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Prenatal phthalate exposure associates with low regulatory T-cell numbers and atopic dermatitis in early childhood: Results from the LINA mother-child study

2016; Elsevier BV; Volume: 139; Issue: 4 Linguagem: Inglês

10.1016/j.jaci.2016.09.034

1097-6825

Gunda Herberth, Arkadiusz Pierzchalski, Ralph Feltens, Mario Bauer, Stefan Röder, Sven Olek, Denise Hinz, Michael Borte, Martin von Bergen�, Irina Lehmann,

Immunotoxicology and immune responses

Phthalates serve as binders and plasticizers in everyday items, including cosmetics, household cleaners, food packaging, personal-care products, toys, and many other consumer products. Recent publications show that exposure to these chemicals may contribute to an increased risk of allergy development including asthma and atopic dermatitis.1Gascon M. Casas M. Morales E. Valvi D. Ballesteros-Gomez A. Luque N. et al.Prenatal exposure to bisphenol A and phthalates and childhood respiratory tract infections and allergy.J Allergy Clin Immunol. 2015; 135: 370-378Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 2Just A.C. Whyatt R.M. Perzanowski M.S. Calafat A.M. Perera F.P. Goldstein I.F. et al.Prenatal exposure to butylbenzyl phthalate and early eczema in an urban cohort.Environ Health Perspect. 2012; 120: 1475-1480Crossref PubMed Scopus (78) Google Scholar An immune modulatory capacity of these compounds is assumed but not fully elucidated by now.3Rogers J.A. Metz L. Yong V.W. Review: endocrine disrupting chemicals and immune responses: a focus on bisphenol-A and its potential mechanisms.Mol Immunol. 2013; 53: 421-430Crossref PubMed Scopus (307) Google Scholar Epidemiological studies provide evidence that exposure to phthalates and their metabolites might be more critical in the prenatal period when the fetal immune system is developing.4Whyatt R.M. Rundle A.G. Perzanowski M.S. Just A.C. Donohue K.M. Calafat A.M. et al.Prenatal phthalate and early childhood bisphenol A exposures increase asthma risk in inner-city children.J Allergy Clin Immunol. 2014; 134: 1195-1197.e2Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar According to the fact that regulatory T (Treg) cells are key players in the modulation of immune responses, we asked whether phthalates may affect the number of these cells leading to the observed increased risk to develop atopic dermatitis.2Just A.C. Whyatt R.M. Perzanowski M.S. Calafat A.M. Perera F.P. Goldstein I.F. et al.Prenatal exposure to butylbenzyl phthalate and early eczema in an urban cohort.Environ Health Perspect. 2012; 120: 1475-1480Crossref PubMed Scopus (78) Google Scholar In a recent study, we demonstrated that low number of Treg cells at birth is associated with a higher risk to develop an atopic dermatitis within the first 3 years of life.5Herberth G. Bauer M. Gasch M. Hinz D. Roder S. Olek S. et al.Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.J Allergy Clin Immunol. 2014; 133: 543-550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar In the present investigation, we aimed to evaluate whether a high maternal phthalate burden impacts the number of Treg cells in pregnancy as well as in early childhood. Associations to children's atopic dermatitis were found with the primary metabolites of diethyl, di-n-butyl, butylbenzyl, or di-2-ethylhexyl phthalate.2Just A.C. Whyatt R.M. Perzanowski M.S. Calafat A.M. Perera F.P. Goldstein I.F. et al.Prenatal exposure to butylbenzyl phthalate and early eczema in an urban cohort.Environ Health Perspect. 2012; 120: 1475-1480Crossref PubMed Scopus (78) Google Scholar Therefore, in our study, we measured the concentration of these metabolites (monoethyl phthalate [MEP], monoisobutyl phthalate [MiBP], mono-n-butyl phthalate [MnBP], monobenzyl phthalate [MBzP], mono-(2-ethylhexyl) phthalate [MEHP]) in maternal urine and assessed the relationship to Treg-cell numbers and the development of atopic dermatitis as well as to hay fever and allergic sensitization to food (fx5) and inhalant (sx1) allergens. Data were gained from our prospective birth cohort study LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk). For this study, 629 mother-child pairs (622 mothers, 7 twin pairs) were recruited from March 2006 until December 2008 in Leipzig, Germany.6Hinz D. Bauer M. Roder S. Olek S. Huehn J. Sack U. et al.Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.Allergy. 2012; 67: 380-389Crossref PubMed Scopus (157) Google Scholar Annually, around the birthday of the child, follow-up investigations were performed with questionnaire evaluations for allergic outcomes (atopic dermatitis, hay fever, asthma) and confounders as well as clinical visits including blood collection. Early morning urine samples were collected during the third trimester (gestational age 36 weeks, n = 610) and analyzed for the primary phthalate metabolites (MEP, MiBP, MnBP, MBzP, and MEHP) using a multianalyte procedure as described by Feltens et al.7Feltens R. Roeder S. Otto W. Borte M. Lehmann I. von Bergen M. et al.Evaluation of population and individual variances of urinary phthalate metabolites in terms of epidemiological studies.J Chromatogr Sep Tech. 2015; 6: 290Google Scholar Absolute concentrations of phthalates were calculated on the basis of calibration curves and normalized to urinary creatinine concentrations. The number of Treg cells was determined by FOXP3 methylation-specific real-time PCR in the Treg-cell–specific demethylated region in blood samples from pregnancy (34th gestational week, n = 607), cord blood (n = 448), and from the second year of children's life (n = 331) as described previously.6Hinz D. Bauer M. Roder S. Olek S. Huehn J. Sack U. et al.Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.Allergy. 2012; 67: 380-389Crossref PubMed Scopus (157) Google Scholar This method enables the identification of cells with stable Treg-cell phenotype and function.8Wieczorek G. Asemissen A. Model F. Turbachova I. Floess S. Liebenberg V. et al.Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.Cancer Res. 2009; 69: 599-608Crossref PubMed Scopus (197) Google Scholar Characteristics of the study population, detailed method description, and median values and interquartile ranges for all phthalate metabolites and Treg-cell numbers are given in this article's Online Repository (Tables E2 and E3 at www.jacionline.org). Associations between maternal urine phthalate metabolite concentrations and the number of Treg cells were calculated using a linear regression model adjusted for maternal atopic dermatitis, maternal smoking and/or ETS exposure at home, maternal education, cat ownership, previous births (presence of older siblings), and in addition (only for children) for sex and breast-feeding until 6 months. Out of the analyzed phthalate metabolites, MEP and MiBP were associated with lower Treg-cell numbers in cord blood (adjusted mean ratios [aMR], 0.90, 95% CI, 0.82-1, P = .046 and aMR, 0.91, 95% CI, 0.82-1, P = .047, respectively) and in children aged 2 years (aMR, 0.85, 95% CI, 0.76-0.96, P = .007 and aMR, 0.88, 95% CI, 0.78-0.99, P = .045, respectively) but not in mothers during pregnancy (aMR, 1.02, 95% CI, 0.94-1.12, P = .529 and aMR, 1.07, 95% CI, 0.98-1.17, P = .095, respectively) (Fig 1). We repeated the analysis on the basis of a subgroup of 208 children with available data for maternal urine phthalate concentrations and Treg-cell numbers during pregnancy, as well as Treg-cell measurements in cord blood and at the age of 2 years and gained similar results. In addition here, at the age of 2 years, a reduced number of Treg cells was observed in association with high concentrations of MnBP during pregnancy (see Fig E1 in this article's Online Repository at www.jacionline.org). Although cord blood Treg-cell numbers are only affected by maternal exposure during pregnancy, we cannot exclude that phthalate exposure after birth also contributed to the observed low Treg-cell numbers at the age of 2 years. Furthermore, the impact of high phthalate metabolite levels in pregnancy on the development of atopic dermatitis during the first 3 years in children's life was assessed by calculating odds ratios adjusted for sex, maternal atopic dermatitis, maternal smoking