BMJ Open. Maternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the 7 year-old children

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1 BMJ Open Maternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the year-old children Journal: BMJ Open Manuscript ID: bmjopen Article Type: Research Date Submitted by the Author: -Nov-0 Complete List of Authors: Christensen, Berit; Aarhus University, Department of Public Health; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre Thulstrup, Ane Marie; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre Hougaard, Karin; The National Research Center for the Working Environment, Skadhauge, Lars; Hospital of South-West Jutland, Department of Occupational Medicine Frydenberg, Morten; Aarhus University, Department of Public Health Hansen, Kirsten; University Hospital Herlev, Department of Paediatrics Schlünssen, Vivi; Aarhus University, Department of Public Health; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre <b>primary Subject Heading</b>: Secondary Subject Heading: Keywords: Occupational and environmental medicine Epidemiology, Occupational and environmental medicine, Reproductive medicine, Respiratory medicine OCCUPATIONAL & INDUSTRIAL MEDICINE, EPIDEMIOLOGY, Community child health < PAEDIATRICS, PREVENTIVE MEDICINE, Allergy < THORACIC MEDICINE, Asthma < THORACIC MEDICINE - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

2 Page of 0 BMJ Open Maternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the year-old children Berit Hvass Christensen MD, ; Ane Marie Thulstrup PhD ; Karin Sørig Hougaard PhD ; Lars R. Skadhauge PhD ; Kirsten Skamstrup Hansen PhD ; Morten FrydenbergPhD ; Vivi Schlünssen PhD,. Department of Public Health, Section of Environment, Occupation and Health, Danish Ramazzini Centre, Aarhus University, Denmark. Department of Occupational Medicine, Danish Ramazzini Centre, Aarhus University Hospital, Denmark. The National Research Center for the Working Environment, Copenhagen, Denmark. Department of Occupational Medicine, Hospital of South-West Jutland, Denmark. Department of Paediatrics, University Hospital Herlev, Denmark. Department of Public Health, Section of Biostatistics, Aarhus University, Denmark. Corresponding author: Vivi Schlünssen Department of Public Health, Section of Environment, Occupation and Health, Danish Ramazzini Centre, Aarhus University, Bartholins Allé, 000 Aarhus C, Denmark. vs@mil.au.dk Phone + Running head: prenatal occupational exposure and asthma Word count:, (text only). Abstract words. Four tables, one figure, online supplement tables. references - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

3 BMJ Open Page of ABSTRACT: Objectives The occurrence of allergic diseases has increased considerably in the last decades. In this prospective follow-up study, we examined whether maternal exposure to asthmogens during pregnancy is associated with the development of asthma in year-old Danish children, taking atopic status into consideration. Methods A total of, women and their children from The Danish National Birth Cohort were categorised according to maternal occupational exposure. Exposure information was obtained by combining job title in pregnancy and months after pregnancy with a commonly used asthma Job Exposure Matrix. Information on asthma in the child was obtained by an internetquestionnaire when the child was years old. Results Prenatal exposure to Low Molecular Weight (LMW) agents was associated to asthma in the children with OR. (0.;.) for atopic and. (0.;.) for non-atopic children. Also maternal postnatal exposure was associated with asthma (OR. (.0;.), but after mutual adjustment only postnatal exposure (OR. (0.;.) and the combined effect of pre- and postnatal exposure (OR, (.;.)) increased the risk of asthma in the children. No significant associations were observed for other prenatal or postnatal exposures. Conclusion Maternal occupational exposures during pregnancy do not seem to be a substantial risk factor for the development of asthma in year old children. Maternal pre- and postnatal exposure to LMW agents may predispose the propensity of the children to develop asthma. Future studies should prioritise the characterisation of the timing exposure in relation to the birth. Keywords Prenatal exposure; postnatal exposure delayed effects; Asthma; Prospective cohort study; Allergy; Maternal exposure; Occupational exposure; pregnancy; epidemiology - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

4 Page of 0 BMJ Open ARTICLE SUMMARY Article focus To study the impact of maternal occupational exposure during pregnancy on the occurrence of asthma among their year-old children Key messages Mothers occupational exposures during pregnancy is not a substantial risk factor for asthma in their year old children Maternal pre- and postnatal exposure to LMW agents may increase the risk for asthma in the year old children Future studies should focus on timing of exposure in relation to the birth Strengths and limitations of this study: This study is the largest prospective study so far addressing the impact on childhood asthma of mothers occupational exposure during pregnancy. It is also the first study where atopic status of the child is taken into account when exploring the importance of prenatal occupational exposures on childhood allergic diseases. A substantial loss to follow-up was seen. We presume that the loss to follow-up did not influence our results to a major degree. The occupational exposure assessment was based on crude information on job titles and a general asthma JEM. Definitions of asthma and atopy were based on self-reported informations from the mothers. Still we were able confirm previously described associations between childhood asthma and several risk factors. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

5 BMJ Open Page of BACKGROUND The occurrence of asthma, rhinitis, and atopic dermatitis has increased in the last decades, from a few per cent in the 0s to -0% in the 0s[, ]. Lifestyle seems to influence the development of allergic diseases, also during childhood[]. Prenatal risk factors for asthma have been investigated and include environmental tobacco smoking[], stress[] and maternal diet during pregnancy[, ]. Thus, the maternal environment during pregnancy might encompass risk factors for asthma in the child. Allergic diseases are hallmarked by childhood immune dysfunction, but surprisingly, little attention has been paid to prenatal influences on immune development, although the foetal immune system is vulnerable to programming[]. In animals, airborne exposure to particles and aerosols of the mother during pregnancy have been shown to increase asthma susceptibility in the offspring[, ], and findings in some human studies corroborate this association[, ]. Maternal exposure to volatile organic compounds in pregnancy may skew the Th-/Th immune response in the foetus, thereby potentially predisposing the children to asthma later in life[]. An increasing number of women work outside the home during pregnancy, and many are occupationally exposed to asthmogens, i.e. chemical and biological agents that are recognised to increase the risk for work-related asthma. It therefore seems pertinent to investigate whether women s working environments during pregnancy interfere with their children s propensity to develop asthma later in life. Two earlier cohort studies have investigated prenatal occupational exposures and the risk of asthma among adolescent[] and 0- year-old children[] with inconclusive results. Asthma can be characterised as allergic (atopic) or non-allergic (non-atopic). These two phenotypes are characterised by e.g. different risk factors. Endotoxin has for example opposite - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

6 Page of 0 BMJ Open effects in atopic and non-atopic asthma[]. In the present study, we hypothesise that maternal exposure to asthmogens during pregnancy impacts on the foetal development of the immune system and predisposes the children to develop asthma later in life. Specifically, we analysed whether maternal occupational exposures during pregnancy are associated with the development of asthma in year-old Danish children, taking atopic status into consideration. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

7 BMJ Open Page of MATERIAL AND METHODS Population The study population was part of the prospective cohort: The Danish National Birth Cohort (DNBC, Better health for mother and child ). The cohort was established in -00 to study pregnancy outcome and disease in children as a function of factors operating in early life[] ). Women were recruited at their initial pregnancy consultation with their general practitioner or midwife and participation implied four telephone interviews; during week to and week 0 to of pregnancy, and when the child was and months old. When the child was seven years of age, the mothers answered an internet questionnaire on behalf of their children. Mothers without internet access were offered a paper version. The first telephone interview comprised questions about work, atopic diseases and risk factors for asthma (e.g. smoking, use of medication, parity). The questionnaire at age dealt with asthma, rhinitis and atopic dermatitis in the child and risk factors (e.g. parental smoking). A total of 0, pregnancies were enrolled in the cohort. The first telephone interview was answered, times. A total of, women were no longer pregnant at the time of the interview and were excluded. Only singletons and only one pregnancy per woman (the first in the sampling period) were included, so,0 siblings and, children from multiple births were excluded. This leaves a total of. mother/child pairs, of which the internet questionnaire at age was answered for, children. Among these,, pairs were excluded due to lack of information on maternal occupational status. Thus, mother/child pairs were eligible for analysis. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

8 Page of 0 BMJ Open Occupational exposure Data on job exposure during pregnancy was obtained by combining reported information on jobtitle (DISCO codes) from telephone interview one during pregnancy and a commonly used asthma Job Exposure Matrix (JEM)[] based on known risk factors for occupational asthma. The job titles were coded according to the Danish International Standard classification of Occupations, the Danish edition of ISCO- (DISCO-code). Seven exposure categories were formed and consisted of the following occupations and typical allergens/irritants:. High molecular weight (HMW) agents (veterinarians, gardeners, and bakers; grass pollen, animal dander, flour/grain). Low molecular weight (LMW) agents or irritants (cooks, cleaners, hairdressers, dentistry and all kinds of manufacturing of dust-producing materials; aerosols, dust from manufacturing of wood/ textile/ stone/ rubber/ plastics etc.). Mixed HMW and LMW agents (health care professionals; antibiotics, latex, cleaning agents). Farmers (organic dust, grass pollen, animal dander, ammonia). Students (no or unknown exposure). Unclassifiable (subjects where the same job title involved several different environments, e.g. waiters, engineering technicians, biologists, shop managers; diverse exposure). Reference (office workers, teachers and journalists; no or low exposure) The applied JEM was slightly modified from[] by Christensen BH and Schlünssen V to comply with working conditions in Denmark based on a priori knowledge of exposures to asthmogens in the Danish working environment. As an example, medical doctors were originally categorised as not being exposed to asthmogens, but were reclassified to the mixed group in the modified version of the JEM. Furthermore, farmers were identified as a special group as early life exposure - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

9 BMJ Open Page of to a farming environment has shown to offer protection against allergic diseases[]. Also, students were considered as a special group due to possible selection related to health. Postnatal occupational exposure groups were established using the -digit DISCO codes months after the birth of the child, i.e. at a time when maternity leave was terminated and the mother would most probably have returned to work. The codes were retrieved from Statistics Denmark, as every adult person ( years) in Denmark is classified annually according to occupation. Maternal postnatal occupation was categorised in the same JEM categories as for the prenatal exposures. Women classified as students during pregnancy, but without a DISCO code indicative of employment after having given birth were not included in the postnatal analyses. Level of education and socioeconomic status (self-employed, employees at the highest level, employees, students, unemployed or not in the work force) were obtained for both the father and the mother (the calendar year preceding the birth year of the child) from Statistics Denmark. Information on the parent with the highest level of education/socioeconomic status defined the level for the family. Outcome We used validated core questions on asthma from the International Study of Asthma and Allergies in Childhood (ISAAC)[0]. Asthma in the child at age was defined as an affirmative answer to one or more of three questions: Has your child had wheezing or whistling in the chest in the last months? ; Has your child ever had asthma? or to Has your child ever been diagnosed with asthma by a doctor? - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

10 Page of 0 BMJ Open Ever atopic dermatitis was used as a proxy for atopy among the children. Information on atopic dermatitis was collected by telephone interview when the child was months old and by questionnaire at age. It was defined as parental report at months of atopic dermatitis ever AND itchy rash in the locations known to be typical for atopic dermatitis AND/OR report of persistent itchy rash in the locations known to be typical for atopic dermatitis at age. Confounders Information on known possible confounders according to prior knowledge from other studies on the effects of asthma in children was obtained primarily from the telephone interview at - weeks of gestation: maternal age, pre-pregnancy BMI, parity, smoking during pregnancy, and furry animals in the home. Maternal atopic disposition was defined as ever reported asthma, rhinitis, and/or atopic dermatitis. Information on maternal use of acetyl salicylic acid, paracetamol, folic acid and antibiotics during pregnancy was retrieved from the second telephone interview at 0- weeks of gestation. Birth weight, gestational age at birth, sex, and singleton status were obtained from the Danish Birth Registry. Statistical methods All analyses were performed in STATA SE.0 (STATA corp., Texas, US). Univariate analyses were performed for categorical variables using χ tests. For comparisons of continuous variables, the independent sample t-test or Kruskal-Wallis test was used. A multivariate unconditional logistic regression analysis was used to evaluate associations between exposure and outcome. A univariate analysis revealed an equal distribution between the exposure groups for some of the - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

11 BMJ Open Page of potential confounders, which was subsequently excluded from the model. The final model contained the following maternal variables: age (-; -; 0-; + years), pre pregnancy BMI ( ; 0-; -; 0+), atopy (yes/no), smoking during pregnancy (no; -; + cig/day), use of medication during pregnancy (yes/no to the use of acetyl salicylic acid, paracetamol, folic acid, and antibiotics), parity (0;-;+) and furry animal ownership during pregnancy (yes/no). For the children, SGA (small for gestational age) (yes/no) and sex were included. SGA was defined as the smallest % of the children born in a specific gestational week based on birth weights from the entire BSMB cohort, and separately for boys and girls. The analysis was repeated after including the children s exposure to environmental tobacco smoke (ETS) in the model according to the information on the parents current smoking. Unless otherwise stated, the level of significance was p < 0.0, two-sided. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

12 Page of 0 BMJ Open RESULTS Table shows the demographic characteristics of the cohort stratified by exposure groups. The women had a mean age of 0 years at inclusion. Students were younger than the other groups, i.e.. % were below 0 years of age. About half (.%) of the women had not given birth previously. Among the students,. % were primiparous. On average,. % of the women had used paracetamol, mostly prevalent in the mixed exposure group (.%), and least prevalent in the farmer group (.%). The majority (.%) of the women were non-smokers during pregnancy. The LMW group had the highest tobacco use with. % smoking + cig/day. A similar pattern was seen at follow up at years of age, but in general fewer mothers smoked at follow up, with a decrease from one in four to one in six. The lowest prevalence of atopy (.%) was seen in the HMW group, whereas the prevalence was highest among students (.%). - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

13 BMJ Open Page of Table Demographics, categorised according to mothers exposure during pregnancy into categories: High molecular weight (HMW), Low molecular weight/irritants (LMW), Mixed (a mixture of HMW and LMW), farmers, unclassifiable, students and references. The numbers are presented as % unless otherwise stated. During pregnancy: Mother: HMW LMW Mixed Farmer Unclassifiable Student Reference Missing, % n = 0 n =,0 n =, n = 0 n =,0 n =, n =, Age, yrs, median (%;0%) (;) (;) 0 (;) 0 (;) (;) (;) 0 (;) 0 BMI, median (%;0%).0 (.;.). (.;0.). (.;.0). (.;.). (.;.).0 (.;.). (.;.). Parity: or Use of medicine: ASA Paracetamol Antibiotics Folic acid Rural residence Furry animals a Smoking: 0.0 no use of tobacco cigarettes/day > 0.0 cigarettes/day Atopy b.,., Child: Boy Birth weight, kg, median (%;0%).0 (.0;.0). (.;.).0 (.;.). (.;.).0 (.;.). (.;.).0 (.;.) Small for gestational age on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

14 Page of 0 BMJ Open At year followup: Maternal smoking Paternal smoking Child atopy d Child ETS exposure on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

15 BMJ Open Page of Table E (online supplement) presents the characteristics among participants and nonparticipants. The distributions of exposure groups were quite similar between participants and non-participants, but with a slightly lower number of LMW-exposed and students among the participating mothers compared to the non-participating mothers. More of the participating mothers were non-smokers during pregnancy, vs. %, they were slightly older (0 vs. years), better educated (academic level, vs. %), belonged to a higher socioeconomic group (employees at the highest level, vs. %), and more were expecting their first child compared to the non-participants, vs. %. Maternal atopic status was similar in the two groups. At birth, children of participating mothers weighed on average 0 g more and the number of SGA children was.0% in participants compared to.% among non-participants. Table : Unadjusted prevalence of asthma stratified for atopy (defined as ever atopic dermatitis) in the year old children (n =,). Exposure group All children n (%) Atopic children n (%*) Non-atopic children n (%*) High molecular (.) (.) (.) Low molecular/ irritants (.) a (.) b (.) c Mixed, (.) (.) (.) Farmer (.0) (.) (.) Unclassifiable 0 (.0) (.) (.) Student (.) (.0) (.) Reference, (.) (.), (.) Total n (%),0 (.), (.) d, (.) *The percentage is the number of atopic (or non-atopic) children within the maternal exposure group. a: P<0.0, group vs. mixed, farmer, unclassifiable, student and reference. b: P<0.0, group vs. mixed, farmer, unclassifiable and reference c: P<0.0, group vs. reference. d: P<0.0 group vs. non-atopic children - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

16 Page of 0 BMJ Open Table shows the prevalence of asthma stratified for atopic status. The overall prevalence of asthma was. %;. % for atopic and. % for non-atopic children. Children from the LMW exposure group had the highest prevalence of asthma, both in total (.%), and for the atopic (.%) and the non-atopic group (.%). The HMW children had similar prevalences of asthma as the LMW children. Farmers children had the lowest prevalence of asthma, although not significantly different from the other exposure groups. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

