnancy, mother's history of bronchial asthma, maternal age at birth, pre-pregnancy body mass index, maternal educational level, child sex, gestational age at birth, mode of delivery, attendance to childcare facility at 1 year of age, respiratory syncytial virus infection at 1 year of age, breastfeeding at 1 year of age, older siblings.
To clarify the association between prenatal maternal smoking status and bronchial asthma at 3 years of age, we examined children who were not exposed to SHS after birth (n = 56,212) ( Table 3). Maternal smoking during pregnancy was signi cantly associated with a higher risk of bronchial asthma at 3 years of age compared to never smoking (cOR 1.93, 95% CI 1.60-2.32; aOR 1.40, 95% CI 1.12-1.75). Mothers who quit smoking before pregnancy were also signi cantly associated with a higher risk of bronchial asthma at 3 years of age compared to never smoking. Mothers who quit before recognizing current pregnancy (cOR 1.17, 95% CI 1.08-1.26; aOR 1.12, 95% CI 1.03-1.22) or who quit after nding out current pregnancy (cOR 1.25, 95% CI 1.12-1.39; aOR 1.16, 95% CI 1.02-1.30) were also signi cantly associated with a higher risk of bronchial asthma at 3 years of age compared to never smoking. n (%) mean no child is exposed to someone's cigarette smoke in each group at the age of one and a half years.
Adjusted for partner's smoking status before birth, frequency of second-hand smoke during pregnancy, mother's history of bronchial asthma, maternal age at birth, pre-pregnancy body mass index, maternal educational level, child sex, gestational age at birth, mode of delivery, attendance to childcare facility at 1 year of age, respiratory syncytial virus infection at 1 year of age, breastfeeding at 1 year of age, older siblings.
Subsequently, to clarify the effects of postnatal SHS exposure, a logistic regression analysis was performed by stratifying the maternal smoking status before childbirth (Table 4). In a group of mothers who had never smoked before childbirth, children exposed to SHS after birth were signi cantly associated with a higher risk of bronchial asthma at 3 years of age compared to unexposed children (aOR 1.23, 95% CI 1.11-1.37). In contrast, there was no signi cant difference in the risk of bronchial asthma at 3 years of age among children exposed to SHS after birth and unexposed children in any group of mothers who smoked before childbirth. Adjusted for partner's smoking status before birth, frequency of second-hand smoke during pregnancy, mother's history of bronchial asthma, maternal age at birth, pre-pregnancy body mass index, maternal educational level, child sex, gestational age at birth, mode of delivery, attendance to childcare facility at 1 year of age, respiratory syncytial virus infection at 1 year of age, breastfeeding at 1 year of age, older sibling.