and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months. In our study, 19.5% of children developed an atopic dermatitis within the first 3 years of life. For children who developed an atopic dermatitis during this time, maternal urine MiBP concentrations were significantly higher compared with those in healthy controls (Table I). Furthermore, high maternal MiBP urine concentrations were associated with a higher risk for the child to develop an atopic dermatitis until the age of 3 years (adjusted odds ratio, 2.21; 95% CI, 1.1-4.45; P = .026; Table II). These results sustain earlier publications2Just A.C. Whyatt R.M. Perzanowski M.S. Calafat A.M. Perera F.P. Goldstein I.F. et al.Prenatal exposure to butylbenzyl phthalate and early eczema in an urban cohort.Environ Health Perspect. 2012; 120: 1475-1480Crossref PubMed Scopus (78) Google Scholar, 9Wang I.J. Lin C.C. Lin Y.J. Hsieh W.S. Chen P.C. Early life phthalate exposure and atopic disorders in children: a prospective birth cohort study.Environ Int. 2014; 62: 48-54Crossref PubMed Scopus (77) Google Scholar presenting a relationship between prenatal exposure to phthalate metabolites and an increased risk for atopic dermatitis in early childhood. However, in our study, the metabolite MiBP, but not MBzP like in the mentioned publications, showed this association. This apparent discrepancy may reflect the fact that the studies have been conducted in different countries/continents, for example, New York (United States), Taiwan (Asia) versus our study, which is located in Leipzig (Germany, Europe), where the composition of plastics or consumer products may differ in the distribution of phthalates compared with that in the other continents. In addition, the consumer behavior in the different countries and continents may also impact the phthalate distribution. At least for MiBP it is obvious that in our study the concentrations found in urine are much higher (65.6 μg/g) compared with the concentrations found in urine in Taiwan (15.2 μg/g), the United States (8.1 μg/g), or Mexico (8.4 μg/g).7Feltens R. Roeder S. Otto W. Borte M. Lehmann I. von Bergen M. et al.Evaluation of population and individual variances of urinary phthalate metabolites in terms of epidemiological studies.J Chromatogr Sep Tech. 2015; 6: 290Google ScholarTable IConcentrations of phthalate metabolites in maternal urine (36th gestational week) in regard to the development of atopic dermatitis (physician diagnosed) in the first 3 years of children's lifePhthalate metabolitesAtopic dermatitis 0-3rd year, median (IQR) (ng/mg)∗Concentrations were adjusted for creatinine (in ng per mg of creatinine) to control for urine dilution.P value†P values from Mann-Whitney U test.Yes (n = 85)‡Cases with maternal phthalate measurements in maternal urine (36th gestational week).No (n = 350)‡Cases with maternal phthalate measurements in maternal urine (36th gestational week).MEP60 (28.3-143.9)50.5 (23.9-99.4).155MiBP73.3 (53.2-102.9)§Significant values are marked in boldface (P < .05).62.4 (45.7-90.9)§Significant values are marked in boldface (P < .05)..036§Significant values are marked in boldface (P < .05).MnBP112.7 (78.8-169.7)96.9 (66.7-143.9).057MBzP6.9 (3.7-11.7)6.4 (3.7-10.6).646MEHP7.7 (5.5-11.5)7.2 (5-10.8).438IQR, Interquartile range.∗ Concentrations were adjusted for creatinine (in ng per mg of creatinine) to control for urine dilution.† P values from Mann-Whitney U test.‡ Cases with maternal phthalate measurements in maternal urine (36th gestational week).§ Significant values are marked in boldface (P < .05). Open table in a new tab Table IIRelationship between phthalate metabolites (concentration in maternal urine 36th gestational week) exposure and the development of atopic dermatitis (physician diagnosed) in the first 3 years of children's lifePhthalate metabolitesAtopic dermatitis 0-3rd year; % (n/N), 19.5% (85/435)∗Cases with maternal phthalate measurements in maternal urine (36th gestational week).Crude OR (95% CI)P valueAdjusted OR (95% CI)†Analysis was performed using a logistic regression model with phthalate values categorized into quartiles; odds ratios (ORs) were