17 BMJ Open Page of Table : Unadjusted and adjusted logistic regression models showing the association between mothers occupational exposure during pregnancy and asthma in the child at age years stratified by atopic status of the child. Crude OR (% CI) Model Model Outcome Atopic children, Non-atopic children, Atopic children, Non-atopic children, Atopic children, Non-atopic children, n =, n=, n =,0 n =, n =, n = 0, Asthma High molecular. (0.;,). (0.;.). (0.;.).0 (0.;.). (0.;.).0 (0.;.) Low molecular/ irritants. (.;.). (.;.). (0.;.). (0.;.). (0.;.).0 (0.;.) Mixed.0 (0.;.).0 (0.;.) 0. (0.;.).00 (0.;.0) 0. (0.;.) 0. (0.;.0) Farmer. (0.;.) 0. (0.;.). (0.;.) 0. (0.0;.) 0. (0.;.) 0. (0.;.0) Unclassifiable 0. (0.;.) 0. (0.;.).0 (0.;.).0 (0.;.).0 (0.;.).00 (0.;.) Student.0 (0.;.).0 (0.;.) 0. (0.;.) 0. (0.;.0) 0. (0.;.0) 0. (0.0;.0) Reference Model includes: the following variables for the mothers: age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy (dichotomous variables for the use of Acetyl salicylic acid, paracetamol, Folic acid and Antibiotics), parity and furry animal ownership during pregnancy. The following variables were included for the children: Birth weight (SGA) and sex of the child. Model includes the adjustments in model and level of education and socioeconomic level. on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

18 Page of 0 BMJ Open Table shows crude and adjusted ORs for the associations between mothers occupational exposure during pregnancy and asthma in the child. In model, levels of education and socioeconomic status were added to the a priori chosen confounders. For HMW agents, ORs were non-significantly increased in the range.-. in both the crude and the adjusted analyses, especially for atopic children. Children of mothers exposed to LMW agents during pregnancy had an increased OR for asthma in the crude analysis for both atopic (OR. (% C.I..;.)) and non-atopic children (OR. (.;.). The same tendency was seen in model with OR. (0.;.) for atopic and. (0.;.) for non-atopic children. The ORs were further decreased in model with OR. (0.;.) for atopic and.0 (0.;.) for non-atopic children. For the other exposure groups, no significant association between maternal exposure and asthma in the children was observed. In the crude analysis a borderline significant association between being student/mixed groups and asthma were seen with OR.0-.0, but without confirmation in model or. The analysis in Table was repeated taking the year-old children s current parental ETS into account. This did not change the results (data not shown). The same was true when paternal atopic status was included. Even though educational level and socioeconomic status (SES) were considered as potential confounders, they were correlated to our exposure variable, partly because the DISCO code was included in the definition of SES. We decided therefore to use model in the ensuing analysis. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

19 BMJ Open Page of We did not find any significant interactions between exposure and atopy when the interaction exposure*atopy was included in the model. In Table E (online supplement), ORs for covariates in model are displayed with asthma as the outcome of interest. The mother s age was inversely associated to asthma in the child. The children of women < years of age had an increased OR for asthma,.0 (.;.), compared to - year-old women (reference). The risk was further decreased for 0- years, OR 0. (0.;0.) and + year old women, OR 0. (0.0;0.). Maternal prepregnancy BMI was positively associated with asthma with ORs,. (.0;.) and. (.;.) for BMI - and above 0, respectively, compared to BMI 0- (reference). Children with maternal atopic disposition had increased OR for asthma (.(.;.0)). The use of paracetamol and antibiotics during pregnancy was associated with asthma in the children, OR.0 (.0;.) and. (.0;.), respectively. Smoking during pregnancy was positively associated with asthma in a dose-dependent manner with OR.0 (.;.) for - cig/day), and OR. (.0;.) for > cig/day compared to non-smokers (reference). Parity was positively associated with asthma, with OR. (.0;.) for - pregnancies, and OR. (0.;.) for or more pregnancies compared to no earlier pregnancy (reference), whereas pet ownership was not associated to childhood asthma, OR 0. (0.;.0). Being a boy, and being born SGA was both positively associated with asthma with ORs. (.;.) and. (.0;.), respectively. The analysis in table E was repeated after stratification for atopic status in the children, and almost identical results were seen for atopic and non-atopic children (data not shown). - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

20 Page of 0 BMJ Open Figure shows the adjusted logistic regression analysis on the association between mothers pre- and postnatal exposure and asthma in the children. For LMW agents, both pre- and postnatal exposure were significantly associated with asthma in the children, ORs. (.00;.) and. (.0-.), respectively. After mutual adjustments only postnatal exposure (OR. (0.;.)) and the combined effect of pre- and postnatal exposure (OR, (.;.)) were associated with asthma in the child. For the other exposure groups, no significant associations were seen for either prenatal or postnatal exposures. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

21 BMJ Open Page 0 of Figure. Adjusted logistic regression analysis on the association between mothers pre- and postnatal exposure and asthma in the year-old children. Reference: reference workers. OR 0.. HMW LMW Mix Farm UC Pre: Prenatal exposure. Post: Postnatal exposure. Post just pre: postnatal exposure adjusted for prenatal exposure. Pre just post: Prenatal exposure adjusted for postnatal exposure. Combined: Additive effect of pre-and postnatal exposure. pre post pre just post post just pre common Included in the model: maternal age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy, furry animal ownership during pregnancy, SGA and sex of the child BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

22 Page of 0 BMJ Open Table : Crude and adjusted logistic regression analyses on associations between mothers LMW jobs and asthma in the year old children. Crude Total Adjusted Total Crude Atopic Adjusted Atopic Crude Non-atopic Adjusted Non-atopic (n=,) (n=,0) Children (n=,) Children (n=,0) Children (n=,) Children (n=0,) LMW OR % CI OR % CI OR % CI OR % CI OR % CI OR % CI Plastic (0).0 (0.;.). (0.;.). (0.;.). (0.;.).00 (0.;.) 0. (0.;.) Paper () 0. (0.;.) 0. (0.;.) 0. (0.;.0) 0. (0.;.) 0. (0.;.) 0.0 (0.;.) Stone (). (0.;.) 0. (0.0;.). (0.;.).0 (0.;.) 0. (0.;.) 0. (0.;.) Wood () 0. (0.;.) 0. (0.;.) 0. (0.;.) 0. (0.;.). (0.;.).0 (0.;.) Food (0). (.;.). (0.;.).0 (.0;.). (0.0;.). (.;.0). (0.;.) Chemical ).0 (.0;.).0 (0.;.) 0. (0.;.) 0. (0.;.). (.0;.0). (0.;.) Cleaning (). (.0;.).0 (0.;.). (.;.). (0.;.). (0.;.) 0. (0.;.) Metal (). (0.;.0). (0.0;.). (0.;.). (0.;.). (0.;.0). (0.;.) Electronics (0). (0.;.) 0. (0.;.0). (0.;.0). (0.;.).0 (0.;.) 0. (0.0;.) Hairdressing (). (.0;.). (0.;.). (0.;.). (0.;.).0 (.0;.). (0.;.) Textile(). (0.;.0). (0.;.). (0.;.). (0.;.). (0.;.0). (0.0;.00) Assembly (). (.00;.). (0.;.). (0.;.).0 (0.;.0). (.0;.).0 (0.;.) Transport (). (.0;.). (0.;.). (0.;.).0 (0.;.). (.0;.0). (0.0;.) Reference (,) Models adjusted for: Maternal age, -BMI, -smoking during pregnancy, -atopic status, -use of medicine during pregnancy, - furry animal ownership during pregnancy, parity, sex of the child and the child being small for gestational age. on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

23 BMJ Open Page of The association between maternal specific LMW occupations and asthma in the children is displayed in Table. In the adjusted analysis, hair dressing was borderline associated with asthma in the child, OR. (0.;.) without any significant difference between atopic and non-atopic children. For atopic children, cleaning was borderline associated with asthma with OR. (0.;.). A similar pattern was observed for non-atopic children and mother s work in the chemical industry, OR. (0.;.). - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

24 Page of 0 BMJ Open DISCUSSION We found, that maternal occupational exposure to LMW agents during and after pregnancy may be associated to asthma in year-old children. Sub analyses suggested associations between a few specific LMW jobs (hairdressing, cleaning and chemical industry) and asthma in the children. Maternal prenatal - and postnatal LMW exposures were associated with childhood asthma, but after mutual adjustments, postnatal exposure seemed more significant. In a Swedish multigenerational register study,,000 year-old children with a st hospitalisation for asthma were identified[]. The children were then linked to national census data on parental (maternal if available, otherwise paternal) occupation prior to birth of the child. Standardised incidence ratios (SIRs) of.0 or higher were found for hospitalisation due to asthma for boys of beverage manufacturers, packers/loaders, cooks/stewards, home helpers and building caretakers/cleaners. We also found that atopic boys of cleaners had an increased OR for asthma (OR.0 (.;.). For girls, SIRs of.0 or higher was found for chemical process workers, cooks/stewards and waiters. In the present study, we could confirm adjusted increased ORs for asthma in year-old girls of chemical workers (OR. (.0;.). Two previous birth cohort studies investigated prenatal occupational exposures and risk of asthma and wheeze in the children. In a study among, adolescents between years of age Magnusson et al.[] concluded that some maternal jobs (e.g. bakers, dental assistants, technicians, librarians, bookbinders) during pregnancy were associated with en elevated risk of wheeze or asthma in the children. In a cohort of,000 children from the - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

25 BMJ Open Page of ALSPAC study, Tagiyeva et al.[] found that maternal occupational exposure to latex and biocides/fungicides was associated with an increased likelihood of asthma in year-old children. Inclusion of maternal postnatal exposure in the statistical model made the prenatal effects disappear. Our results even suggest that prenatal exposure attenuated the otherwise positive association between postnatal exposure and asthma. Furthermore, paternal occupational exposure to flour dust after birth was associated with an increased likelihood of childhood wheeze and asthma. These findings are in line with our results, indicating that postnatal maternal exposure seems to influence childhood asthma more than prenatal exposure. These results should however be interpreted with caution, since pre- and postnatal exposures are closely correlated. Most mothers worked within the same occupation during the toddler period, i.e. only one fifth of the included women changed occupational exposure group from early pregnancy and until months after childbirth. The effect of postnatal parental occupation on childhood asthma has been suggested to occur due to a carrier home effect[], implicating that the parents carry home components of the work environment leading to indirect exposure of the child from the parental work environment. Effects of maternal exposure during pregnancy are also biologically plausible. Antigens may be transferred from the placenta or via the amniotic fluid and cause the sensitisation of the foetus to take place[, ]. Allergen exposure during pregnancy can also promote an excessive or prolonged Th-skewed immunity after birth. This exaggerates the normal dominance of Th during and after pregnancy, and increases the propensity of developing allergy[, ]. Also other postnatal modifying factors are to be considered, such as breastfeeding, early life day care, number of siblings and ETS. In our study, only 0.% of the women had never - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

26 Page of 0 BMJ Open breastfed their children, and more than 0% of the children attended day care from a very early age, making it unlikely that these potential risk factors have influenced our results considerably. Adjusting for postnatal ETS and parity did not change our estimates. As far as we know, this is the first study to take atopic status in the children into consideration when studying the impact of maternal work on asthma in children. We did not find convincing differences between atopic and non-atopic children for associations between maternal occupational exposures and asthma in the children. The present study thus corroborates the results from our study on hay fever[] in the same mother/child pairs. We did however notice non-significantly increased ORs for atopic children in mothers exposed to HMW agents and mothers with cleaning jobs compared to ORs close to for the non-atopic children. We also noticed an insignificantly increased OR for non-atopic children among mothers working in the chemical industry compared to atopic children. The Impact of offspring atopic status has in earlier studies been emphasised for domestic exposures. Others have found that exposure to endotoxin early in life may protect against the development of atopic asthma, which is not true for non-atopic asthma[, ]. Exposure to maternal domestic cleaning has been found to increase the risk for persistent wheeze only in non-atopic children in the ALSPAC study[]. Even though educational level and SES were considered as potential confounders, they were correlated to our exposure variable, and furthermore SES and educational level were positively associated to asthma with OR. (.-.) for asthma among unemployed compared to self-employed and OR 0. (0.-0.) for women with an academic education - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

27 BMJ Open Page of compared to compulsory schooling. We therefore decided to exclude educational level and SES from the final model due to risk for over adjustment. To our knowledge, the present study is the largest of its kind so far, regarding maternal occupational exposure to asthmogens during pregnancy and asthma in the children. The DNBC data-collection was performed prospectively, i.e. exposure status was collected before the outcome was known. Our results confirm previously described associations between childhood asthma and several risk factors, i.e. maternal atopic disposition[], prepregnancy BMI[], age[], gestational smoking[0], and use of medication[, ] as well as offspring sex[], and SGA[]. A number of limitations have to be considered. In The DNBC cohort, non-smoking, older and well-educated women are overrepresented compared to the general Danish population[]. A substantial loss to follow-up was seen, i.e. nearly 0% of the original cohort was not included in our analyses. We found the same loss to follow-up in the current study, i.e. more of the participants were better educated, smoked less and belonged to a higher socioeconomic group. Otherwise, only small differences were seen according to age, parity and birth weight. For more outcomes, for example childhood asthma, register data has indicated that associations in DNBC are not biased by non-participation[, ]. Furthermore, we have adjusted for some of the obvious confounders in the analysis, and we found similar associations between maternal exposure and asthma for smokers and nonsmokers. For smokers, the association between LMW and childhood asthma was, however mostly seen among non-atopic children,. (.0-.) whereas for non-smokers the association was strongest for atopic children, OR. (0.-.). The asthma prevalence - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

28 Page of 0 BMJ Open found in this study is comparable to other Danish prevalence data on childhood asthma with a similar asthma definition[]. We are aware that mothers with allergic diseases may avoid certain types of occupations, and employees with work-related asthma or allergy may be advised to avoid or reduce occupational exposure leaving non-atopics in risk occupations (healthy worker effect). However, the proportion of women with atopy did not differ between the exposure groups (except in students) and did not differ between participants and non-participants. All in all, we presume that the loss to follow-up did not influence our results to a major degree. The occupational exposure assessment was based on the crude information on job titles and the overall probability of exposures for each job type. This lack of access to detailed individual exposure information implies a risk of non-differential exposure misclassification. Another limitation of the current study is the lack of objective and specific measures for atopy, e.g. positive skin prick tests or analyses of specific IgE. Ever atopic dermatitis in the children was used as proxy for atopy, as this condition is linked to elevated serum IgE and often also to specific sensitisation[]. Misclassification is however inherently present. We defined atopic dermatitis by a combination of maternally reported atopic dermatitis and eczema in locations typical for atopic dermatitis. These questions have been validated against a clinical investigation for atopic dermatitis and were deemed suitable for defining atopic dermatitis in large scale epidemiological studies[]. Our definition is not entirely identical to the wording suggested by Benn et al.[], eczema at year was therefore also included and a slightly different combination of questions applied. Overall, this was expected to increase the sensitivity at the expense of decreased specificity. Diagnosis of asthma in the - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

29 BMJ Open Page of children was also based on maternal reports. Although standardised and validated asthma questions were used[0], some non-differential misclassification would still be expected, and our results might underestimate a true effect. Our study aimed to add to the current limited available knowledge on the impact of prenatal maternal occupational exposure and asthma in children. It was based on a large population cohort of women and their children. The women had a variety of occupational job titles, making exposure contrast possible, even with a trivial exposure misclassification. In conclusion, maternal occupational exposures during pregnancy do not seem to be a substantial risk factor for the development of asthma in year-old children. Maternal occupational exposure to low molecular weight agents early in the child s life may predispose for asthma. Future studies should prioritise exposure characterisation during the both pre- and postnatal period in order to disentangle the importance of the timing of maternal exposure for development of asthma in the children. Funding This study is part of the Danish collaborative MINERVA project ( addressing occupational risks to human reproduction, supported by a grant from the Danish Working Environment Research Fund (contract 0000). Additional funding was obtained from Aarhus University and by the Danish Graduate School of Public Health Science (GRASPH). Competing interests None. Data sharing statement Documentation for how the analysis has been performed can be provided by contact to the corresponding author. The data has been analysed in Statistics Denmark after an application on the specific variables of interest. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

30 Page of 0 BMJ Open CONTRIBUTORSHIP Al authors have contributed to the idea and the analysis plan of the study Berit H Christensen has performed the collection of register data and has performed the data analysis Berit H Christensen and Vivi Schlünssen has performed the first draft of the paper, and all other authors have actively and substantially commented on the manuscript REFERENCE LIST James AL, Knuiman MW, Divitini ML, et al. Changes in the prevalence of asthma in adults since : the Busselton health study. Eur Respir J 0; ():-. Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 00; ():-. Asher MI, Montefort S, Bjorksten B, et al. Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys. Lancet 00; ():. Alati R, Al MA, O'Callaghan M, et al. In utero and postnatal maternal smoking and asthma in adolescence. Epidemiology 00; ():-. Wright RJ. Prenatal maternal stress and early caregiving experiences: implications for childhood asthma risk. Paediatr Perinat Epidemiol 00; Suppl :-. Haberg SE, London SJ, Stigum H, et al. Folic acid supplements in pregnancy and early childhood respiratory health. Arch Dis Child 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

