Prenatal maternal anxiety & respiratory infection 1 Effect of Perinatal

Prenatal maternal anxiety & respiratory infection

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Effect of Perinatal Anxiety on Bronchiolitis Modified by ROS-Related Genes

3 Eun Lee 1 *, MD, Hyoung Yoon Chang 2 *, MD, MS, MPH, Kyung-Sook Lee 3 , PhD, Dong In

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Suh

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, MD, Ho-Sung Yu

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, MS, Mi-Jin Kang

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, MS, In Ae Choi

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, MA, Jinah Park

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, PhD, Kyung

5 Won Kim

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, MD, PhD, Youn Ho Shin

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, MD, PhD, Kang Mo Ahn

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, MD, PhD, Ja-Young

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Kwon

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, MD, PhD, Suk-Joo Choi

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, MD, PhD, Kyung-Ju Lee

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, MD, PhD, Hye-Sung Won

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,

7 MD, PhD, Song I Yang

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, MD, Young-ho Jung

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, MD, Hyung Young Kim

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, MD, Ju-Hee

8 Seo 14 , MD, Ji-Won Kwon 15 , MD, Byoung-Ju Kim 16 , MD, PhD, Hyo-Bin Kim 17 , MD, PhD,

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So-Yeon Lee

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, MD, PhD, Eun-Jin Kim

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, PhD, Joo-Shil Lee

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, PhD, Katherine M. Keyes

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PhD, MPH, Yee-Jin Shin

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, MD, PhD

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, Soo-Jong Hong

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, MD, PhD, and the COCOA study group

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*Eun Lee and Hyoung Yoon Chang contributed equally to this work as co-first authors.

** Soo-Jong Hong and Yee-Jin Shin contributed equally to this work as co-corresponding

15 authors.

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Department of Pediatrics, Childhood Asthma Atopy Center, Research Center for

Standardization of Allergic Diseases, Asan Medical Center, University of Ulsan College of

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Medicine, Seoul, Korea

Department of Psychiatry, Ajou University College of Medicine, Gyeonggi-do, Korea

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Department of Rehabilitation, Hanshin University, Gyeonggi-do, Korea

Department of Pediatrics, Seoul National University College of Medicine, Seoul, Korea

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Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Korea

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Sewon Infant Child Development Center, Seoul, Korea


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Department of Pediatrics, Severance Children’s Hospital, College of Medicine, Yonsei

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University, Seoul, Korea

8 Department of Pediatrics, CHA Medical Center, CHA University School of Medicine, Seoul,

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Korea

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Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of

Medicine, Seoul, Korea

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Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul,

Korea

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Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan

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University School of Medicine, Seoul, Korea

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Department of Obstetrics and Gynecology, Asan Medical Center, University of Ulsan

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College of Medicine, Seoul, Korea

13 Department of Pediatrics, Pusan National University Yangsan Hospital, Yangsan, Korea

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Department of Pediatrics, Korea Cancer Center Hospital, Seoul, Korea

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Department of Pediatrics, Seoul National University Bundang Hospital, Seongnam, Korea

Department of Pediatrics, Inje University Haeundae Paik Hospital, Busan, Korea

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Department of Pediatrics, Inje University Sanggye Paik Hospital, Seoul, Korea

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Department of Pediatrics, Hallym University Sacred Heart Hospital, Hallym University

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College of Medicine, Anyang, Korea

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Allergy TF, Department of Immunology and Pathology, Korea National Institute of Health,

Cheongwon, Korea

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Department of Epidemiology, Columbia University Mailman School of Public Health, New

York, USA

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Department of Psychiatry, Gangnam Severance Hospital, Yonsei University College of

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Correspondence to:

Soo-Jong Hong, MD, PhD

Department of Pediatrics, Childhood Asthma Atopy Center, Research Center for

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Standardization of Allergic Diseases, University of Ulsan College of Medicine, 88 Olympicro 43-gil, Songpa-gu, Seoul 138-736, Korea

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Tel: +82.2-3010-3379, Fax: +82.2-473-3725

E-mail: sjhong@amc.seoul.kr

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Co-corresponding author

Yee-Jin Shin, MD, PhD

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Department of Psychiatry, Gangnam Severance Hospital, Yonsei University College of

Medicine, 250 Seongsandong, Seodaumnu-gu, Seoul, 120-752, Korea

Tel: 82-2-2019-3340, Fax: 82-2-3462-4304

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E-mail: yjshin@yuhs.ac


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Abstract

Objectives : Although perinatal anxiety affects the developing immune system and susceptibility to diseases in offspring, studies on gene-environment interactions with respect to exposure to perinatal anxiety are lacking. This study aimed to elucidate the effect and

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71 interaction between perinatal anxiety and polymorphisms in antioxidant defenses and innate immunity genes on the development of respiratory tract infections (RTIs) during the first year

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74 of life.

Study design: Trait anxiety levels in 440 women were assessed by the State-Trait Anxiety

Inventory (STAI) during late gestation. The occurrence of RTIs including bronchiolitis during

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76 the first year of life was assessed by parent-reported doctor diagnosis. Polymorphisms in glutathione S -transferase P-1 ( GSTP1 , rs1695) and CD14 (rs2569190) were genotyped using

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79 the TaqMan assay. Copy number variations of GSTT1 were measured by real-time polymerase chain reaction.