adjusted for sex, maternal atopic dermatitis, maternal smoking, and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months.P valueMEP1.6 (0.84-3.04).1451.45 (0.75-2.83).268MiBP2.15 (1.11-4.14)‡Significant values are marked in boldface (P < .05)..022‡Significant values are marked in boldface (P < .05).2.21 (1.1-4.45)‡Significant values are marked in boldface (P < .05)..026‡Significant values are marked in boldface (P < .05).MnBP1.79 (0.95-3.37).0721.79 (0.91-3.52).090MBzP1.29 (0.68-2.47).4271.28 (0.65-2.52).470MEHP1.47 (0.77-2.79).2421.5 (0.76-2.98).238∗ Cases with maternal phthalate measurements in maternal urine (36th gestational week).† Analysis was performed using a logistic regression model with phthalate values categorized into quartiles; odds ratios (ORs) were adjusted for sex, maternal atopic dermatitis, maternal smoking, and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months.‡ Significant values are marked in boldface (P < .05). Open table in a new tab IQR, Interquartile range. In addition to the above-described link between MiBP and atopic dermatitis, we could show that exposure to high concentrations of MEP and MnBP during the fetal period was associated with an increased risk to sensitization to food allergens (fx5) at the age of 2 years and in trend also at the age of 1 year (see Table E4 in this article's Online Repository at www.jacionline.org). We furthermore could demonstrate that a high number of Treg cells at birth is protective against sensitization to food allergens at the age of 1 year and a high number of Treg cells at the age of 2 years was associated with a lower risk for sensitization to inhalant allergens at this age (see Table E5 in this article's Online Repository at www.jacionline.org). As Treg cells are keeping immune responses in balance, lower numbers of these cells may facilitate the development of allergic diseases, as we could show earlier for atopic dermatitis.5Herberth G. Bauer M. Gasch M. Hinz D. Roder S. Olek S. et al.Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.J Allergy Clin Immunol. 2014; 133: 543-550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 6Hinz D. Bauer M. Roder S. Olek S. Huehn J. Sack U. et al.Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.Allergy. 2012; 67: 380-389Crossref PubMed Scopus (157) Google Scholar Interestingly, maternal Treg-cell numbers in pregnancy were not associated with phthalate metabolite concentrations, pointing out that the immature immune system of the fetus and toddler is more vulnerable to these chemicals. Although we are not able to provide a mechanistic explanation for the impact of MEP and MiBP on Treg-cell numbers (which might implicate the differentiation and/or proliferation of these cells), our epidemiological findings may encourage for the performance of in vitro assays in this direction in future studies. Taken together, our data suggest that MiBP being associated with reduced numbers of Treg cells may facilitate the development of an atopic dermatitis in early childhood. Because DiBP, the parent compound from which MiBP originates, is widely used in food packages, adhesives, and cosmetics, the usage of these products especially during pregnancy should be avoided. Six-hundred twenty-nine mother-child pairs were recruited within the prospective birth cohort study LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) from March 2006 until December 2008 in Leipzig, Germany. Blood samples were obtained during pregnancy (mother, 34th week of gestation), at birth (venous umbilical cord blood), and annually around the child's birthday. Early morning urine samples were obtained from mothers in the 36th week of gestation. Data on confounding variables, prenatal exposure, lifestyle factors, and children's disease outcomes were gained from questionnaires filled in by the parents 4 weeks before birth and annually thereafter. The present investigation comprises of mothers having urine sample analyses and measurement of Treg cells in blood samples as well as children with Treg-cell measurements in cord blood and at the age of 2 years. Participation was voluntary and