31 BMJ Open Page 0 of Willers SM, Wijga AH, Brunekreef B, et al. Maternal food consumption during pregnancy and the longitudinal development of childhood asthma. Am J Respir Crit Care Med 00; ():-. Dietert RR, Zelikoff JT. Early-life environment, developmental immunotoxicology, and the risk of pediatric allergic disease including asthma. Birth Defects Res B Dev Reprod Toxicol 00; ():-0. Fedulov AV, Leme A, Yang Z, et al. Pulmonary exposure to particles during pregnancy causes increased neonatal asthma susceptibility. Am J Respir Cell Mol Biol 00; ():-. Hamada K, Suzaki Y, Leme A, et al. Exposure of pregnant mice to an air pollutant aerosol increases asthma susceptibility in offspring. J Toxicol Environ Health A 00;0 ():-. Peters JL, Suglia SF, Platts-Mills TA, et al. Relationships among prenatal aeroallergen exposure and maternal and cord blood IgE: project ACCESS. J Allergy Clin Immunol 00; ():-. Henderson J, Sherriff A, Farrow A, et al. Household chemicals, persistent wheezing and lung function: effect modification by atopy? Eur Respir J 00; ():-. Lehmann I, Thoelke A, Rehwagen M, et al. The influence of maternal exposure to volatile organic compounds on the cytokine secretion profile of neonatal T cells. Environ Toxicol 00; ():0-. Magnusson LL, Wennborg H, Bonde JP, et al. Wheezing, asthma, hay fever, and atopic eczema in relation to maternal occupations in pregnancy. Occup Environ Med 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

32 Page of 0 BMJ Open Tagiyeva N, Devereux G, Semple S, Sherriff A, Henderson J, Elias P, Ayres JG. Parental occupation is a risk factor for childhood wheeze and asthma. Eur Respir J. 0;():-. Braun-Fahrlander C, Riedler J, Herz U, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 00; ():-. Olsen J, Melbye M, Olsen SF, et al. The Danish National Birth Cohort--its background, structure and aim. Scand J Public Health 00; ():00-. Kennedy SM, Le MN, Choudat D, et al. Development of an asthma specific job exposure matrix and its application in the epidemiological study of genetics and environment in asthma (EGEA). Occup Environ Med 000; ():-. Karvonen AM, Hyvarinen A, Gehring U, et al. Exposure to microbial agents in house dust and wheezing, atopic dermatitis and atopic sensitization in early childhood: a birth cohort study in rural areas. Clin Exp Allergy 0; ():-. 0 Asher MI, Keil U, Anderson HR, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J ; ():-. Li X, Sundquist K, Sundquist J. Parental occupation and risk of hospitalization for asthma in children and adolescents. J Asthma 00; ():-. Tagiyeva N, Anua SM, Semple S, et al. The 'take home' burden of workplace sensitizers: flour contamination in bakers' families. Environ Int 0;:-. Holloway JA, Warner JO, Vance GH, et al. Detection of house-dust-mite allergen in amniotic fluid and umbilical-cord blood. Lancet 000; ():00-. Lim RH, Arredouani MS, Fedulov A, et al. Maternal allergic contact dermatitis causes increased asthma risk in offspring. Respir Res 00;:. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

33 BMJ Open Page of Christensen BH, Thulstrup AM, Hougaard KS, et al. Occupational exposure during pregnancy and the risk of hay fever in -year-old children. Clin Respir J 0. Basinas I, Schlünssen V, Heederik D, Sigsgaard T, Smit LA, Samadi S, et al. Sensitisation to common allergens and respiratory symptoms in endotoxin exposed workers: a pooled analysis. Occup Environ Med 0 Feb;():-] Jenkins MA, Hopper JL, Flander LB, et al. The associations between childhood asthma and atopy, and parental asthma, hay fever and smoking. Paediatr Perinat Epidemiol ; ():-. Scholtens S, Wijga AH, Brunekreef B, et al. Maternal overweight before pregnancy and asthma in offspring followed for years. Int J Obes (Lond) 0; ():0-. Laerum BN, Svanes C, Wentzel-Larsen T, et al. Young maternal age at delivery is associated with asthma in adult offspring. Respir Med 00; ():-. 0 Lodrup Carlsen KC, Carlsen KH. Effects of maternal and early tobacco exposure on the development of asthma and airway hyperreactivity. Curr Opin Allergy Clin Immunol 00; ():-. Benn CS, Thorsen P, Jensen JS, et al. Maternal vaginal microflora during pregnancy and the risk of asthma hospitalization and use of antiasthma medication in early childhood. J Allergy Clin Immunol 00;0 ():-. Shaheen SO, Newson RB, Henderson AJ, et al. Prenatal paracetamol exposure and risk of asthma and elevated immunoglobulin E in childhood. Clin Exp Allergy 00; ():-. Lowe AJ, Carlin JB, Bennett CM, et al. Do boys do the atopic march while girls dawdle? J Allergy Clin Immunol 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

34 Page of 0 BMJ Open Svanes C, Omenaas E, Heuch JM, et al. Birth characteristics and asthma symptoms in young adults: results from a population-based cohort study in Norway. Eur Respir J ; ():-0. Nohr EA, Frydenberg M, Henriksen TB, et al. Does low participation in cohort studies induce bias? Epidemiology 00; ():-. Greene N, Greenland S, Olsen J, et al. Estimating bias from loss to follow-up in the Danish National Birth Cohort. Epidemiology 0; ():-. Hermann C, De Fine ON, Host A, et al. Prevalence, severity and determinants of asthma in Danish five-year-olds. Acta Paediatr 00; ():-0. Bergmann RL, Edenharter G, Bergmann KE, Forster J, Bauer CP, Wahn V, Zepp F, Wahn U Atopic dermatitis in early infancy predicts allergic airway disease at years. Clin Exp Allergy. Aug;():-0.) Benn CS, Benfeldt E, Andersen PK, et al. Atopic dermatitis in young children: diagnostic criteria for use in epidemiological studies based on telephone interviews. Acta Derm Venereol 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

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36 Page of 0 BMJ Open Table E. Comparison of participants and non-participants, the latter defined as women and children (first pregnancy and singleton) included in BMSB with valid information from the st questionnaire, but not included in the final study population Participants Non-participants RR Total number..0 Mothers: Exposure groups: (n=,) (n=,0) high molecular (0.;.) low molecular/ irritants.. 0. (0.;0.0) mixed...0 (.0;.0) farmer (0.;.0) unclassifiable..0. (.0;.) student.. 0. (0.;0.) reference...0 (.00;.0) Smoking during pregnancy: no use of tobacco...0 (.0;.) - cigarettes/day (0.;0.) > cigarettes/day.. 0. (0.;0.) Rural residence (0.;0.) Furry animals.. 0. (0.0;0.) Age, median (%;0%) 0 (;) (;) BMI, median (%;0%). (.;.). (.;.) Atopic (0.;.00) Parity: (.0;.0) (0.;0.) (0.;0.) Level of education: no or unknown (0.;0.) compulsory schooling.. 0. (0.;0.) beyond compulsory schooling...0 (.0;.0) academic level..0. (.;.) Socioeoconomic level: (n=,) (n=,0) self-employed.. 0. (0.;0.) employees at the highest level.0.. (.;.) employees (0.;0.) students (0.;0.) unemployed, not in work force (0.;0.) Fathers: Atopic...0 (.0;.0) Child: Boy..0.0 (0.;.0) Birth weight, kg, median (%;0%).0 (.;.). (.;.) Small For Gestational Age (0.;0.) - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

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38 Page of 0 BMJ Open Table E: Adjusted logistic regression models on mothers occupational exposure and other potential risk factors, and asthma in the child at age. N =, Asthma risk, adjusted OR (%CI). Exposure: high molecular. (0.;.) low molecular/ irritants. (.00;.) mixed.00 (0.;.0) farmer 0. (0.;.) unclassifiable.0 (0.0;.) student 0. (0.;.0) reference Age: -.0 (.;.) (0.;0.) + 0. (0.0;0.) Pre-pregnancy BMI: 0. (0.;.00) (.0;.) 0+. (.;.) Maternal atopy:. (.;.0) Furry animals during pregnancy: 0. (0.;.0) Parity: 0 -. (.0;.) +. (0.-.) Use of medicine during pregnancy: folic acid.0 (0.;.) paracetamol.0 (.0;.) acetyl salicylic acid 0. (0.;.0) antibiotics. (.0;.) Smoking: no smoking - cigarettes/day.0 (.;.) > cigarettes/day. (.0;.) Sex (male):. (.;.) SGA. (.0;.) Included in the model: maternal age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy, parity, furry animal ownership during pregnancy, parity, SGA and sex of the child. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

39 BMJ Open Page of Section/Topic Item # STROBE 00 (v) Statement Checklist of items that should be included in reports of cohort studies Recommendation Reported on page # Title and abstract (a) Indicate the study s design with a commonly used term in the title or the abstract Introduction (b) Provide in the abstract an informative and balanced summary of what was done and what was found Background/rationale Explain the scientific background and rationale for the investigation being reported - Objectives State specific objectives, including any prespecified hypotheses Methods Study design Present key elements of study design early in the paper Setting Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection Participants (a) Give the eligibility criteria, and the sources and methods of selection of participants. Describe methods of follow-up - (b) For matched studies, give matching criteria and number of exposed and unexposed - Variables Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if Data sources/ measurement applicable * For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability of assessment methods if there is more than one group Bias Describe any efforts to address potential sources of bias -; Table E Study size Explain how the study size was arrived at Quantitative variables Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why Statistical methods (a) Describe all statistical methods, including those used to control for confounding - on December 0 by guest. Protected by copyright (online supplement) (b) Describe any methods used to examine subgroups and interactions -;- (c) Explain how missing data were addressed Table, page - (d) If applicable, explain how loss to follow-up was addressed Table E (online supplement); page BMJ Open: first published as./bmjopen on April 0. Downloaded from

40 Page of 0 BMJ Open (e) Describe any sensitivity analyses -;- Results Participants Descriptive data * (a) Report numbers of individuals at each stage of study eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (b) Give reasons for non-participation at each stage (c) Consider use of a flow diagram - * (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential Table, page - confounders (b) Indicate number of participants with missing data for each variable of interest Table (c) Summarise follow-up time (eg, average and total amount) Outcome data * Report numbers of outcome events or summary measures over time Table, page - Main results (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, % confidence interval). Make clear which confounders were adjusted for and why they were included Table, figure, page -0 (b) Report category boundaries when continuous variables were categorized (c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period - Other analyses Report other analyses done eg analyses of subgroups and interactions, and sensitivity analyses -;- Discussion Key results Summarise key results with reference to study objectives Limitations Interpretation 0 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar studies, and other relevant evidence Generalisability Discuss the generalisability (external validity) of the study results - Other information Funding Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the present article is based *Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

41 BMJ Open Page 0 of Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at Annals of Internal Medicine at and Epidemiology at Information on the STROBE Initiative is available at BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

42 BMJ Open A prospective study on maternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the year-old children. Journal: BMJ Open Manuscript ID: bmjopen r Article Type: Research Date Submitted by the Author: 0-Feb-0 Complete List of Authors: Christensen, Berit; Aarhus University, Department of Public Health; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre Thulstrup, Ane Marie; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre Hougaard, Karin; The National Research Center for the Working Environment, Skadhauge, Lars; Hospital of South-West Jutland, Department of Occupational Medicine Frydenberg, Morten; Aarhus University, Department of Public Health Hansen, Kirsten; University Hospital Herlev, Department of Paediatrics Schlünssen, Vivi; Aarhus University, Department of Public Health; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre <b>primary Subject Heading</b>: Secondary Subject Heading: Keywords: Occupational and environmental medicine Epidemiology, Occupational and environmental medicine, Reproductive medicine, Respiratory medicine OCCUPATIONAL & INDUSTRIAL MEDICINE, EPIDEMIOLOGY, Community child health < PAEDIATRICS, PREVENTIVE MEDICINE, Allergy < THORACIC MEDICINE, Asthma < THORACIC MEDICINE - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

43 Page of BMJ Open A prospective study on maternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the year-old children Berit Hvass Christensen MD, ; Ane Marie Thulstrup PhD ; Karin Sørig Hougaard PhD ; Lars R. Skadhauge PhD ; Kirsten Skamstrup Hansen PhD ; Morten FrydenbergPhD ; Vivi Schlünssen PhD,. Department of Public Health, Section of Environment, Occupation and Health, Danish Ramazzini Centre, Aarhus University, Denmark. Department of Occupational Medicine, Danish Ramazzini Centre, Aarhus University Hospital, Denmark. The National Research Center for the Working Environment, Copenhagen, Denmark. Department of Occupational Medicine, Hospital of South-West Jutland, Denmark. Department of Paediatrics, University Hospital Herlev, Denmark. Department of Public Health, Section of Biostatistics, Aarhus University, Denmark. Corresponding author: Vivi Schlünssen Department of Public Health, Section of Environment, Occupation and Health, Danish Ramazzini Centre, Aarhus University, Bartholins Allé, 000 Aarhus C, Denmark. vs@mil.au.dk Phone + Running head: prenatal occupational exposure and asthma Word count:, (text only). Abstract words. Four tables, one figure, online supplement tables. references - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

44 BMJ Open Page of ABSTRACT: Objectives The occurrence of allergic diseases has increased considerably in the last decades. In this prospective follow-up study, we examined whether maternal exposure to asthmogens during pregnancy is associated with the development of asthma in year-old Danish children, taking atopic status into consideration. Methods A total of, women and their children from The Danish National Birth Cohort were categorised according to maternal occupational exposure. Exposure information was obtained by combining job title in pregnancy and months after pregnancy with a commonly used asthma Job Exposure Matrix. Information on asthma in the child was obtained by an internetquestionnaire when the child was years old. Results Prenatal exposure to Low Molecular Weight (LMW) agents was borderline associated to asthma in the children with OR. (0.;.) for atopic and. (0.;.) for non-atopic children. Maternal postnatal exposure was associated with asthma (OR. (.0;.). After mutual adjustment postnatal exposure (OR. (0.;.) and the combined effect of pre- and postnatal exposure (OR, (.;.)) seems to increas the risk of asthma in the children. No significant associations were observed for other prenatal or postnatal exposures. Conclusion Maternal occupational exposures during pregnancy do not seem to be a substantial risk factor for the development of asthma in year old children. Maternal pre- and postnatal exposure to LMW agents may predispose the propensity of the children to develop asthma. Future studies should prioritise the characterisation of the timing exposure in relation to the birth. Keywords Prenatal exposure; postnatal exposure delayed effects; Asthma; Prospective cohort study; Allergy; Maternal exposure; Occupational exposure; pregnancy; epidemiology ARTICLE SUMMARY Article focus - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

45 Page of BMJ Open To study the impact of maternal occupational exposure during pregnancy on the occurrence of asthma among their year-old children Key messages Mothers occupational exposures during pregnancy is not a substantial risk factor for asthma in their year old children Maternal pre- and postnatal exposure to LMW agents may increase the risk for asthma in the year old children Future studies should focus on timing of exposure in relation to the birth Strengths and limitations of this study: This study is the largest prospective study so far addressing the impact on childhood asthma of mothers occupational exposure during pregnancy. It is also the first study where atopic status of the child is taken into account when exploring the importance of prenatal occupational exposures on childhood allergic diseases. A substantial loss to follow-up was seen. We presume that the loss to follow-up did not influence our results to a major degree. The occupational exposure assessment was based on crude information on job titles and a general asthma JEM. Definitions of asthma and atopy were based on self-reported informations from the mothers. Still we were able confirm previously described associations between childhood asthma and several risk factors. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

46 BMJ Open Page of BACKGROUND The occurrence of asthma, rhinitis, and atopic dermatitis has increased in the last decades, from a few per cent in the 0s to -0% in the 0s[, ]. Lifestyle seems to influence the development of allergic diseases, also during childhood[]. Prenatal risk factors for asthma have been investigated and include environmental tobacco smoking[], stress[] and maternal diet during pregnancy[, ]. Thus, the maternal environment during pregnancy might encompass risk factors for asthma in the child. Allergic diseases are hallmarked by childhood immune dysfunction, but surprisingly, little attention has been paid to prenatal influences on immune development, although the foetal immune system is vulnerable to programming[]. In animals, airborne exposure to particles and aerosols of the mother during pregnancy have been shown to increase asthma susceptibility in the offspring[, ], and findings in some human studies corroborate this association[, ]. Maternal exposure to volatile organic compounds in pregnancy may skew the Th-/Th immune response in the foetus, thereby potentially predisposing the children to asthma later in life[]. An increasing number of women work outside the home during pregnancy, and many are occupationally exposed to asthmogens, i.e. chemical and biological agents that are recognised to increase the risk for work-related asthma. It therefore seems pertinent to investigate whether women s working environments during pregnancy interfere with their children s propensity to develop asthma later in life. Two earlier cohort studies have investigated prenatal occupational exposures and the risk of asthma among adolescent[] and 0- year-old children[] with inconclusive results. Asthma can be characterised as allergic (atopic) or non-allergic (non-atopic). These two phenotypes are characterised by e.g. different risk factors. Endotoxin has for example opposite - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

47 Page of BMJ Open effects in atopic and non-atopic asthma[]. In the present study, we hypothesise that maternal exposure to asthmogens during pregnancy impacts on the foetal development of the immune system and predisposes the children to develop asthma later in life. Specifically, we analysed whether maternal occupational exposures during pregnancy are associated with the development of asthma in year-old Danish children, taking atopic status into consideration. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