Discussion
In a large birth cohort study, we found strong evidence that maternal smoking during pregnancy was associated with an increased risk of bronchial asthma at 3 years of age even when adjusted for postnatal covariates such as attendance to nursery school, breastfeeding, and RS virus infection. Additionally, we found that mothers who had smoked in early pregnancy and before pregnancy were also signi cantly associated with an increased risk of bronchial asthma at 3 years of age.
Several cohort studies have previously reported that maternal smoking during pregnancy increases the risk of asthma in children. In a meta-analysis of eight European birth cohorts [8], maternal smoking during pregnancy is associated with asthma at preschool age, with an adjusted odds ratio of 1.65 (95% CI 1.18-2.31). In a large-scale cohort study (n=60,254) in Finland [19], both maternal light (<10 cigarettes per day) and heavy (>10 cigarettes per day) smoking during pregnancy had signi cantly increased risk of asthma by 7 years of age with odds ratios of 1.23 (95% CI 1.07-1.42) and 1.35 (95% CI 1.13-1.62), respectively, compared to never smoking. Our large birth cohort study found that the risk of bronchial asthma at 3 years of age increases not only with the number of cigarettes smoked per day, but also with the duration of smoking ( Figure S1). The association between pre-and postnatal SHS exposure and the development of asthma in children is controversial [10,13,20]. Our results found that mothers who were frequently exposed to SHS at home, workplace, or any other indoor places during pregnancy were associated with an increased risk of bronchial asthma in 3-year-old children. Additionally, postnatal SHS exposure signi cantly increased the risk of bronchial asthma at 3 years of age even after prenatal smoking exposure and adjusting for other covariates.
We found that prenatal smoking exposure was associated with an increased risk of bronchial asthma at 3 years of age, even without the effects of postnatal smoking exposure. The concept of Developmental Origins of Health and Disease (DOHaD) is considered a molecular mechanism that links prenatal smoking exposure and bronchial asthma. DOHaD evaluates how the early life environment can impact the risk of non-communicable diseases from childhood to adulthood [21]. Epigenetic modi cations such as DNA methylation are considered the molecular mechanism of DOHaD [22][23][24]. Maternal smoking during pregnancy has been shown to alter umbilical cord blood DNA methylation in various genes such as aryl-hydrocarbon receptor repressor (AHRR), cytochrome P450 family 1 subfamily A member 1 (CYP1A1), growth factor-independent 1 transcriptional repressor (GFI1), and myosin IG (MYO1G) [25,26].
It has been suggested that this DNA methylation change is maintained throughout the life course [27,28]. Neophytou et al. showed that it suggests a potential mediation of AHRR methylation in the association between maternal smoking during pregnancy and asthma in Latino children [29]. Gao also showed a synergistic effect of prenatal maternal smoking and AXL receptor tyrosine kinase (AXL) methylation on the risk of childhood bronchitis symptoms [30]. Thus, although methylation of several genes has been reported to be associated with maternal smoking exposure and the risk of childhood asthma, it is necessary to elucidate the molecular mechanisms underlying the development of asthma in the future.
The present study is the rst report that mothers who quit before recognizing current pregnancy or quit after nding out about current pregnancy also had a signi cantly increased risk of bronchial asthma at 3 years of age even without the effects of SHS exposure. It has been suggested that pre-conception exposure to tobacco, chemicals, and stress can cause epigenomic changes in germ cells, adversely affecting the health of the next generation [31]. Pre-pregnancy female mouse exposure to cyclophosphamide, a widely used drug in the treatment of breast cancer, has been shown to alter DNA methylation in F1 and F2 mouse oocytes [32]. Wu et al. recently reported that prenatal paternal smoking exposure increased DNA methylation of immune-related genes, such as LMO2 and IL-10 and correlated with the development of asthma in children [33]. Furthermore, a three-generation cohort study suggests that maternal smoking before conception is associated with an increased risk of childhood asthma [34,35]. Therefore, it is suggested that epigenetic changes due to the parental smoking history may be transmitted on to the next generation through germ lines even if they are not smoking during pregnancy. It is important for mothers to quit smoking early before pregnancy, as we have shown that the longer the mother quits smoking, the lower the risk of bronchial asthma at 3 years of age.
This study has several limitations. First, the smoking status of parents was self-reported and may be misclassi ed. Previous reports using the same dataset reported that 78% of passive smokers might be misclassi ed as non-smokers [36]. However, we analysed based on previously reported cut-offs based on urinary cotinine concentration and found no signi cant association between SHS and increased risk of bronchial asthma at 3 years of age (Table S1). Second, the effects of SHS on children after birth are conducted using a one-and-a-half-year-old questionnaire. Passive smoking exposure of postnatal children was investigated using a questionnaire at the age of one and a half years. It is unknown how long a child was exposed to SHS from birth to age 3 years. Finally, we have adjusted for the essential confounding factors identi ed in previous studies, but observational studies cannot exclude the possibility of residual confounding.

Conclusions
Our results strengthened the existing evidence that maternal smoking during pregnancy increases the risk of childhood bronchial asthma in a large birth cohort study, further identifying new features in this association. We have suggested that the effects of maternal smoking even before pregnancy may increase the risk of asthma in children. Therefore, it is important for both parents to quit smoking early to reduce the risk of future health hazards to their children.  Flow chart of the study participants.

Supplementary Files
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