informed consent was given by the parents. This study was approved by the Ethics Committees of the University of Leipzig (file references 046-2006, 160-2008). Genomic DNA (gDNA) was isolated from whole blood using the DNA Blood Mini Kit according to manufacturer's instructions (Qiagen, Hilden, Germany). Briefly, 200 μL whole blood was treated with 20 μL Proteinase K (activity, 600 mAU/mL) and incubated with Buffer AL in a ratio of 1:1 (10 minutes, 56°C, 350 rpm). Cell lysis was stopped by adding 200 μL ethanol. The complete sample volume was transferred onto a QIAamp Mini spin column and centrifuged (12,000 rpm). DNA bound to the QIAamp membrane was washed twice using Buffers AW1 and AW2 (12,000 rpm, 1 minute; 13,000 rpm, 5 minutes). DNA was eluted by adding Buffer AE and stored at 4°C until subsequent analysis, applying the mean of the 2 reference genes for normalizing. Bisulfite treatment of gDNA was conducted using EpiTect 96 Bisulfite Kit (Qiagen, Hilden, Germany) as described by the manufacturer. Briefly, 1 μg of gDNA, 85 μL of the Bisulfite Mix, and 35 μL of the DNA Protect Buffer were mixed in a 96-well EpiTect Conversion Plate, and bisulphite conversion was performed in a thermal cycler according to manufacturer's instruction. Converted gDNA was stored at −20°C until further analysis. Quantification of demethylation in Treg-cell–specific demethylated region was performed by real-time PCR by Epiontis (Berlin, Germany; for details, see data in this article's Online Repository at www.jacionline.org). As described previously, the number of Treg cells in blood is presented as percentage corresponding to the measured amount of Treg-cell–specific demethylated region demethylation in the FOXP3 gene.E1Wieczorek G. Asemissen A. Model F. Turbachova I. Floess S. Liebenberg V. et al.Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.Cancer Res. 2009; 69: 599-608Crossref PubMed Scopus (267) Google Scholar Phthalate quantification was carried out for 610 early morning maternal urine samples. In the present study, data for 542 mothers, the subset with Treg-cell measurements in their blood, are presented. Urine samples were collected at 36th weeks' gestation and stored in polypropylene tubes at −80°C until further analysis. Primary phthalate metabolite (MEP, MiBP, MnBP, MBzP, MEHP) quantification was carried out for all samples using a multianalyte procedure as described by Feltens et al.E2Feltens R. Roeder S. Otto W. Borte M. Lehmann I. von Bergen M. et al.Evaluation of population and individual variances of urinary phthalate metabolites in terms of epidemiological studies.J Chromatogr Sep Tech. 2015; 6: 290Google Scholar Absolute concentrations of phthalate metabolites were calculated on the basis of calibration curves and normalized to urinary creatinine concentrations as previously described.E3Remane D. Grunwald S. Hoeke H. Mueller A. Roeder S. von Bergen M. et al.Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid in human urine.J Chromatogr B Analyt Technol Biomed Life Sci. 2015; 998-9: 40-44Crossref Scopus (28) Google Scholar Concentrations are given in ng per mg creatinine. Information on allergic outcomes was collected using questionnaires self-administered by the parents. Atopic dermatitis and hay fever were recorded as physician diagnoses ("Has a doctor diagnosed your child with atopic dermatitis in the last 12 months?"; "Has a doctor diagnosed your child with hay fever in the last 12 months?"). The lifetime prevalence within the first 3 years of life was determined by adding the information of having doctor's-diagnosed atopic dermatitis or hay fever from each year. The control group was defined including children having never developed symptoms or being diagnosed for this disease until the age of 3 years. Allergic sensitization was assessed by the measurement of specific IgE against food allergens (fx5) and inhalant allergens (sx1). Samples were categorized as positive by the cutoff value of more than 0.35 kU/L. The concentrations of specific IgE against food (fx5) and inhalant allergens (sx1) in sera of 1-year-old children were determined by the Phadia CAP