48 BMJ Open Page of MATERIAL AND METHODS Population The study population was part of the prospective cohort: The Danish National Birth Cohort (DNBC, Better health for mother and child ). The cohort was established in -00 to study pregnancy outcome and disease in children as a function of factors operating in early life[] ). Women were recruited at their initial pregnancy consultation with their general practitioner or midwife and participation implied four telephone interviews; during week to and week 0 to of pregnancy, and when the child was and months old. When the child was seven years of age, the mothers answered an internet questionnaire on behalf of their children. Mothers without internet access were offered a paper version. The first telephone interview comprised questions about work, atopic diseases and risk factors for asthma (e.g. smoking, use of medication, parity). The questionnaire at age dealt with asthma, rhinitis and atopic dermatitis in the child and risk factors (e.g. parental smoking). A total of 0, pregnancies were enrolled in the cohort. The first telephone interview was answered, times. A total of, women were no longer pregnant at the time of the interview and were excluded. Only singletons and only one pregnancy per woman (the first in the sampling period) were included, so,0 siblings and, children from multiple births were excluded. This leaves a total of. mother/child pairs, of which the internet questionnaire at age was answered for, children. Among these,, pairs were excluded due to lack of information on maternal occupational status. Thus, mother/child pairs were eligible for analysis. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

49 Page of BMJ Open Occupational exposure Data on job exposure during pregnancy was obtained by combining reported information on jobtitle (DISCO codes) from telephone interview one during - week of pregnancy and a commonly used asthma Job Exposure Matrix (JEM)[] based on known risk factors for occupational asthma. The job titles were coded according to the Danish International Standard classification of Occupations, the Danish edition of ISCO- (DISCO-code). Seven exposure categories were formed and consisted of the following occupations and typical allergens/irritants:. High molecular weight (HMW) agents (veterinarians, gardeners, and bakers; grass pollen, animal dander, flour/grain). Low molecular weight (LMW) agents or irritants (cooks, cleaners, hairdressers, dentistry and all kinds of manufacturing of dust-producing materials; aerosols, dust from manufacturing of wood/ textile/ stone/ rubber/ plastics etc.). Mixed HMW and LMW agents (health care professionals; antibiotics, latex, cleaning agents). Farmers (organic dust, grass pollen, animal dander, ammonia). Students (no or unknown exposure). Unclassifiable (subjects where the same job title involved several different environments, e.g. waiters, engineering technicians, biologists, shop managers; diverse exposure). Reference (office workers, teachers and journalists; no or low exposure) The applied JEM was slightly modified from[] by Christensen BH and Schlünssen V to comply with working conditions in Denmark based on a priori knowledge of exposures to asthmogens in the Danish working environment. As an example, medical doctors were originally categorised as not being exposed to asthmogens, but were reclassified to the mixed group in the modified version of the JEM. Furthermore, farmers were identified as a special group as early life exposure - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

50 BMJ Open Page of to a farming environment has shown to offer protection against allergic diseases[]. Also, students were considered as a special group due to possible selection related to health. Postnatal occupational exposure groups were established using the -digit DISCO codes months after the birth of the child, i.e. at a time when maternity leave was terminated and the mother would most probably have returned to work. The codes were retrieved from Statistics Denmark, as every adult person ( years) in Denmark is classified annually according to occupation. Maternal postnatal occupation was categorised in the same JEM categories as for the prenatal exposures. Women classified as students during pregnancy, but without a DISCO code indicative of employment after having given birth were not included in the postnatal analyses. Level of education and socioeconomic status (self-employed, employees at the highest level, employees, students, unemployed or not in the work force) were obtained for both the father and the mother (the calendar year preceding the birth year of the child) from Statistics Denmark. Information on the parent with the highest level of education/socioeconomic status defined the level for the family. Outcome We used validated core questions on asthma from the International Study of Asthma and Allergies in Childhood (ISAAC)[0]. Asthma in the child at age was defined as an affirmative answer to one or more of three questions: Has your child had wheezing or whistling in the chest in the last months? ; Has your child ever had asthma? or to Has your child ever been diagnosed with asthma by a doctor? - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

51 Page of BMJ Open Ever atopic dermatitis was used as a proxy for atopy among the children. Information on atopic dermatitis was collected by telephone interview when the child was months old and by questionnaire at age. It was defined as parental report at months of atopic dermatitis ever AND itchy rash in the locations known to be typical for atopic dermatitis AND/OR report of persistent itchy rash in the locations known to be typical for atopic dermatitis at age. Confounders Information on known possible confounders according to prior knowledge from other studies on the effects of asthma in children was obtained primarily from the telephone interview at - weeks of gestation: maternal age, pre-pregnancy BMI, parity, smoking during pregnancy, and furry animals in the home. Maternal atopic disposition was defined as ever reported asthma, rhinitis, and/or atopic dermatitis. Information on maternal use of acetyl salicylic acid, paracetamol, folic acid and antibiotics during pregnancy was retrieved from the second telephone interview at 0- weeks of gestation. Birth weight, gestational age at birth, sex, and singleton status were obtained from the Danish Birth Registry. Statistical methods All analyses were performed in STATA SE.0 (STATA corp., Texas, US). Univariate analyses were performed for categorical variables using χ tests. For comparisons of continuous variables, the independent sample t-test or Kruskal-Wallis test was used. A multivariate unconditional logistic regression analysis was used to evaluate associations between exposure and outcome. A univariate analysis revealed an equal distribution between the exposure groups for some of the - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

52 BMJ Open Page of potential confounders, which was subsequently excluded from the model. The final model contained the following maternal variables: age (-; -; 0-; + years), pre pregnancy BMI ( ; 0-; -; 0+), atopy (yes/no), smoking during pregnancy (no; -; + cig/day), use of medication during pregnancy (yes/no to the use of acetyl salicylic acid, paracetamol, folic acid, and antibiotics), parity (0;-;+) and furry animal ownership during pregnancy (yes/no). For the children, SGA (small for gestational age) (yes/no) and sex were included. SGA was defined as the smallest % of the children born in a specific gestational week based on birth weights from the entire BSMB cohort, and separately for boys and girls. The analysis was repeated after including the children s exposure to environmental tobacco smoke (ETS) in the model according to the information on the parents current smoking. Unless otherwise stated, the level of significance was p < 0.0, two-sided. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

53 Page of BMJ Open RESULTS Table shows the demographic characteristics of the cohort stratified by exposure groups. The women had a mean age of 0 years at inclusion. Students were younger than the other groups, i.e.. % were below 0 years of age. About half (.%) of the women had not given birth previously. Among the students,. % were primiparous. On average,. % of the women had used paracetamol, mostly prevalent in the mixed exposure group (.%), and least prevalent in the farmer group (.%). The majority (.%) of the women were non-smokers during pregnancy. The LMW group had the highest tobacco use with. % smoking + cig/day. A similar pattern was seen at follow up at years of age, but in general fewer mothers smoked at follow up, with a decrease from one in four to one in six. The lowest prevalence of atopy (.%) was seen in the HMW group, whereas the prevalence was highest among students (.%). - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

54 BMJ Open Page of Table Demographics, categorised according to mothers exposure during pregnancy into categories: High molecular weight (HMW), Low molecular weight/irritants (LMW), Mixed (a mixture of HMW and LMW), farmers, unclassifiable, students and references. The numbers are presented as % unless otherwise stated. During pregnancy: Mother: HMW LMW Mixed Farmer Unclassifiable Student Reference Missing, % n = 0 n =,0 n =, n = 0 n =,0 n =, n =, Age, yrs, median (%;0%) (;) (;) 0 (;) 0 (;) (;) (;) 0 (;) 0 BMI, median (%;0%).0 (.;.). (.;0.). (.;.0). (.;.). (.;.).0 (.;.). (.;.). Parity: or Use of medicine: ASA Paracetamol Antibiotics Folic acid Rural residence Furry animals a Smoking: 0.0 no use of tobacco cigarettes/day > 0.0 cigarettes/day Atopy b.,., Child: Boy Birth weight, kg, median (%;0%).0 (.0;.0). (.;.).0 (.;.). (.;.).0 (.;.). (.;.).0 (.;.) Small for gestational age on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

55 Page of BMJ Open At year followup: Maternal smoking Paternal smoking Child atopy d Child ETS exposure on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

56 BMJ Open Page of Table E (online supplement) presents the characteristics among participants and nonparticipants. The distributions of exposure groups were quite similar between participants and non-participants, but with a slightly lower number of LMW-exposed and students among the participating mothers compared to the non-participating mothers. More of the participating mothers were non-smokers during pregnancy, vs. %, they were slightly older (0 vs. years), better educated (academic level, vs. %), belonged to a higher socioeconomic group (employees at the highest level, vs. %), and more were expecting their first child compared to the non-participants, vs. %. Maternal atopic status was similar in the two groups. At birth, children of participating mothers weighed on average 0 g more and the number of SGA children was.0% in participants compared to.% among non-participants. Table : Unadjusted prevalence of asthma stratified for atopy defined as ever atopic dermatitis in the year old children (n =,). Exposure group All children n (%) Atopic children n (%*) Non-atopic children n (%*) High molecular (.) (.) (.) Low molecular/ irritants (.) a (.) b (.) c Mixed, (.) (.) (.) Farmer (.0) (.) (.) Unclassifiable 0 (.0) (.) (.) Student (.) (.0) (.) Reference, (.) (.), (.) Total n (%),0 (.), (.) d, (.) *The percentage is the number of atopic (or non-atopic) children within the maternal exposure group. a: P<0.0, group vs. mixed, farmer, unclassifiable, student and reference. b: P<0.0, group vs. mixed, farmer, unclassifiable and reference c: P<0.0, group vs. reference. d: P<0.0 group vs. non-atopic children - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

57 Page of BMJ Open Table shows the prevalence of asthma stratified for atopic status defined as ever atopic dermatitis. The overall prevalence of asthma was %; % for atopic and % for non-atopic children. Children from the LMW and the HMW exposure group had the highest prevalence of asthma, both in total (-%), and for the atopic (-%) and the non-atopic group (-%). Farmers children had the lowest prevalence of asthma, although not significantly different from the other exposure groups. In Table E (online supplement) the prevalence of asthma was further stratified by sex. The prevalence of asthma was higher among boys (%) compared to girls (%), and for both sexes the overall prevalence of asthma was higher for atopics compared to nonatopic. Children from the LMW and HMW exposure groups had the highest prevalence of asthma in boys, for girls this was only true for LMW. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

58 BMJ Open Page of Table : Unadjusted and adjusted logistic regression models showing the association between mothers occupational exposure during pregnancy and asthma in the child at age years stratified by atopic status of the child. Crude OR (% CI) Model Model Atopic children, Non-atopic children, Atopic children, Non-atopic children, Atopic children, Non-atopic children, n =, n=, n =,0 n =, n =, n = 0, Exposure group High molecular. (0.;,). (0.;.). (0.;.).0 (0.;.). (0.;.).0 (0.;.) Low molecular/ irritants. (.;.). (.;.). (0.;.). (0.;.). (0.;.).0 (0.;.) Mixed.0 (0.;.).0 (0.;.) 0. (0.;.).00 (0.;.0) 0. (0.;.) 0. (0.;.0) Farmer. (0.;.) 0. (0.;.). (0.;.) 0. (0.0;.) 0. (0.;.) 0. (0.;.0) Unclassifiable 0. (0.;.) 0. (0.;.).0 (0.;.).0 (0.;.).0 (0.;.).00 (0.;.) Student.0 (0.;.).0 (0.;.) 0. (0.;.) 0. (0.;.0) 0. (0.;.0) 0. (0.0;.0) Reference Model includes: the following variables for the mothers: age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy (dichotomous variables for the use of Acetyl salicylic acid, paracetamol, Folic acid and Antibiotics), parity and furry animal ownership during pregnancy. The following variables were included for the children: Birth weight (SGA) and sex of the child. Model includes the adjustments in model and level of education and socioeconomic level. on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

59 Page of BMJ Open Table shows crude and adjusted ORs for the associations between mothers occupational exposure during pregnancy and asthma in the child. In model, levels of education and socioeconomic status were added to the a priori chosen confounders. For HMW agents, ORs were non-significantly increased in the range.-. in both the crude and the adjusted analyses, especially for atopic children. Children of mothers exposed to LMW agents during pregnancy had an increased OR for asthma in the crude analysis for both atopic (OR. (% C.I..;.)) and non-atopic children (OR. (.;.). The same tendency was seen in model with OR. (0.;.) for atopic and. (0.;.) for non-atopic children. The ORs were further decreased in model with OR. (0.;.) for atopic and.0 (0.;.) for non-atopic children. For the other exposure groups, no significant association between maternal exposure and asthma in the children was observed. In the crude analysis a borderline significant association between being student/mixed groups and asthma were seen with OR.0-.0, but without confirmation in model or. The analysis in Table was repeated taking the year-old children s current parental ETS into account. This did not change the results (data not shown). The same was true when paternal atopic status was included. Even though educational level and socioeconomic status (SES) were considered as potential confounders, they were correlated to our exposure variable, partly because the DISCO code was included in the definition of SES. We decided therefore to use model in the ensuing analysis. We did not find any significant interactions between exposure and atopy when the interaction exposure*atopy was included in the model. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

60 BMJ Open Page of Model was repeated after stratification for sex, please see Table E, online supplement. In general similar results were found for boys and girls, i.e. Children of mothers exposed to LMW agents during pregnancy had a borderline increased OR for asthma in both girls (. (0.;.)) and boys (.0(0.;.)). For girls, the association was significant for atopic children,. (.0;.), where for boys this was true for non-atopic children,. (.0;.). In Table E (online supplement), ORs for covariates in model are displayed with asthma as the outcome of interest. The mother s age was inversely associated to asthma in the child. The children of women < years of age had an increased OR for asthma,.0 (.;.), compared to - year-old women (reference). The risk was further decreased for 0- years, OR 0. (0.;0.) and + year old women, OR 0. (0.0;0.). Maternal prepregnancy BMI was positively associated with asthma with ORs,. (.0;.) and. (.;.) for BMI - and above 0, respectively, compared to BMI 0- (reference). Children with maternal atopic disposition had increased OR for asthma (.(.;.0)). The use of paracetamol and antibiotics during pregnancy was associated with asthma in the children, OR.0 (.0;.) and. (.0;.), respectively. Smoking during pregnancy was positively associated with asthma in a dose-dependent manner with OR.0 (.;.) for - cig/day), and OR. (.0;.) for > cig/day compared to non-smokers (reference). Parity was positively associated with asthma, with OR. (.0;.) for - pregnancies, and OR. (0.;.) for or more pregnancies compared to no earlier pregnancy (reference), whereas pet ownership was not associated to childhood asthma, OR 0. (0.;.0). - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

61 Page of BMJ Open Being a boy, and being born SGA was both positively associated with asthma with ORs. (.;.) and. (.0;.), respectively. The analysis in table E was repeated after stratification for atopic status in the children, and almost identical results were seen for atopic and non-atopic children (data not shown). Figure shows the adjusted logistic regression analysis on the association between mothers pre- and postnatal exposure and asthma in the children. For LMW agents, both pre- and postnatal exposure were significantly associated with asthma in the children, ORs. (.00;.) and. (.0-.), respectively. After mutual adjustments only postnatal exposure (OR. (0.;.)) and the combined effect of pre- and postnatal exposure (OR, (.;.)) were associated with asthma in the child. For the other exposure groups, no significant associations were seen for either prenatal or postnatal exposures. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

62 BMJ Open Page 0 of Figure. Adjusted logistic regression analysis on the association between mothers pre- and postnatal exposure and asthma in the year-old children. Reference: reference workers.. OR. 0 HMW LMW Mix Farm UC pre post pre adjusted post post adjusted pre combined Pre: Prenatal exposure. Post: Postnatal exposure. Post just pre: postnatal exposure adjusted for prenatal exposure. Pre just post: Prenatal exposure adjusted for postnatal exposure. Combined: Additive effect of pre-and postnatal exposure. Included in the model: maternal age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy, furry animal ownership during pregnancy, SGA and sex of the child BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