System (Phadia GmbH, Freiburg, Germany). The allergen multipanel fx5 consists of hen's egg, cow's milk, wheat, fish, peanut, and soy. Sx1 includes timothy, rye, mugwort, birch, house dust mite (Dermatophagoides pteronyssinus), cat, and dog. Samples with a specific IgE concentration of more than 0.35 kU/L were regarded as positive. Information on maternal history of atopy and prenatal exposure was assessed by detailed questionnaires answered by parents during the third trimester of pregnancy and annually after delivery. To address maternal atopy history, we included asthma, hay fever, and atopic dermatitis ("Did you ever suffer from asthma, allergic rhinitis, atopic dermatitis?"). Smoking or exposure to environmental tobacco smoke at home during pregnancy was recorded with the question "Did you or anybody else smoke inside your dwelling during the last twelve months?" If yes; occasionally, once per week, or daily. Maternal education was assessed by asking "Which is the highest education level you have?" "low, 9 yr of schooling or less" "Hauptschulabschluss"; "intermediate, 10 yrs of schooling" "Mittlere Reife"; high, 12 yrs of schooling or more "(Fach-)hochschulreife." Furthermore, the number of present siblings, the presence of pets (including cats) during pregnancy, and the duration of breast-feeding were recorded. Statistical analyses were performed using Statistica for Windows Version 10.0 (StatSoft Inc [Europe], Hamburg, Germany). The chi-square test for cross-relationship was performed to ensure the equal distribution of parameters in the analyzed subgroups and the entire LINA cohort. All P values of less than .05 were considered to be significant. Because measured phthalate metabolite concentrations and Treg-cell numbers were not normally distributed, a logarithmic transformation was performed. Furthermore, the data were categorized into quartiles and used in the regression models. To analyze the relationship between maternal urine phthalate metabolite concentrations from pregnancy and the number of Treg cells in blood samples from pregnancy, cord blood, and at children's age of 2 years, linear regression models were used. The linear regression models were adjusted for the possible confounding factors maternal atopic dermatitis, maternal smoking and/or ETS exposure at home, maternal education, cat ownership, previous births (presence of older siblings), and also (only for children) for sex and breast-feeding until 6 months. Data are presented as mean ratios, which are the back-transformed effects from the regression model of the logarithmically transformed outcome. Odds ratios were calculated to show the relationship between prenatal phthalate concentrations in maternal urine and the development of atopic dermatitis (physician diagnosed) within the first 3 years of life. The logistic regression models were adjusted for sex, maternal atopic dermatitis, maternal smoking and/or ETS exposure at home, presence of older siblings, maternal education, cat ownership, and breast-feeding until 6 months. Characteristics of the study population are listed in Table E1. There were no differences in the distribution of considered parameters in the analyzed subgroups compared with the entire LINA cohort. Out of the 622 mothers participating in the study, 542 had urinary samples with phthalate metabolite measurements as well as Treg-cell measurements in blood. Out of the 470 children with cord blood samples, in 448 the number of Treg cells was assessed whereas at the age of 2 years, Treg-cell numbers were measured in 331 out of 339 blood samples. In the analyzed subgroup of children with prenatal maternal urine phthalate measurements, 85 (19.5%) developed an atopic dermatitis until the age of 3 years compared with 92 (18.5%) children from the entire LINA cohort.Table E1Characteristics of the analyzed subgroups and the entire LINA cohortParametersEntire LINA cohort, n (%), N = 629Analyzed subgroupsP value∗P value from chi-square test for cross-relationship.Maternal urine samples in pregnancy, n (%), N = 542†Mothers with phthalate and Treg-cell measurements in pregnancy.Cord blood samples