63 Page of BMJ Open Table : Crude and adjusted logistic regression analyses on associations between mothers LMW jobs and asthma in the year old children. Crude Total Adjusted Total Crude Atopic Adjusted Atopic Crude Non-atopic Adjusted Non-atopic (n=,) (n=,0) Children (n=,) Children (n=,0) Children (n=,) Children (n=0,) LMW OR % CI OR % CI OR % CI OR % CI OR % CI OR % CI Plastic work (0).0 (0.;.). (0.;.). (0.;.). (0.;.).00 (0.;.) 0. (0.;.) Paper work () 0. (0.;.) 0. (0.;.) 0. (0.;.0) 0. (0.;.) 0. (0.;.) 0.0 (0.;.) Stone work (). (0.;.) 0. (0.0;.). (0.;.).0 (0.;.) 0. (0.;.) 0. (0.;.) Wood work () 0. (0.;.) 0. (0.;.) 0. (0.;.) 0. (0.;.). (0.;.).0 (0.;.) Food work (0). (.;.). (0.;.).0 (.0;.). (0.0;.). (.;.0). (0.;.) Chemical work ).0 (.0;.).0 (0.;.) 0. (0.;.) 0. (0.;.). (.0;.0). (0.;.) Cleaning (). (.0;.).0 (0.;.). (.;.). (0.;.). (0.;.) 0. (0.;.) Metal work (). (0.;.0). (0.0;.). (0.;.). (0.;.). (0.;.0). (0.;.) Electronic work (0). (0.;.) 0. (0.;.0). (0.;.0). (0.;.).0 (0.;.) 0. (0.0;.) Hairdressing (). (.0;.). (0.;.). (0.;.). (0.;.).0 (.0;.). (0.;.) Textilework (). (0.;.0). (0.;.). (0.;.). (0.;.). (0.;.0). (0.0;.00) Assembly work (). (.00;.). (0.;.). (0.;.).0 (0.;.0). (.0;.).0 (0.;.) Transport work (). (.0;.). (0.;.). (0.;.).0 (0.;.). (.0;.0). (0.0;.) Reference (,) Models adjusted for: Maternal age, -BMI, -smoking during pregnancy, -atopic status, -use of medicine during pregnancy, - furry animal ownership during pregnancy, parity, sex of the child and the child being small for gestational age. on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

64 BMJ Open Page of The association between maternal specific occupations (included in the LMW exposure group) and asthma in the children is displayed in Table. In the adjusted analysis, hair dressing was borderline associated with asthma in the child, OR. (0.;.) without any significant difference between atopic and non-atopic children. For atopic children, cleaning was borderline associated with asthma with OR. (0.;.). A similar pattern was observed for non-atopic children and mother s work in the chemical industry, OR. (0.;.). The above analysis was repeated after stratification for sex, please see Table E, online supplement. Atopic boys of mothers working as cleaners during pregnancy (.(.;.) and non-atopic girls of women working in the chemical industry (OR. (.;.0) had increased OR for asthma. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

65 Page of BMJ Open DISCUSSION Key findings We found, that maternal occupational exposure to LMW agents during and after pregnancy may be associated to asthma in year-old children. Sub analyses suggested associations between a few specific LMW jobs (hairdressing, cleaning and chemical industry) and asthma in the children. Maternal prenatal - and postnatal LMW exposures were associated with childhood asthma, but after mutual adjustments, postnatal exposure seemed more significant. How do the findings fit with earlier study results? In a Swedish multigenerational register study,,000 year-old children with a st hospitalisation for asthma were identified[]. The children were linked to national census data on parental (maternal if available, otherwise paternal) occupation prior to birth of the child. Standardised incidence ratios (SIRs) of.0 or higher were found for hospitalisation due to asthma for boys of beverage manufacturers, packers/loaders, cooks/stewards, home helpers and building caretakers/cleaners. We confirmed that atopic boys of cleaners had an increased OR for asthma. For girls, SIRs of.0 or higher was found for chemical process workers, cooks/stewards and waiters. In the present study, we could confirm adjusted increased ORs for asthma in year-old girls of chemical workers. Two previous birth cohort studies investigated prenatal occupational exposures and risk of asthma and wheeze in the children. In a study among, year old adolescents Magnusson et al.[] concluded that some maternal jobs (e.g. bakers, dental assistants, - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

66 BMJ Open Page of technicians, librarians, bookbinders) during pregnancy were associated with en elevated risk of wheeze or asthma in the children. Prenatal vs. postnatal effects In a cohort of,000 children from the ALSPAC study, Tagiyeva et al.[] found that maternal occupational exposure to latex and biocides/fungicides was associated with an increased likelihood of asthma in year-old children. Adjusting for maternal postnatal exposure made the prenatal effects disappear. Furthermore, paternal occupational exposure to flour dust after birth was associated with an increased likelihood of childhood wheeze and asthma. These findings are in line with our results, indicating that postnatal maternal exposure seems to influence childhood asthma more than prenatal exposure. These results should however be interpreted with caution, since pre- and postnatal exposures are closely correlated. Most mothers worked within the same occupation during the toddler period, i.e. only one fifth of the included women changed occupational exposure group from early pregnancy and until months after childbirth. The effect of postnatal parental occupation on childhood asthma has been suggested to occur due to a carrier home effect[], implicating that the parents carry home components of the work environment leading to indirect exposure of the child from the parental work environment. Effects of maternal exposure during pregnancy are also biologically plausible. Antigens may be transferred from the placenta or via the amniotic fluid and cause the sensitisation of the foetus to take place[, ]. Allergen exposure during pregnancy can also promote an excessive or prolonged Th-skewed - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

67 Page of BMJ Open immunity after birth. This exaggerates the normal dominance of Th during and after pregnancy, and increases the propensity of developing allergy[, ]. Also other postnatal modifying factors are to be considered, such as breastfeeding, early life day care, number of siblings and ETS. In our study, only 0.% of the women had never breastfed their children, and more than 0% of the children attended day care from a very early age, making it unlikely that these potential risk factors have influenced our results considerably. Adjusting for postnatal ETS and parity did not change our estimates. Implications of atopic status and sex of the child As far as we know, this is the first study to take atopic status in the children into consideration when studying the impact of maternal work on asthma in children. We did not find convincing differences between atopic and non-atopic children for associations between maternal occupational exposures and asthma in the children. The present study thus corroborates the results from our study on hay fever[] in the same mother/child pairs. We did however notice non-significantly increased ORs for atopic children in mothers exposed to HMW agents and mothers with cleaning jobs compared to ORs close to for the non-atopic children. We also noticed an insignificantly increased OR for non-atopic children among mothers working in the chemical industry compared to atopic children. The Impact of atopic status on associations between environmental exposures and asthma has been emphasised before, i.e. others have found that exposure to endotoxin early in life may protect against the development of atopic asthma, but not non-atopic asthma[, ]. Exposure to maternal domestic cleaning has been found to increase the risk for persistent wheeze only in non-atopic children in the ALSPAC study[]. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

68 BMJ Open Page of As expected, we found a higher prevalence of asthma among boys compared to girls, but in general sex did not modify the effect of maternal work during pregnancy on the development of offspring asthma. In a few analyses stratified by both atopy and sex differences were found for boys and girls, but our results are too vague to support a specific gender effect. Implications of socioeconomic status Even though educational level and SES were considered as potential confounders, they were correlated to our exposure variable, and furthermore SES and educational level were positively associated to asthma with OR. (.-.) for asthma among unemployed compared to self-employed and OR 0. (0.-0.) for women with an academic education compared to compulsory schooling. We therefore decided to exclude educational level and SES from the final model due to risk for over adjustment. Strengths and limitations of the study To our knowledge, the present study is the largest of its kind so far, regarding maternal occupational exposure to asthmogens during pregnancy and asthma in the children. The DNBC data-collection was performed prospectively. Our results confirm previously described associations between childhood asthma and several risk factors, i.e. maternal atopic disposition[], pre- pregnancy BMI[], age[], gestational smoking[0], use of medication[, ] as well as offspring sex[], and SGA[]. A number of limitations have to be considered as well. In The DNBC cohort, non-smoking, older and well-educated women are overrepresented compared to the general Danish population[]. A substantial loss to follow-up is present,, i.e. nearly 0% of the original - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

69 Page of BMJ Open cohort was not included in our analyses, and only % (, out of. eligible mother/child pairs) were included in the current analysis. We found the same characteristics among the loss to follow-up subjects in the current study compared to the DNBC cohort in general, i.e. more of the participants were better educated, smoked less and belonged to a higher socioeconomic group. Otherwise, only small differences were seen according to age, parity and birth weight. For more outcomes, for example childhood asthma, register data has indicated that associations in DNBC are not biased by non-participation[, ]. Furthermore, we have adjusted for some of the obvious confounders in the analysis, and we found similar associations between maternal exposure and asthma for smokers and nonsmokers. The asthma prevalence found in this study is comparable to other Danish prevalence data on childhood asthma with a similar asthma definition[]. We are aware that mothers with allergic diseases may avoid certain types of occupations, and employees with work-related asthma or allergy may be advised to avoid or reduce occupational exposure leaving nonatopics in risk occupations (healthy worker effect). However, the proportion of women with atopy did not differ between the exposure groups (except in students) and did not differ between participants and non-participants. All in all, we presume that the loss to follow-up did not influence our results to a major degree, but it cannot be excluded that it has attenuated or overestimated the presented associations to some extent. The occupational exposure assessment was based on crude information on job titles and the overall probability of exposures for each job type based in a specific asthma JEM. This lack of access to detailed individual exposure information implies a substantial risk of non- - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

70 BMJ Open Page of differential exposure misclassification. Furthermore we have only job information from one time point in pregnancy (- week), and change in job during pregnancy was not accounted for, which might add to the exposure misclassification as well. Based on information about postnatal exposure where only one fifth of the women changed job between - week of pregnancy and month after birth we will expect that relatively few women change job within the pregnancy, but we cannot say that with certainty. Prenatal exposures were derived from the interview-based job descriptions, where postnatal codes originated from register data from Statistics Denmark based on obligatory information handed in by the employer to the Danish tax authorities. Some inconsistency between the two data sources are to be expected. The self-reported data is probably more precise, but also prone to possible reporting bias. As the exposure group was identical in the interview and the register data for more than 0%, we do not think the different data sources had a major impact of the result. Another clear limitation of this epidemiological study is the lack of specific measures for atopy, e.g. positive skin prick tests or specific IgE. Ever atopic dermatitis in the children was used as proxy for atopy, as this condition is relatively closely linked to elevated serum IgE and often also to specific sensitisation[]. Misclassification is however inherently present. Many atopic subjects (children and adults) defined by a positive SPT or elevated serum IgE, do not have atopic dermatitis but have other manifestations of allergy, and atopy does not always result in atopic dermatitis.. It can be argued that allergic rhinitis is even closer related to IgE mediated sensitisation than atopic dermatitis, but due to the age of the children ( year) only few have developed allergic rhinitis, and therefore allergic rhinitis was not a good - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

71 Page of BMJ Open predictor for atopy in this study. Still, with these weaknesses in mind, we think it is justified to use the presence of atopic dermatitis as a reasonable proxy for atopy among young children. We defined atopic dermatitis by a combination of maternally reported atopic dermatitis and eczema in locations typical for atopic dermatitis. These questions have been validated against a clinical investigation for atopic dermatitis and were deemed suitable for defining atopic dermatitis in large scale epidemiological studies[]. Our definition is not entirely identical to the wording suggested by Benn et al.[]., eczema at year was therefore also included and a slightly different combination of questions applied. Overall, this was expected to increase the sensitivity at the expense of decreased specificity. Diagnosis of asthma in the children was based on maternal reports. Although standardised and validated asthma questions were used[0], some non-differential misclassification would still be expected, and our results might underestimate a true effect. Concluding remarks Our study aimed to add to the current limited available knowledge on the impact of prenatal maternal occupational exposure and asthma in children. It was based on a large population cohort of women and their children. The women had a variety of occupational job titles, making exposure contrast possible, even with a trivial exposure misclassification. In conclusion, maternal occupational exposures during pregnancy do not seem to be a substantial risk factor for the development of asthma in year-old children. Maternal occupational exposure to low molecular weight agents early in the child s life may - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

72 BMJ Open Page 0 of predispose for asthma. Future studies should prioritise exposure characterisation during the both pre- and postnatal period in order to disentangle the importance of the timing of maternal exposure for development of asthma in the children. Funding This study is part of the Danish collaborative MINERVA project ( addressing occupational risks to human reproduction, supported by a grant from the Danish Working Environment Research Fund (contract 0000). Additional funding was obtained from Aarhus University and by the Danish Graduate School of Public Health Science (GRASPH). Competing interests None. Data sharing statement Documentation for how the analysis has been performed can be provided by contact to the corresponding author. The data has been analysed by the authors on a data platform in Statistics Denmark, where register data and DNBC data were merged after an application on the specific register variables of interest were approved by Statistics Denmark. REFERENCE LIST James AL, Knuiman MW, Divitini ML, et al. Changes in the prevalence of asthma in adults since : the Busselton health study. Eur Respir J 0; ():-. Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 00; ():-. Asher MI, Montefort S, Bjorksten B, et al. Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys. Lancet 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

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74 BMJ Open Page of Lehmann I, Thoelke A, Rehwagen M, et al. The influence of maternal exposure to volatile organic compounds on the cytokine secretion profile of neonatal T cells. Environ Toxicol 00; ():0-. Magnusson LL, Wennborg H, Bonde JP, et al. Wheezing, asthma, hay fever, and atopic eczema in relation to maternal occupations in pregnancy. Occup Environ Med 00; ():0-. Tagiyeva N, Devereux G, Semple S, Sherriff A, Henderson J, Elias P, Ayres JG. Parental occupation is a risk factor for childhood wheeze and asthma. Eur Respir J. 0;():-. Braun-Fahrlander C, Riedler J, Herz U, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 00; ():-. Olsen J, Melbye M, Olsen SF, et al. The Danish National Birth Cohort--its background, structure and aim. Scand J Public Health 00; ():00-. Kennedy SM, Le MN, Choudat D, et al. Development of an asthma specific job exposure matrix and its application in the epidemiological study of genetics and environment in asthma (EGEA). Occup Environ Med 000; ():-. Karvonen AM, Hyvarinen A, Gehring U, et al. Exposure to microbial agents in house dust and wheezing, atopic dermatitis and atopic sensitization in early childhood: a birth cohort study in rural areas. Clin Exp Allergy 0; ():-. 0 Asher MI, Keil U, Anderson HR, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J ; ():-. Li X, Sundquist K, Sundquist J. Parental occupation and risk of hospitalization for asthma in children and adolescents. J Asthma 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

75 Page of BMJ Open Tagiyeva N, Anua SM, Semple S, et al. The 'take home' burden of workplace sensitizers: flour contamination in bakers' families. Environ Int 0;:-. Holloway JA, Warner JO, Vance GH, et al. Detection of house-dust-mite allergen in amniotic fluid and umbilical-cord blood. Lancet 000; ():00-. Lim RH, Arredouani MS, Fedulov A, et al. Maternal allergic contact dermatitis causes increased asthma risk in offspring. Respir Res 00;:. Christensen BH, Thulstrup AM, Hougaard KS, et al. Occupational exposure during pregnancy and the risk of hay fever in -year-old children. Clin Respir J 0. Basinas I, Schlünssen V, Heederik D, Sigsgaard T, Smit LA, Samadi S, et al. Sensitisation to common allergens and respiratory symptoms in endotoxin exposed workers: a pooled analysis. Occup Environ Med 0 Feb;():-] Jenkins MA, Hopper JL, Flander LB, et al. The associations between childhood asthma and atopy, and parental asthma, hay fever and smoking. Paediatr Perinat Epidemiol ; ():-. Scholtens S, Wijga AH, Brunekreef B, et al. Maternal overweight before pregnancy and asthma in offspring followed for years. Int J Obes (Lond) 0; ():0-. Laerum BN, Svanes C, Wentzel-Larsen T, et al. Young maternal age at delivery is associated with asthma in adult offspring. Respir Med 00; ():-. 0 Lodrup Carlsen KC, Carlsen KH. Effects of maternal and early tobacco exposure on the development of asthma and airway hyperreactivity. Curr Opin Allergy Clin Immunol 00; ():-. Benn CS, Thorsen P, Jensen JS, et al. Maternal vaginal microflora during pregnancy and the risk of asthma hospitalization and use of antiasthma medication in early childhood. J - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

76 BMJ Open Page of Allergy Clin Immunol 00;0 ():-. Shaheen SO, Newson RB, Henderson AJ, et al. Prenatal paracetamol exposure and risk of asthma and elevated immunoglobulin E in childhood. Clin Exp Allergy 00; ():-. Lowe AJ, Carlin JB, Bennett CM, et al. Do boys do the atopic march while girls dawdle? J Allergy Clin Immunol 00; ():-. Svanes C, Omenaas E, Heuch JM, et al. Birth characteristics and asthma symptoms in young adults: results from a population-based cohort study in Norway. Eur Respir J ; ():-0. Nohr EA, Frydenberg M, Henriksen TB, et al. Does low participation in cohort studies induce bias? Epidemiology 00; ():-. Greene N, Greenland S, Olsen J, et al. Estimating bias from loss to follow-up in the Danish National Birth Cohort. Epidemiology 0; ():-. Hermann C, De Fine ON, Host A, et al. Prevalence, severity and determinants of asthma in Danish five-year-olds. Acta Paediatr 00; ():-0. Bergmann RL, Edenharter G, Bergmann KE, Forster J, Bauer CP, Wahn V, Zepp F, Wahn U Atopic dermatitis in early infancy predicts allergic airway disease at years. Clin Exp Allergy. Aug;():-0.) Benn CS, Benfeldt E, Andersen PK, et al. Atopic dermatitis in young children: diagnostic criteria for use in epidemiological studies based on telephone interviews. Acta Derm Venereol 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

77 Page of BMJ Open A prospective study on mmaternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the year-old children Berit Hvass Christensen MD, ; Ane Marie Thulstrup PhD ; Karin Sørig Hougaard PhD ; Lars R. Skadhauge PhD ; Kirsten Skamstrup Hansen PhD ; Morten FrydenbergPhD ; Vivi Schlünssen PhD,. Department of Public Health, Section of Environment, Occupation and Health, Danish Ramazzini Centre, Aarhus University, Denmark. Department of Occupational Medicine, Danish Ramazzini Centre, Aarhus University Hospital, Denmark. The National Research Center for the Working Environment, Copenhagen, Denmark. Department of Occupational Medicine, Hospital of South-West Jutland, Denmark. Department of Paediatrics, University Hospital Herlev, Denmark. Department of Public Health, Section of Biostatistics, Aarhus University, Denmark. Corresponding author: Vivi Schlünssen Department of Public Health, Section of Environment, Occupation and Health, Danish Ramazzini Centre, Aarhus University, Bartholins Allé, 000 Aarhus C, Denmark. vs@mil.au.dk Phone + Running head: prenatal occupational exposure and asthma Word count:, (text only). Abstract words. Four tables, one figure, online supplement tables. references - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

78 BMJ Open Page of ABSTRACT: Objectives The occurrence of allergic diseases has increased considerably in the last decades. In this prospective follow-up study, we examined whether maternal exposure to asthmogens during pregnancy is associated with the development of asthma in year-old Danish children, taking atopic status into consideration. Methods A total of, women and their children from The Danish National Birth Cohort were categorised according to maternal occupational exposure. Exposure information was obtained by combining job title in pregnancy and months after pregnancy with a commonly used asthma Job Exposure Matrix. Information on asthma in the child was obtained by an internetquestionnaire when the child was years old. Results Prenatal exposure to Low Molecular Weight (LMW) agents was borderline associated to asthma in the children with OR. (0.;.) for atopic and. (0.;.) for non-atopic children. Also Mmaternal postnatal exposure was associated with asthma (OR. (.0;.). After, but after mutual adjustment only postnatal exposure (OR. (0.;.) and the combined effect of pre- and postnatal exposure (OR, (.;.)) seems to increased the risk of asthma in the children. No significant associations were observed for other prenatal or postnatal exposures. Conclusion Maternal occupational exposures during pregnancy do not seem to be a substantial risk factor for the development of asthma in year old children. Maternal pre- and postnatal exposure to LMW agents may predispose the propensity of the children to develop asthma. Future studies should prioritise the characterisation of the timing exposure in relation to the birth. Keywords Prenatal exposure; postnatal exposure delayed effects; Asthma; Prospective cohort study; Allergy; Maternal exposure; Occupational exposure; pregnancy; epidemiology - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

79 Page of BMJ Open ARTICLE SUMMARY Article focus To study the impact of maternal occupational exposure during pregnancy on the Key messages occurrence of asthma among their year-old children Mothers occupational exposures during pregnancy is not a substantial risk factor for asthma in their year old children Maternal pre- and postnatal exposure to LMW agents may increase the risk for asthma in the year old children Future studies should focus on timing of exposure in relation to the birth Strengths and limitations of this study: This study is the largest prospective study so far addressing the impact on childhood asthma of mothers occupational exposure during pregnancy. It is also the first study where atopic status of the child is taken into account when exploring the importance of prenatal occupational exposures on childhood allergic diseases. A substantial loss to follow-up was seen. We presume that the loss to follow-up did not influence our results to a major degree. The occupational exposure assessment was based on crude information on job titles and a general asthma JEM. Definitions of asthma and atopy were based on self-reported informations from the mothers. Still we were able confirm previously described associations between childhood asthma and several risk factors. - Formatted: Left, Space After: pt, Line spacing: Multiple. li BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

80 BMJ Open Page of BACKGROUND The occurrence of asthma, rhinitis, and atopic dermatitis has increased in the last decades, from a few per cent in the 0s to -0% in the 0s[, ]. Lifestyle seems to influence the development of allergic diseases, also during childhood[]. Prenatal risk factors for asthma have been investigated and include environmental tobacco smoking[], stress[] and maternal diet during pregnancy[, ]. Thus, the maternal environment during pregnancy might encompass risk factors for asthma in the child. Allergic diseases are hallmarked by childhood immune dysfunction, but surprisingly, little attention has been paid to prenatal influences on immune development, although the foetal immune system is vulnerable to programming[]. In animals, airborne exposure to particles and aerosols of the mother during pregnancy have been shown to increase asthma susceptibility in the offspring[, ], and findings in some human studies corroborate this association[, ]. Maternal exposure to volatile organic compounds in pregnancy may skew the Th-/Th immune response in the foetus, thereby potentially predisposing the children to asthma later in life[]. An increasing number of women work outside the home during pregnancy, and many are occupationally exposed to asthmogens, i.e. chemical and biological agents that are recognised to increase the risk for work-related asthma. It therefore seems pertinent to investigate whether women s working environments during pregnancy interfere with their children s propensity to develop asthma later in life. Two earlier cohort studies have investigated prenatal occupational exposures and the risk of asthma among adolescent[] and 0- year-old children[] with inconclusive results. Asthma can be characterised as allergic (atopic) or non-allergic (non-atopic). These two phenotypes are characterised by e.g. different risk factors. Endotoxin has for example opposite - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

81 Page of BMJ Open effects in atopic and non-atopic asthma[]. In the present study, we hypothesise that maternal exposure to asthmogens during pregnancy impacts on the foetal development of the immune system and predisposes the children to develop asthma later in life. Specifically, we analysed whether maternal occupational exposures during pregnancy are associated with the development of asthma in year-old Danish children, taking atopic status into consideration. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

82 BMJ Open Page 0 of MATERIAL AND METHODS Population The study population was part of the prospective cohort: The Danish National Birth Cohort (DNBC, Better health for mother and child ). The cohort was established in -00 to study pregnancy outcome and disease in children as a function of factors operating in early life[] ). Women were recruited at their initial pregnancy consultation with their general practitioner or midwife and participation implied four telephone interviews; during week to and week 0 to of pregnancy, and when the child was and months old. When the child was seven years of age, the mothers answered an internet questionnaire on behalf of their children. Mothers without internet access were offered a paper version. The first telephone interview comprised questions about work, atopic diseases and risk factors for asthma (e.g. smoking, use of medication, parity). The questionnaire at age dealt with asthma, rhinitis and atopic dermatitis in the child and risk factors (e.g. parental smoking). A total of 0, pregnancies were enrolled in the cohort. The first telephone interview was answered, times. A total of, women were no longer pregnant at the time of the interview and were excluded. Only singletons and only one pregnancy per woman (the first in the sampling period) were included, so,0 siblings and, children from multiple births were excluded. This leaves a total of. mother/child pairs, of which the internet questionnaire at age was answered for, children. Among these,, pairs were excluded due to lack of information on maternal occupational status. Thus, mother/child pairs were eligible for analysis. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

83 Page of BMJ Open Occupational exposure Data on job exposure during pregnancy was obtained by combining reported information on jobtitle (DISCO codes) from telephone interview one during - week of pregnancy and a commonly used asthma Job Exposure Matrix (JEM)[] based on known risk factors for occupational asthma. The job titles were coded according to the Danish International Standard classification of Occupations, the Danish edition of ISCO- (DISCO-code). Seven exposure categories were formed and consisted of the following occupations and typical allergens/irritants:. High molecular weight (HMW) agents (veterinarians, gardeners, and bakers; grass pollen, animal dander, flour/grain). Low molecular weight (LMW) agents or irritants (cooks, cleaners, hairdressers, dentistry and all kinds of manufacturing of dust-producing materials; aerosols, dust from manufacturing of wood/ textile/ stone/ rubber/ plastics etc.). Mixed HMW and LMW agents (health care professionals; antibiotics, latex, cleaning agents). Farmers (organic dust, grass pollen, animal dander, ammonia). Students (no or unknown exposure). Unclassifiable (subjects where the same job title involved several different environments, e.g. waiters, engineering technicians, biologists, shop managers; diverse exposure). Reference (office workers, teachers and journalists; no or low exposure) The applied JEM was slightly modified from[] by Christensen BH and Schlünssen V to comply with working conditions in Denmark based on a priori knowledge of exposures to asthmogens in the Danish working environment. As an example, medical doctors were originally categorised as not being exposed to asthmogens, but were reclassified to the mixed group in the modified version of the JEM. Furthermore, farmers were identified as a special group as early life exposure - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

84 BMJ Open Page of to a farming environment has shown to offer protection against allergic diseases[]. Also, students were considered as a special group due to possible selection related to health. Postnatal occupational exposure groups were established using the -digit DISCO codes months after the birth of the child, i.e. at a time when maternity leave was terminated and the mother would most probably have returned to work. The codes were retrieved from Statistics Denmark, as every adult person ( years) in Denmark is classified annually according to occupation. Maternal postnatal occupation was categorised in the same JEM categories as for the prenatal exposures. Women classified as students during pregnancy, but without a DISCO code indicative of employment after having given birth were not included in the postnatal analyses. Level of education and socioeconomic status (self-employed, employees at the highest level, employees, students, unemployed or not in the work force) were obtained for both the father and the mother (the calendar year preceding the birth year of the child) from Statistics Denmark. Information on the parent with the highest level of education/socioeconomic status defined the level for the family. Outcome We used validated core questions on asthma from the International Study of Asthma and Allergies in Childhood (ISAAC)[0]. Asthma in the child at age was defined as an affirmative answer to one or more of three questions: Has your child had wheezing or whistling in the chest in the last months? ; Has your child ever had asthma? or to Has your child ever been diagnosed with asthma by a doctor? - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

85 Page of BMJ Open Ever atopic dermatitis was used as a proxy for atopy among the children. Information on atopic dermatitis was collected by telephone interview when the child was months old and by questionnaire at age. It was defined as parental report at months of atopic dermatitis ever AND itchy rash in the locations known to be typical for atopic dermatitis AND/OR report of persistent itchy rash in the locations known to be typical for atopic dermatitis at age. Confounders Information on known possible confounders according to prior knowledge from other studies on the effects of asthma in children was obtained primarily from the telephone interview at - weeks of gestation: maternal age, pre-pregnancy BMI, parity, smoking during pregnancy, and furry animals in the home. Maternal atopic disposition was defined as ever reported asthma, rhinitis, and/or atopic dermatitis. Information on maternal use of acetyl salicylic acid, paracetamol, folic acid and antibiotics during pregnancy was retrieved from the second telephone interview at 0- weeks of gestation. Birth weight, gestational age at birth, sex, and singleton status were obtained from the Danish Birth Registry. Statistical methods All analyses were performed in STATA SE.0 (STATA corp., Texas, US). Univariate analyses were performed for categorical variables using χ tests. For comparisons of continuous variables, the independent sample t-test or Kruskal-Wallis test was used. A multivariate unconditional logistic regression analysis was used to evaluate associations between exposure and outcome. A univariate analysis revealed an equal distribution between the exposure groups for some of the - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

86 BMJ Open Page of potential confounders, which was subsequently excluded from the model. The final model contained the following maternal variables: age (-; -; 0-; + years), pre pregnancy BMI ( ; 0-; -; 0+), atopy (yes/no), smoking during pregnancy (no; -; + cig/day), use of medication during pregnancy (yes/no to the use of acetyl salicylic acid, paracetamol, folic acid, and antibiotics), parity (0;-;+) and furry animal ownership during pregnancy (yes/no). For the children, SGA (small for gestational age) (yes/no) and sex were included. SGA was defined as the smallest % of the children born in a specific gestational week based on birth weights from the entire BSMB cohort, and separately for boys and girls. The analysis was repeated after including the children s exposure to environmental tobacco smoke (ETS) in the model according to the information on the parents current smoking. Unless otherwise stated, the level of significance was p < 0.0, two-sided. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

87 Page of BMJ Open RESULTS Table shows the demographic characteristics of the cohort stratified by exposure groups. The women had a mean age of 0 years at inclusion. Students were younger than the other groups, i.e.. % were below 0 years of age. About half (. %) of the women had not given birth previously. Among the students,. % were primiparous. On average,. % of the women had used paracetamol, mostly prevalent in the mixed exposure group (.%), and least prevalent in the farmer group (.%). The majority (.%) of the women were non-smokers during pregnancy. The LMW group had the highest tobacco use with. % smoking + cig/day. A similar pattern was seen at follow up at years of age, but in general fewer mothers smoked at follow up, with a decrease from one in four to one in six. The lowest prevalence of atopy (.%) was seen in the HMW group, whereas the prevalence was highest among students (.%). - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

88 BMJ Open Page of Table Demographics, categorised according to mothers exposure during pregnancy into categories: High molecular weight (HMW), Low molecular weight/irritants (LMW), Mixed (a mixture of HMW and LMW), farmers, unclassifiable, students and references. The numbers are presented as % unless otherwise stated. During pregnancy: HMW LMW Mixed Farmer Unclassifiable Student Reference Missing, % Mother: n = 0 n =,0 n =, n = 0 n =,0 n =, n =, Age, yrs, median (%;0%) (;) (;) 0 (;) 0 (;) (;) (;) 0 (;) 0 BMI, median (%;0%).0 (.;.). (.;0.). (.;.0). (.;.). (.;.).0 (.;.). (.;.). Parity: or Use of medicine: ASA Paracetamol Antibiotics Folic acid Rural residence Furry animals a Smoking: 0.0 no use of tobacco cigarettes/day > 0.0 cigarettes/day Atopy b.,., Child: Boy Birth weight, kg, median (%;0%).0 (.0;.0). (.;.).0 (.;.). (.;.).0 (.;.). (.;.).0 (.;.) Small for gestational age on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

89 Page of BMJ Open At year followup: Maternal smoking Paternal smoking Child atopy d Child ETS exposure on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

90 BMJ Open Page of Table E (online supplement) presents the characteristics among participants and nonparticipants. The distributions of exposure groups were quite similar between participants and non-participants, but with a slightly lower number of LMW-exposed and students among the participating mothers compared to the non-participating mothers. More of the participating mothers were non-smokers during pregnancy, vs. %, they were slightly older (0 vs. years), better educated (academic level, vs. %), belonged to a higher socioeconomic group (employees at the highest level, vs. %), and more were expecting their first child compared to the non-participants, vs. %. Maternal atopic status was similar in the two groups. At birth, children of participating mothers weighed on average 0 g more and the number of SGA children was.0% in participants compared to.% among non-participants. Table : Unadjusted prevalence of asthma stratified for atopy (defined as ever atopic dermatitis) in the year old children (n =,). Exposure group All children n (%) Atopic children n (%*) Non-atopic children n (%*) High molecular (.) (.) (.) Low molecular/ irritants (.) a (.) b (.) c Mixed, (.) (.) (.) Farmer (.0) (.) (.) Unclassifiable 0 (.0) (.) (.) Student (.) (.0) (.) Reference, (.) (.), (.) Total n (%),0 (.), (.) d, (.) *The percentage is the number of atopic (or non-atopic) children within the maternal exposure group. a: P<0.0, group vs. mixed, farmer, unclassifiable, student and reference. b: P<0.0, group vs. mixed, farmer, unclassifiable and reference c: P<0.0, group vs. reference. d: P<0.0 group vs. non-atopic children - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

91 Page of BMJ Open Table shows the prevalence of asthma stratified for atopic status defined as ever atopic dermatitis. The overall prevalence of asthma was. %;. % for atopic and. % for nonatopic children. Children from the LMW and the HMW exposure group had the highest prevalence of asthma, both in total (-%. %), and for the atopic (-. %) and the non-atopic group (-. %). The HMW children had similar prevalences of asthma as the LMW children. Farmers children had the lowest prevalence of asthma, although not significantly different from the other exposure groups. In Table E (online supplement) the prevalence of asthma was further stratified by sex. The prevalence of asthma was higher among boys (%) compared to girls (%), and for both sexes the overall prevalence of asthma was higher for atopics compared to non-atopic. Children from the LMW and HMW exposure groups had the highest prevalence of asthma in boys, for girls this was only true for LMW. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

92 BMJ Open Page 0 of Table : Unadjusted and adjusted logistic regression models showing the association between mothers occupational exposure during pregnancy and asthma in the child at age years stratified by atopic status of the child. Crude OR (% CI) Model Model Outcome Atopic children, Non-atopic children, Atopic children, Non-atopic children, Atopic children, Non-atopic children, n =, n=, n =,0 n =, n =, n = 0, Exposure groupasthma High molecular. (0.;,). (0.;.). (0.;.).0 (0.;.). (0.;.).0 (0.;.) Low molecular/ irritants. (.;.). (.;.). (0.;.). (0.;.). (0.;.).0 (0.;.) Mixed.0 (0.;.).0 (0.;.) 0. (0.;.).00 (0.;.0) 0. (0.;.) 0. (0.;.0) Farmer. (0.;.) 0. (0.;.). (0.;.) 0. (0.0;.) 0. (0.;.) 0. (0.;.0) Unclassifiable 0. (0.;.) 0. (0.;.).0 (0.;.).0 (0.;.).0 (0.;.).00 (0.;.) Student.0 (0.;.).0 (0.;.) 0. (0.;.) 0. (0.;.0) 0. (0.;.0) 0. (0.0;.0) Reference Model includes: the following variables for the mothers: age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy (dichotomous variables for the use of Acetyl salicylic acid, paracetamol, Folic acid and Antibiotics), parity and furry animal ownership during pregnancy. The following variables were included for the children: Birth weight (SGA) and sex of the child. Model includes the adjustments in model and level of education and socioeconomic level. on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