at birth, n (%), N = 448‡Children with Treg-cell measurements in cord blood.Blood samples at children's age of 2 y, n (%), N = 331§Children with Treg-cell measurements at the age of 2 years.Sex of the child Male327 (52)282 (52)211 (47.1)165 (49.8).885 Female302 (48.0)260 (48)237 (52.9)166 (50.2)Maternal history of atopyHistory of atopy is defined as occurrence of asthma or atopic dermatitis or hay fever. No330 (53.1)280 (51.7)242 (54)166 (50.2).954 Yes292 (46.9)262 (48.3)206 (46)165 (49.8)Maternal atopic dermatitis No517 (82.2)443 (81.7)373 (83.3)265 (80.1).947 Yes111 (17.6)99 (18.3)75 (16.7)66 (19.9)Maternal education¶Low, 9 years of schooling or less "Hauptschulabschluss"; intermediate, 10 years of schooling "Mittlere Reife"; high, 12 years of schooling or more "(Fach-)hochschulreife." Low24 (3.8)20 (3.7)13 (2.9)6 (1.8).98 Intermediate194 (30.8)165 (30.4)143 (31.9)113 (34.1) High411 (65.3)357 (65.9)292 (65.2)212 (64)Breast-feeding until 6 mo No139 (23.92)112 (20.7)102 (22.8)77 (23.3).987 Yes442 (76.07)386 (71.2)311 (69.4)242 (73.1)Cat ownership during pregnancy No516 (82)443 (81.7)364 (81.2)262 (79.2).957 Yes113 (18)99 (18.2)84 (18.8)69 (20.8)Presence of older siblings No420 (66.8)359 (66.2)298 (66.5)218 (65.9).999 Yes209 (33.2)183 (33.8)150 (33.5)113 (34.1)Smoking during pregnancy#Maternal smoking and/or ETS exposure during pregnancy at home. Never527 (84.7)465 (85.8)383 (85.5)287 (86.7)1 Occasionally43 (6.9)37 (6.8)23 (5.8)20 (6) Once per week4 (0.7)4 (0.7)3 (0.7)1 (0.3) Daily48 (7.7)36 (6.6)36 (8)23 (6.9)Because of missing data, case number may vary for some variables.∗ P value from chi-square test for cross-relationship.† Mothers with phthalate and Treg-cell measurements in pregnancy.‡ Children with Treg-cell measurements in cord blood.§ Children with Treg-cell measurements at the age of 2 years.|| History of atopy is defined as occurrence of asthma or atopic dermatitis or hay fever.¶ Low, 9 years of schooling or less "Hauptschulabschluss"; intermediate, 10 years of schooling "Mittlere Reife"; high, 12 years of schooling or more "(Fach-)hochschulreife."# Maternal smoking and/or ETS exposure during pregnancy at home. Open table in a new tab Table E2Maternal urine phthalate metabolite concentrations (36th gestational week, n = 542, samples with Treg-cell measurements)Phthalate metaboliteMedian (IQR) (ng/mg)∗Concentrations were adjusted for creatinine (in ng per mg of creatinine) to control for urine dilution.MEP50 (24.8-102.3)MiBP66.6 (48.9-99.8)MnBP104.9 (69.9-146.8)MBzP6.7 (3.8-11.6)MEHP7.3 (5.1-11.4)IQR, Interquartile range.∗ Concentrations were adjusted for creatinine (in ng per mg of creatinine) to control for urine dilution. Open table in a new tab Table E3Treg-cell numbers in samples with phthalate measurements as detected by means of demethylation in the FOXP3 geneTreg cellMedian (IQR) (%)∗The number of Treg cells is presented as percentage of Treg cells in whole blood corresponding to the measured amount of TSDR demethylation in the FOXP3 gene. The detection limit of the FOXP3 TSDR demethylation assay is 0.03%; all values were above this limit.Maternal blood (34th gestational week) n = 5420.91 (0.56-1.34)Birth (cord blood) n = 4481.18 (0.75-1.71)Children aged 2 y n = 3313.31 (2.49-4.39)IQR, Interquartile range; TSDR, Treg-cell–specific demethylated region.∗ The number of Treg cells is presented as percentage of Treg cells in whole blood corresponding to the measured amount of TSDR demethylation in the FOXP3 gene. The detection limit of the FOXP3 TSDR demethylation assay is 0.03%; all values were above this limit. Open table in a new tab Table E4Relationship between maternal urine phthalate metabolite concentrations (36th gestational week) and allergic outcomes in the first 3 years of children's lifePhthalate metabolitesAllergic outcomes, adjusted OR (95% CI)∗Analysis was performed using a logistic regression model with phthalate values categorized into quartiles; odds ratios (ORs) were adjusted for sex, parental history of atopy, maternal smoking and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months.Hay feverSensitization to food allergens (fx5)†According to the Pharmacia CAP System, concentrations of >0.35 kU/L were regarded as positive.Sensitization to inhalant allergens (sx1)†According to the Pharmacia CAP System, concentrations of >0.35 kU/L were regarded as positive.0-3rd year,3.6% (17 of 475)First year,15.3% (79 of 515)Second year,12.1% (41 of 340)Third year,13.8% (40 of 289)First year,3.3% (17 of 515)Second year,5.3% (18 of 340)Third year,12.1% (35 of 289)MEP1.05 (0.62-1.76)P = .8571.23 (0.97-1.57)P = .0911.49 (1.05-2.13)‡Significant values are marked in boldface (P < .05).P = .0241.23 (0.88-1.72)P = .2170.95 (0.59-1.53)P = .8561.14 (0.69-1.85)P = .6021.06 (0.75-1.51)P = .729MiBP1.09 (0.66-1.80)P = .7340.97 (0.77-1.23)P = .8351.08 (0.77-1.52)P = .6300.95 (0.69-1.32)P = .7751.16 (0.74-1.83)P = .5121.16 (0.71-1.88)P = .5511.17 (0.82-1.66)P = .370MnBP1.01 (0.61-1.66)P = .9721.24 (0.98-1.58)P = .0651.47 (1.05-2.07)‡Significant values are marked in boldface (P < .05).P = .0251.16 (0.84-1.58)P = .3571.07 (0.68-1.69)P = .7710.94 (0.59-1.50)P = .8031.02 (0.73-1.44)P = .879MBzP0.98 (0.58-1.67)P = .9430.83 (0.65-1.05)P = .1270.95 (0.68-1.33)P = .7800.92 (0.66-1.27)P = .6071.03 (0.65-1.62)P = .9070.97 (0.59-1.58)P = .9020.94 (0.66-1.33)P = .731MEHP1.34 (0.79-2.26)P = .2670.83 (0.65-1.05)P = .6041.23 (0.88-1.72)P = .2121.18 (0.85-1.64)P = .3271.12 (0.72-1.76)P = .5991.29 (0.78-2.11)P = .3141.32 (0.92-1.88)P = .127Note the reduced case numbers for allergic sensitization because blood samples were not available for all children participating in the follow-up investigation.∗ Analysis was performed using a logistic regression model with phthalate values categorized into quartiles; odds ratios (ORs) were adjusted for sex, parental history of atopy, maternal smoking and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months.† According to the Pharmacia CAP System, concentrations of >0.35 kU/L were regarded as positive.‡ Significant values are marked in boldface (P < .05). Open table in a new tab Table E5Relationship between Treg-cell numbers and allergic outcomes in the first 3 years of children's lifeTreg cellsAllergic outcomes, adjusted OR (95% CI)∗Analysis was performed using a logistic regression model with Treg-cell values categorized into quartiles; odds ratios (ORs) were adjusted for sex, parental history of atopy, maternal smoking and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months.Hay feverSensitization to food allergens (fx5)†According to the Pharmacia CAP System, concentrations of >0.35 kU/L were regarded as positive.Sensitization to inhalant allergens (sx1)†According to the Pharmacia CAP System, concentrations of >0.35 kU/L were regarded as positive.0-3rd year,3.6% (17 of 475)First year,15.3% (79 of 515)Second year,12.1% (41 of 340)Third year,13.8% (40 of 289)First year,3.3% (17 of 515)Second year,5.3% (18 of 340)Third year,12.1% (35 of 289)Treg-cellcord blood1.59 (0.72-3.54)P = .2480.72 (0.54-0.96)‡Significant values are marked in boldface (P < .05).P = .0240.97 (0.67-1.41)P = .8820.83 (0.55-1.21)P = .3610.97 (0.54-1.73)P = .9260.71 (0.37-1.39)P = .3211.02 (0.68-1.53)P = .904Treg-cellsecond year0.89 (0.51-1.57)P = .695NA1.02 (0.75-1.39)P = .8690.84 (0.61-1.16)P = .292NA0.71 (0.43-1.15)P = .1690.68 (0.46-0.99)‡Significant values are marked in boldface (P < .05).P = .046NA, Not applicable.∗ Analysis was performed using a logistic regression model with Treg-cell values categorized into quartiles; odds ratios (ORs) were adjusted for sex, parental history of atopy, maternal smoking and/or ETS exposure at home, siblings, maternal education, cat ownership, and breast-feeding until 6 months.† According to the Pharmacia CAP System, concentrations of >0.35 kU/L were regarded as positive.‡ Significant values are marked in boldface (P < .05). Open table in a new tab Because of missing data, case number may vary for some variables. IQR, Interquartile range. IQR, Interquartile range; TSDR, Treg-cell–specific demethylated region. Note the reduced case numbers for allergic sensitization because blood samples were not available for all children participating in the follow-up investigation. NA, Not applicable.