93 Page of BMJ Open Table shows crude and adjusted ORs for the associations between mothers occupational exposure during pregnancy and asthma in the child. In model, levels of education and socioeconomic status were added to the a priori chosen confounders. For HMW agents, ORs were non-significantly increased in the range.-. in both the crude and the adjusted analyses, especially for atopic children. Children of mothers exposed to LMW agents during pregnancy had an increased OR for asthma in the crude analysis for both atopic (OR. (% C.I..;.)) and non-atopic children (OR. (.;.). The same tendency was seen in model with OR. (0.;.) for atopic and. (0.;.) for non-atopic children. The ORs were further decreased in model with OR. (0.;.) for atopic and.0 (0.;.) for non-atopic children. For the other exposure groups, no significant association between maternal exposure and asthma in the children was observed. In the crude analysis a borderline significant association between being student/mixed groups and asthma were seen with OR.0-.0, but without confirmation in model or. The analysis in Table was repeated taking the year-old children s current parental ETS into account. This did not change the results (data not shown). The same was true when paternal atopic status was included. Even though educational level and socioeconomic status (SES) were considered as potential confounders, they were correlated to our exposure variable, partly because the DISCO code was included in the definition of SES. We decided therefore to use model in the ensuing analysis. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

94 BMJ Open Page of We did not find any significant interactions between exposure and atopy when the interaction exposure*atopy was included in the model. Model was repeated after stratification for sex, please see Table E, online supplement. In general similar results were found for boys and girls, i.e. Children of mothers exposed to LMW agents during pregnancy had a borderline increased OR for asthma in both girls (. (0.;.)) and boys (.0 (0.;.)). For girls, the association was significant for atopic children,. (.0;.), where for boys this was true for non-atopic children,. (.0;.). In Table E (online supplement), ORs for covariates in model are displayed with asthma as the outcome of interest. The mother s age was inversely associated to asthma in the child. The children of women < years of age had an increased OR for asthma,.0 (.;.), compared to - year-old women (reference). The risk was further decreased for 0- years, OR 0. (0.;0.) and + year old women, OR 0. (0.0;0.). Maternal prepregnancy BMI was positively associated with asthma with ORs,. (.0;.) and. (.;.) for BMI - and above 0, respectively, compared to BMI 0- (reference). Children with maternal atopic disposition had increased OR for asthma (. (.;.0)). The use of paracetamol and antibiotics during pregnancy was associated with asthma in the children, OR.0 (.0;.) and. (.0;.), respectively. Smoking during pregnancy was positively associated with asthma in a dose-dependent manner with OR.0 (.;.) for - cig/day), and OR. (.0;.) for > cig/day compared to non-smokers (reference). Parity was positively associated with asthma, with OR. (.0;.) for Formatted: Justified, Space After: pt Formatted: Justified, Space After: pt BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

95 Page of BMJ Open pregnancies, and OR. (0.;.) for or more pregnancies compared to no earlier pregnancy (reference), whereas pet ownership was not associated to childhood asthma, OR 0. (0.;.0). Being a boy, and being born SGA was both positively associated with asthma with ORs. (.;.) and. (.0;.), respectively. The analysis in table E was repeated after stratification for atopic status in the children, and almost identical results were seen for atopic and non-atopic children (data not shown). Figure shows the adjusted logistic regression analysis on the association between mothers pre- and postnatal exposure and asthma in the children. For LMW agents, both pre- and postnatal exposure were significantly associated with asthma in the children, ORs. (.00;.) and. (.0-.), respectively. After mutual adjustments only postnatal exposure (OR. (0.;.)) and the combined effect of pre- and postnatal exposure (OR, (.;.)) were associated with asthma in the child. For the other exposure groups, no significant associations were seen for either prenatal or postnatal exposures. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

96 BMJ Open Page of Figure. Adjusted logistic regression analysis on the association between mothers pre- and postnatal exposure and asthma in the year-old children. Reference: reference workers.. OR. 0 HMW LMW Mix Farm UC pre post pre adjusted post post adjusted pre combined Pre: Prenatal exposure. Post: Postnatal exposure. Post just pre: postnatal exposure adjusted for prenatal exposure. Pre just post: Prenatal exposure adjusted for postnatal exposure. Combined: Additive effect of pre-and postnatal exposure. Included in the model: maternal age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy, furry animal ownership during pregnancy, SGA and sex of the child BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

97 Page of BMJ Open Table : Crude and adjusted logistic regression analyses on associations between mothers LMW jobs and asthma in the year old children. Crude Total Adjusted Total Crude Atopic Adjusted Atopic Crude Non-atopic Adjusted Non-atopic (n=,) (n=,0) Children (n=,) Children (n=,0) Children (n=,) Children (n=0,) LMW OR % CI OR % CI OR % CI OR % CI OR % CI OR % CI Plastic work (0).0 (0.;.). (0.;.). (0.;.). (0.;.).00 (0.;.) 0. (0.;.) Paper work () 0. (0.;.) 0. (0.;.) 0. (0.;.0) 0. (0.;.) 0. (0.;.) 0.0 (0.;.) Stone work (). (0.;.) 0. (0.0;.). (0.;.).0 (0.;.) 0. (0.;.) 0. (0.;.) Wood work () 0. (0.;.) 0. (0.;.) 0. (0.;.) 0. (0.;.). (0.;.).0 (0.;.) Food work (0). (.;.). (0.;.).0 (.0;.). (0.0;.). (.;.0). (0.;.) Chemical work ).0 (.0;.).0 (0.;.) 0. (0.;.) 0. (0.;.). (.0;.0). (0.;.) Cleaning (). (.0;.).0 (0.;.). (.;.). (0.;.). (0.;.) 0. (0.;.) Metal work (). (0.;.0). (0.0;.). (0.;.). (0.;.). (0.;.0). (0.;.) Electronic works (0). (0.;.) 0. (0.;.0). (0.;.0). (0.;.).0 (0.;.) 0. (0.0;.) Hairdressing (). (.0;.). (0.;.). (0.;.). (0.;.).0 (.0;.). (0.;.) Textilework (). (0.;.0). (0.;.). (0.;.). (0.;.). (0.;.0). (0.0;.00) Assembly work (). (.00;.). (0.;.). (0.;.).0 (0.;.0). (.0;.).0 (0.;.) Transport work (). (.0;.). (0.;.). (0.;.).0 (0.;.). (.0;.0). (0.0;.) Reference (,) Models adjusted for: Maternal age, -BMI, -smoking during pregnancy, -atopic status, -use of medicine during pregnancy, - furry animal ownership during pregnancy, parity, sex of the child and the child being small for gestational age. on December 0 by guest. Protected by copyright. - Formatted Table Formatted Table BMJ Open: first published as./bmjopen on April 0. Downloaded from

98 BMJ Open Page of The association between maternal specific LMW occupations (included in the LMW exposure group) and asthma in the children is displayed in Table. In the adjusted analysis, hair dressing was borderline associated with asthma in the child, OR. (0.;.) without any significant difference between atopic and non-atopic children. For atopic children, cleaning was borderline associated with asthma with OR. (0.;.). A similar pattern was observed for non-atopic children and mother s work in the chemical industry, OR. (0.;.). The above analysis was repeated after stratification for sex, please see Table E, online supplement. Atopic boys of mothers working as cleaners during pregnancy (.(.;.) and non-atopic girls of women working in the chemical industry (OR. (.;.0) had increased OR for asthma. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

99 Page of BMJ Open DISCUSSION Key findings We found, that maternal occupational exposure to LMW agents during and after pregnancy may be associated to asthma in year-old children. Sub analyses suggested associations between a few specific LMW jobs (hairdressing, cleaning and chemical industry) and asthma in the children. Maternal prenatal - and postnatal LMW exposures were associated with childhood asthma, but after mutual adjustments, postnatal exposure seemed more significant. How do the findings fit with earlier study results? In a Swedish multigenerational register study,,000 year-old children with a st hospitalisation for asthma were identified[]. The children were then linked to national census data on parental (maternal if available, otherwise paternal) occupation prior to birth of the child. Standardised incidence ratios (SIRs) of.0 or higher were found for hospitalisation due to asthma for boys of beverage manufacturers, packers/loaders, cooks/stewards, home helpers and building caretakers/cleaners. We confirmedalso found that atopic boys of cleaners had an increased OR for asthma (OR.0 (.;.). For girls, SIRs of.0 or higher was found for chemical process workers, cooks/stewards and waiters. In the present study, we could confirm adjusted increased ORs for asthma in year-old girls of chemical workers (OR. (.0;.). Two previous birth cohort studies investigated prenatal occupational exposures and risk of asthma and wheeze in the children. In a study among, year old adolescents between years of age Magnusson et al.[] concluded that some maternal jobs (e.g. - Formatted: Font: Bold Formatted: Space After: 0 pt Formatted: Font: Bold Formatted: Space After: 0 pt Formatted: Font: Bold Formatted: Font: Bold BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

100 BMJ Open Page of bakers, dental assistants, technicians, librarians, bookbinders) during pregnancy were associated with en elevated risk of wheeze or asthma in the children. Prenatal vs. postnatal effects In a cohort of,000 children from the ALSPAC study, Tagiyeva et al.[] found that maternal occupational exposure to latex and biocides/fungicides was associated with an increased likelihood of asthma in year-old children. Adjusting for Inclusion of maternal postnatal exposure in the statistical model made the prenatal effects disappear. Our results even suggest that prenatal exposure attenuated the otherwise positive association between postnatal exposure and asthma. Furthermore, paternal occupational exposure to flour dust after birth was associated with an increased likelihood of childhood wheeze and asthma. These findings are in line with our results, indicating that postnatal maternal exposure seems to influence childhood asthma more than prenatal exposure. These results should however be interpreted with caution, since pre- and postnatal exposures are closely correlated. Most mothers worked within the same occupation during the toddler period, i.e. only one fifth of the included women changed occupational exposure group from early pregnancy and until months after childbirth. The effect of postnatal parental occupation on childhood asthma has been suggested to occur due to a carrier home effect[], implicating that the parents carry home components of the work environment leading to indirect exposure of the child from the parental work environment. Effects of maternal exposure during pregnancy are also biologically plausible. Antigens may be transferred from the placenta or via the amniotic fluid and cause the sensitisation of the foetus to take place[, ]. Allergen - Formatted: Font: Bold Formatted: Space After: 0 pt BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

101 Page of BMJ Open exposure during pregnancy can also promote an excessive or prolonged Th-skewed immunity after birth. This exaggerates the normal dominance of Th during and after pregnancy, and increases the propensity of developing allergy[, ]. Also other postnatal modifying factors are to be considered, such as breastfeeding, early life day care, number of siblings and ETS. In our study, only 0.% of the women had never breastfed their children, and more than 0% of the children attended day care from a very early age, making it unlikely that these potential risk factors have influenced our results considerably. Adjusting for postnatal ETS and parity did not change our estimates. Implications of atopic status and sex of the child As far as we know, this is the first study to take atopic status in the children into consideration when studying the impact of maternal work on asthma in children. We did not find convincing differences between atopic and non-atopic children for associations between maternal occupational exposures and asthma in the children. The present study thus corroborates the results from our study on hay fever[] in the same mother/child pairs. We did however notice non-significantly increased ORs for atopic children in mothers exposed to HMW agents and mothers with cleaning jobs compared to ORs close to for the non-atopic children. We also noticed an insignificantly increased OR for non-atopic children among mothers working in the chemical industry compared to atopic children. The Impact of offspring atopic status on associations between environmental exposures and asthma has in been earlier studies been emphasised before, i.e. for domestic exposures. oothers have found that exposure to endotoxin early in life may protect against the development of atopic asthma, but not which is not true for non-atopic asthma[, ]. - Formatted: Font: Bold Formatted: Font: Bold BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

102 BMJ Open Page 0 of Exposure to maternal domestic cleaning has been found to increase the risk for persistent wheeze only in non-atopic children in the ALSPAC study[]. As expected, we found a higher prevalence of asthma among boys compared to girls, but in general sex did not modify the effect of maternal work during pregnancy on the development of offspring asthma. In a few analyses stratified by both atopy and sex differences were found for boys and girls, but our results are too vague to support a specific gender effect. Implications of socioeconomic status Even though educational level and SES were considered as potential confounders, they were correlated to our exposure variable, and furthermore SES and educational level were positively associated to asthma with OR. (.-.) for asthma among unemployed compared to self-employed and OR 0. (0.-0.) for women with an academic education compared to compulsory schooling. We therefore decided to exclude educational level and SES from the final model due to risk for over adjustment. Strengths and limitations of the study To our knowledge, the present study is the largest of its kind so far, regarding maternal occupational exposure to asthmogens during pregnancy and asthma in the children. The DNBC data-collection was performed prospectively., i.e. exposure status was collected before the outcome was known. Our results confirm previously described associations between childhood asthma and several risk factors, i.e. maternal atopic disposition[], pre- - Formatted: Font: Bold Formatted: Font: Bold BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

103 Page of BMJ Open pregnancy BMI[], age[], gestational smoking[0], and use of medication[, ] as well as offspring sex[], and SGA[]. A number of limitations have to be considered as well. In The DNBC cohort, non-smoking, older and well-educated women are overrepresented compared to the general Danish population[]. A substantial loss to follow-up is present, was seen, i.e. nearly 0% of the original cohort was not included in our analyses, and only % (, out of. eligible mother/child pairs) were included in the current analysis. We found the same characteristics among the loss to follow-up subjects in the current study compared to the DNBC cohort in general,, i.e. more of the participants were better educated, smoked less and belonged to a higher socioeconomic group. Otherwise, only small differences were seen according to age, parity and birth weight. For more outcomes, for example childhood asthma, register data has indicated that associations in DNBC are not biased by non-participation[, ]. Furthermore, we have adjusted for some of the obvious confounders in the analysis, and we found similar associations between maternal exposure and asthma for smokers and nonsmokers. For smokers, the association between LMW and childhood asthma was, however mostly seen among non-atopic children,. (.0-.) whereas for non-smokers the association was strongest for atopic children, OR. (0.-.). The asthma prevalence found in this study is comparable to other Danish prevalence data on childhood asthma with a similar asthma definition[]. We are aware that mothers with allergic diseases may avoid certain types of occupations, and employees with work-related asthma or allergy may be advised to avoid or reduce occupational exposure leaving nonatopics in risk occupations (healthy worker effect). However, the proportion of women with atopy did not differ between the exposure groups (except in students) and did not differ - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

104 BMJ Open Page of between participants and non-participants. All in all, we presume that the loss to follow-up did not influence our results to a major degree, but it cannot be excluded that it has attenuated or overestimated the presented associations to some extent.. The occupational exposure assessment was based on the crude information on job titles and the overall probability of exposures for each job type based in a specific asthma JEM. This lack of access to detailed individual exposure information implies a substantial risk of nondifferential exposure misclassification. Furthermore we have only job information from one time point in pregnancy (- week), and change in job during pregnancy was not accounted for, which might add to the exposure misclassification as well. Based on information about postnatal exposure where only one fifth of the women changed job between - week of pregnancy and month after birth we will expect that relatively few women change job within the pregnancy, but we cannot say that with certainty. Prenatal exposures were derived from the interview-based job descriptions, where postnatal codes originated from register data from Statistics Denmark based on obligatory information handed in by the employer to the Danish tax authorities. Some inconsistency between the two data sources are to be expected. The self-reported data is probably more precise, but also prone to possible reporting bias. As the exposure group was identical in the interview and the register data for more than 0%, we do not think the different data sources had a major impact of the result. Another clear limitation of thise epidemiological current study is the lack of objective and specific measures for atopy, e.g. positive skin prick tests or analyses of specific IgE. Ever atopic dermatitis in the children was used as proxy for atopy, as this condition is relatively - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

105 Page of BMJ Open closely linked to elevated serum IgE and often also to specific sensitisation[]. Misclassification is however inherently present. Many atopic subjects (children and adults) defined by a positive SPT or elevated serum IgE, do not have atopic dermatitis but have other manifestations of allergy, and atopy does not always result in atopic dermatitis.. It can be argued that allergic rhinitis is even closer related to IgE mediated sensitisation than atopic dermatitis, but due to the age of the children ( year) only few have developed allergic rhinitis, and therefore allergic rhinitis was not a good predictor for atopy in this study. Still, with these weaknesses in mind, we think it is justified to use the presence of atopic dermatitis as a reasonable proxy for atopy among young children. We defined atopic dermatitis by a combination of maternally reported atopic dermatitis and eczema in locations typical for atopic dermatitis. These questions have been validated against a clinical investigation for atopic dermatitis and were deemed suitable for defining atopic dermatitis in large scale epidemiological studies[]. Our definition is not entirely identical to the wording suggested by Benn et al.[]., eczema at year was therefore also included and a slightly different combination of questions applied. Overall, this was expected to increase the sensitivity at the expense of decreased specificity. Diagnosis of asthma in the children was also based on maternal reports. Although standardised and validated asthma questions were used[0], some non-differential misclassification would still be expected, and our results might underestimate a true effect. Concluding remarks - Formatted: Space After: 0 pt Formatted: Font: Bold BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

106 BMJ Open Page of Our study aimed to add to the current limited available knowledge on the impact of prenatal maternal occupational exposure and asthma in children. It was based on a large population cohort of women and their children. The women had a variety of occupational job titles, making exposure contrast possible, even with a trivial exposure misclassification. In conclusion, maternal occupational exposures during pregnancy do not seem to be a substantial risk factor for the development of asthma in year-old children. Maternal occupational exposure to low molecular weight agents early in the child s life may predispose for asthma. Future studies should prioritise exposure characterisation during the both pre- and postnatal period in order to disentangle the importance of the timing of maternal exposure for development of asthma in the children. Funding This study is part of the Danish collaborative MINERVA project ( addressing occupational risks to human reproduction, supported by a grant from the Danish Working Environment Research Fund (contract 0000). Additional funding was obtained from Aarhus University and by the Danish Graduate School of Public Health Science (GRASPH). Competing interests None. Data sharing statement Documentation for how the analysis has been performed can be provided by contact to the corresponding author. The data has been analysed by the authors on a data platform in Statistics Denmark, where register data and DNBC data were merged after an application on the specific register variables of interest were approved by Statistics Denmark. REFERENCE LIST Field Code Changed BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

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109 Page of BMJ Open Karvonen AM, Hyvarinen A, Gehring U, et al. Exposure to microbial agents in house dust and wheezing, atopic dermatitis and atopic sensitization in early childhood: a birth cohort study in rural areas. Clin Exp Allergy 0; ():-. 0 Asher MI, Keil U, Anderson HR, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J ; ():-. Li X, Sundquist K, Sundquist J. Parental occupation and risk of hospitalization for asthma in children and adolescents. J Asthma 00; ():-. Tagiyeva N, Anua SM, Semple S, et al. The 'take home' burden of workplace sensitizers: flour contamination in bakers' families. Environ Int 0;:-. Holloway JA, Warner JO, Vance GH, et al. Detection of house-dust-mite allergen in amniotic fluid and umbilical-cord blood. Lancet 000; ():00-. Lim RH, Arredouani MS, Fedulov A, et al. Maternal allergic contact dermatitis causes increased asthma risk in offspring. Respir Res 00;:. Christensen BH, Thulstrup AM, Hougaard KS, et al. Occupational exposure during pregnancy and the risk of hay fever in -year-old children. Clin Respir J 0. Basinas I, Schlünssen V, Heederik D, Sigsgaard T, Smit LA, Samadi S, et al. Sensitisation to common allergens and respiratory symptoms in endotoxin exposed workers: a pooled analysis. Occup Environ Med 0 Feb;():-] Jenkins MA, Hopper JL, Flander LB, et al. The associations between childhood asthma and atopy, and parental asthma, hay fever and smoking. Paediatr Perinat Epidemiol ; ():-. Scholtens S, Wijga AH, Brunekreef B, et al. Maternal overweight before pregnancy and asthma in offspring followed for years. Int J Obes (Lond) 0; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

110 BMJ Open Page of Laerum BN, Svanes C, Wentzel-Larsen T, et al. Young maternal age at delivery is associated with asthma in adult offspring. Respir Med 00; ():-. 0 Lodrup Carlsen KC, Carlsen KH. Effects of maternal and early tobacco exposure on the development of asthma and airway hyperreactivity. Curr Opin Allergy Clin Immunol 00; ():-. Benn CS, Thorsen P, Jensen JS, et al. Maternal vaginal microflora during pregnancy and the risk of asthma hospitalization and use of antiasthma medication in early childhood. J Allergy Clin Immunol 00;0 ():-. Shaheen SO, Newson RB, Henderson AJ, et al. Prenatal paracetamol exposure and risk of asthma and elevated immunoglobulin E in childhood. Clin Exp Allergy 00; ():-. Lowe AJ, Carlin JB, Bennett CM, et al. Do boys do the atopic march while girls dawdle? J Allergy Clin Immunol 00; ():-. Svanes C, Omenaas E, Heuch JM, et al. Birth characteristics and asthma symptoms in young adults: results from a population-based cohort study in Norway. Eur Respir J ; ():-0. Nohr EA, Frydenberg M, Henriksen TB, et al. Does low participation in cohort studies induce bias? Epidemiology 00; ():-. Greene N, Greenland S, Olsen J, et al. Estimating bias from loss to follow-up in the Danish National Birth Cohort. Epidemiology 0; ():-. Hermann C, De Fine ON, Host A, et al. Prevalence, severity and determinants of asthma in Danish five-year-olds. Acta Paediatr 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

111 Page of BMJ Open Bergmann RL, Edenharter G, Bergmann KE, Forster J, Bauer CP, Wahn V, Zepp F, Wahn U Atopic dermatitis in early infancy predicts allergic airway disease at years. Clin Exp Allergy. Aug;():-0.) Benn CS, Benfeldt E, Andersen PK, et al. Atopic dermatitis in young children: diagnostic criteria for use in epidemiological studies based on telephone interviews. Acta Derm Venereol 00; (): BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

112 BMJ Open Page 0 of BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

113 Page of BMJ Open Table E. Comparison of participants and non-participants, the latter defined as women and children (first pregnancy and singleton) included in BMSB with valid information from the st questionnaire, but not included in the final study population Participants Non-participants RR Total number..0 Mothers: Exposure groups: (n=,) (n=,0) high molecular (0.;.) low molecular/ irritants.. 0. (0.;0.0) mixed...0 (.0;.0) farmer (0.;.0) unclassifiable..0. (.0;.) student.. 0. (0.;0.) reference...0 (.00;.0) Smoking during pregnancy: no use of tobacco...0 (.0;.) - cigarettes/day (0.;0.) > cigarettes/day.. 0. (0.;0.) Rural residence (0.;0.) Furry animals.. 0. (0.0;0.) Age, median (%;0%) 0 (;) (;) BMI, median (%;0%). (.;.). (.;.) Atopic (0.;.00) Parity: (.0;.0) (0.;0.) (0.;0.) Level of education: no or unknown (0.;0.) compulsory schooling.. 0. (0.;0.) beyond compulsory schooling...0 (.0;.0) academic level..0. (.;.) Socioeoconomic level: (n=,) (n=,0) self-employed.. 0. (0.;0.) employees at the highest level.0.. (.;.) employees (0.;0.) students (0.;0.) unemployed, not in work force (0.;0.) Fathers: Atopic...0 (.0;.0) Child: Boy..0.0 (0.;.0) Birth weight, kg, median (%;0%).0 (.;.). (.;.) Small For Gestational Age (0.;0.) - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

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115 Page of BMJ Open Table E: Unadjusted prevalence of asthma stratified by sex and atopy (defined as ever atopic dermatitis) in the year old children (n =,). Boys Girls Total n(%) Atopic n(%) Non-atopic n(%) Total n(%) Atopic n(%) Non-atopic n(%) High molecular (.) (.) (0.) (.) (.) (.) Low molecular/ irritants 0 (.) 0 (.) (.) (.) (0.) (.) Mixed (.) 0 (.) (.) 0 (.) (.) 0 (.) Farmer (.) (.) 0 (.) (.) (.0) (.) Unclassifiable (.) (.0) (.) (.) (.) (.) Student (.) (.) 0 (.) (.) (.) (.) Reference, (.) (.), (.), (.) 0 (.) (.) Total n (%), (.),0 (.),0 (.), (.) (.), (.) : P<0.0 group vs. unclassifiable and reference : P<0.0 group vs. mixed, unclassifiable and reference : P<0.0 group vs. reference : P<0.0 group vs. mixed, unclassifiable, students and reference : P<0.0 group vs. farmer, unclassifiable, students and reference : P<0.0 group vs. farmer, unclassifiable and reference on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

116 BMJ Open Page of Table E: Unadjusted and adjusted logistic regression models (OR (% C.I.) showing the association between mothers occupational exposure during pregnancy and asthma in the child at age years stratified by atopic status and sex of the child. BOYS Total Atopic Non-atopic Crude (n=,0) Adjusted (n=,) Crude (n=,) Adjusted (n=,) Crude (n=,) Adjusted (n=,) High molecular. (.00;.). (0.;.). (.00;.). (.0;.). (0.;.). (0.;.) Low molecular/ irritants. (.;.0).0 (0.;.). (.0;.). (.0;.). (.0;.).00 (0.;.) Mixed.0 (0.;.) 0. (0.;.0). (0.;.).0 (0.;.).0 (0.0;.) 0. (0.;.0) Farmer 0. (0.;.) 0.0 (0.;.0) 0. (0.0;.). (0.;.) 0. (0.;.) 0. (0.;.) Unclassifiable 0. (0.;.) 0. (0.;.) 0. (0.;.) 0. (0.;.).00 (0.;.).0 (0.;.) Student. (0.;.) 0. (0.;.).0 (0.;.) 0. (0.;.). (0.;.0) 0. (0.;.) Reference GIRLS Total Atopic Non-atopic Crude (n=0,) Adjusted (n=,) Crude (n=,) Adjusted (n=,) Crude (n=,0) Adjusted (n=,) High molecular 0. (0.;.) 0.0 (0.;.0) 0. (0.;.) 0. (0.0;.).0 (0.;.) 0. (0.;.) Low molecular/ irritants. (.;.0). (0.;.).0 (0.;.) 0.0 (0.;.). (.0;.). (.0;.) Mixed. (.0;.).0 (0.;.0).0 (0.;.) 0. (0.;.). (.0;.). (0.;.) Farmer 0. (0.;.0) 0. (0.;.).0 (0.;.). (0.0;.0) 0.0 (0.0;.) 0. (0.;.) Unclassifiable 0. (0.;.).0 (0.;.). (0.;.). (0.;.) 0. (0.;.).0 (0.0;.) Student.0 (0.;.) 0. (0.;.0).0 (0.;.) 0. (0.;.).0 (0.;.0) 0. (0.;.) Reference on December 0 by guest. Protected by copyright. - BMJ Open: first published as./bmjopen on April 0. Downloaded from

117 Page of BMJ Open Table E: Adjusted logistic regression models on mothers occupational exposure and other potential risk factors, and asthma in the child at age. N =, Asthma risk, adjusted OR (%CI). Exposure: high molecular. (0.;.) low molecular/ irritants. (.00;.) mixed.00 (0.;.0) farmer 0. (0.;.) unclassifiable.0 (0.0;.) student 0. (0.;.0) reference Age: -.0 (.;.) (0.;0.) + 0. (0.0;0.) Pre-pregnancy BMI: 0. (0.;.00) (.0;.) 0+. (.;.) Maternal atopy:. (.;.0) Furry animals during pregnancy: 0. (0.;.0) Parity: 0 -. (.0;.) +. (0.-.) Use of medicine during pregnancy: folic acid.0 (0.;.) paracetamol.0 (.0;.) acetyl salicylic acid 0. (0.;.0) antibiotics. (.0;.) Smoking: no smoking - cigarettes/day.0 (.;.) > cigarettes/day. (.0;.) Sex (male):. (.;.) SGA. (.0;.) Included in the model: maternal age, pre-pregnancy BMI, atopy, smoking during pregnancy, use of medication during pregnancy, parity, furry animal ownership during pregnancy, parity, SGA and sex of the child. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

118 BMJ Open Page of Table E. Crude and adjusted logistic regression analyses on associations between mothers LMW jobs and asthma in the year old children stratified by sex. OR (% C.I.) BOYS Total, n=, Atopic, n=, Non-atopic, n=, Plastic work.0 (0.;.). (0.;.) 0. (0.;.) Paper work 0. (0.;.) 0. (0.;.) 0. (0.;.) Stone work 0. (0.;.). (0.;.0) 0.0 (0.;.0) Wood work 0. (0.;.) 0. (0.0;.0) 0. (0.;.) Food work.0 (0.0;.). (0.;.).0 (0.;.) Chemical work 0. (0.;.) 0. (0.;.). (0.0;.) Cleaning. (0.;.). (.;.).0 (0.;.) Metal work. (0.;.). (0.;.).00 (0.;.) Electronic work 0. (0.0;.). (0.;.) 0. (0.0;.) Hairdressing. (0.;.). (0.;.). (0.;.) Textile work 0. (0.;.). (0.;.) 0.0 (0.;.) Manufacturing work. (0.;.). (0.;.0). (0.;.) Transport work. (0.;.). (0.;.0). (0.;.) Reference GIRLS Total, n=.0 Atopic, n=, Non-atopic, n=, Plastic work. (0.0;.) 0. (0.;.). (0.;.) Paper work 0. (0.;.) NA NA. (0.;.) Stone work. (0.;.) 0. (0.0;.). (0.;.) Wood work. (0.;.0) 0.0 (0.0;.0). (0.;.0) Food work. (0.;.). (0.;.). (0.;.) Chemical work. (.0;.).0 (0.;.). (.;.0) Cleaning 0.0 (0.;.) 0. (0.;.) 0. (0.;.) Metal work. (0.;.) 0. (0.;.). (0.;.) Electronic work 0.0 (0.;.) 0. (0.;.0) 0. (0.;.0) Hairdressing. (0.;.0) 0. (0.;.). (0.;.) Textile work. (0.;.0) 0. (0.;.). (0.;.) Manufacturing work.0 (0.;.) 0. (0.;.0). (0.;.0) Transport work. (0.;.0) 0. (0.;.). (0.;.) Reference - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

119 Page of BMJ Open Section/Topic Item # STROBE 00 (v) Statement Checklist of items that should be included in reports of cohort studies Recommendation Reported on page # Title and abstract (a) Indicate the study s design with a commonly used term in the title or the abstract Introduction (b) Provide in the abstract an informative and balanced summary of what was done and what was found Background/rationale Explain the scientific background and rationale for the investigation being reported - Objectives State specific objectives, including any prespecified hypotheses Methods Study design Present key elements of study design early in the paper Setting Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection Participants (a) Give the eligibility criteria, and the sources and methods of selection of participants. Describe methods of follow-up - (b) For matched studies, give matching criteria and number of exposed and unexposed - Variables Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if Data sources/ measurement applicable * For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability of assessment methods if there is more than one group Bias Describe any efforts to address potential sources of bias -; Table E Study size Explain how the study size was arrived at Quantitative variables Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why Statistical methods (a) Describe all statistical methods, including those used to control for confounding - on December 0 by guest. Protected by copyright (online supplement) (b) Describe any methods used to examine subgroups and interactions -;;-; (c) Explain how missing data were addressed Table, page - (d) If applicable, explain how loss to follow-up was addressed Table E (online supplement); page BMJ Open: first published as./bmjopen on April 0. Downloaded from

120 BMJ Open Page of and page (e) Describe any sensitivity analyses -;;-; Results Participants Descriptive data * (a) Report numbers of individuals at each stage of study eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (b) Give reasons for non-participation at each stage (c) Consider use of a flow diagram - * (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential Table, page - confounders (b) Indicate number of participants with missing data for each variable of interest Table (c) Summarise follow-up time (eg, average and total amount) Outcome data * Report numbers of outcome events or summary measures over time Table, table E, page Main results (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, % confidence interval). Make clear which confounders were adjusted for and why they were included Table, figure, Table E, page -0 (b) Report category boundaries when continuous variables were categorized (c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period - Other analyses Report other analyses done eg analyses of subgroups and interactions, and sensitivity analyses -;;-; Discussion Key results Summarise key results with reference to study objectives Limitations Interpretation 0 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from 0 similar studies, and other relevant evidence Generalisability Discuss the generalisability (external validity) of the study results - Other information Funding Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on 0 which the present article is based *Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies. - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

121 Page of BMJ Open Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at Annals of Internal Medicine at and Epidemiology at Information on the STROBE Initiative is available at BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.

122 BMJ Open A prospective study on maternal occupational exposure to asthmogens during pregnancy and the risk of asthma in the year-old children. Journal: BMJ Open Manuscript ID: bmjopen r Article Type: Research Date Submitted by the Author: -Feb-0 Complete List of Authors: Christensen, Berit; Aarhus University, Department of Public Health; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre Thulstrup, Ane Marie; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre Hougaard, Karin; The National Research Center for the Working Environment, Skadhauge, Lars; Hospital of South-West Jutland, Department of Occupational Medicine Frydenberg, Morten; Aarhus University, Department of Public Health Hansen, Kirsten; University Hospital Herlev, Department of Paediatrics Schlünssen, Vivi; Aarhus University, Department of Public Health; Aarhus University Hospital, Department of Occupational Medicine, Danish Ramazzini Centre <b>primary Subject Heading</b>: Occupational and environmental medicine Secondary Subject Heading: Epidemiology, Paediatrics, Reproductive medicine, Respiratory medicine Keywords: OCCUPATIONAL & INDUSTRIAL MEDICINE, EPIDEMIOLOGY, Community child health < PAEDIATRICS, PREVENTIVE MEDICINE, Allergy < THORACIC MEDICINE, Asthma < THORACIC MEDICINE - BMJ Open: first published as./bmjopen on April 0. Downloaded from on December 0 by guest. Protected by copyright.