Results: Exposure to higher levels of perinatal anxiety increased the risk of lower RTI

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82 bronchiolitis (adjusted odds ratio [aOR] 1.30, 95% confidence intervals [CI] 1.00–1.80).

Perinatal anxiety increased bronchiolitis risk in infants with AG+GG genotype of GSTP1 or

GSTT1 null genotype (aOR 3.36 and 2.79). In the aspect of TC+CC genotype of CD14 ,

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84 higher levels of perinatal anxiety were associated with the increased risk of upper RTI, lower

RTI, and bronchiolitis (aOR 2.51, 4.60, and 4.31, respectively).

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Conclusions: Perinatal maternal anxiety levels affect the occurrence of bronchiolitis in offspring. The effect of perinatal anxiety on the occurrence of bronchiolitis during infancy

87 was modified by genetic polymorphisms in antioxidant defense and innate immunity genes.

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Keywords: anxiety, bronchiolitis, CD14, perinatal, glutathione S -transferase, polymorphism,

90 respiratory tract infection

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Introduction

Environmental factors during early life can influence the development of physiologic and

immune system and modify disease susceptibility later in life [1]. Recent studies revealed that

perinatal maternal stress and anxiety may affect susceptibility to infectious diseases in later

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life [2]. In particular, respiratory tract infection (RTI), the majority of morbidity and mortality

during infancy, poses a worldwide burden that can be prevented by modification of

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environmental factors [3, 4].

The mechanisms underlying the associations between exposure to perinatal maternal stress and an increased risk of RTIs have not been fully elucidated. Perinatal maternal stress can

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102 cause increases in glucocorticoid levels in pregnant women, which can cross the placenta

barrier into the fetus. This might affect fetal programming through immunomodulation [5]

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105 and perinatal anxiety might cause epigenetic changes in offspring with increased vulnerability

to neurodevelopmental diseases in later life [6]. However, no study has directly demonstrated

the possible mechanisms underlying the association between exposure to perinatal maternal

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108 anxiety and development of RTIs in offspring.

The health effects of perinatal anxiety and RTIs share a common pathway of oxidative stress

[7, 8]. Exposure to perinatal anxiety increases the serum levels of glucocorticoid in offspring

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[9], which generate higher levels of ROS. During RTIs, higher levels of ROS are generated in

the epithelium of the respiratory tract. The increased oxidative stress is associated with

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alterations in immune response and affects fetal programming [10, 11].

Glutathione S-transferase (GST) subfamily plays an important role in the protection against

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115 oxidative stress by catalyzing the conjugation of many compounds with reduced glutathione

[12]. Polymorphisms of

GSTP1 (rs1695) and GSTT1 affect the ability to deal with the

excessive oxidative stress due to its altered enzyme activity [12]. In subjects with AG+GG

116 genotype of GSTP1 (rs1695), enzymatic activity of GSTP1 is partially reduced, compared to


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the subjects with AA genotype [12]. The null genotype of

GSTT1 is associated with an

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absence of enzyme activity [12]. CD14 is essential for the host defense as a receptor for microbial ligands in an innate immune response [13]. A functional polymorphism of

CD14

(rs2569190) has been known to be associated with enhanced immune response and thereby

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increases the risk of RTIs [14, 15]. This suggests that these genetic variants may affect the

development of RTI and environmental factors may exaggerate the effects of these genetic

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125 variations.

Therefore, we hypothesized that there are possible associations between exposure to perinatal anxiety and genetic variants involved in antioxidant defense and innate immunity and these

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127 interactions influence RTI risk in offspring especially during early life. First, we examined the association between perinatal anxiety and RTI occurrence during the first year of life.

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Second, we explored the effect of gene-environment interactions between exposure to perinatal anxiety and genetic variants of some of GST subfamily genes and CD14

(rs2569190) on the development of RTIs.

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Methods

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Participants

We used data from the COhort for Childhood Origin of Asthma and allergic disease

(COCOA), a prospective birth cohort study that aims to investigate the effects of

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137 environmental and genetic factors during early life on the development of allergic diseases

and children’s health [16-18]. Women in their third trimester of pregnancy were enrolled in

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140 this study from January 2009 to September 2011, when the questionnaire included State trait anxiety inventory (STAI) score. The exclusion criteria included pregnant women with pregnancy associated complications (gestational diabetes and pregnancy-induced

141 hypertension), high risk for early delivery, and delivery earlier than 36 weeks. Neonates were


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142 excluded if they needed oxygen therapy; had congenital anomalies including congenital

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144 diaphragmatic hernia, congenital heart disease, congenital lung diseases or severe systemic

diseases. Additional information on this ongoing cohort study are described elsewhere [19].

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Among 734 eligible women, 71 refused to participate, 4 were lost to follow-up, and 28 were

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147 excluded due to preterm delivery. Among a total of 631 (86%), 440 (69.7%) completed the

STAI questionnaire. Missing data on anxiety was unrelated to key demographic covariates,

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150 except for the season of offspring’s birth (Table I). Information on potential confounders was obtained from a parental-reported questionnaire at the 36th week of pregnancy and medical record at child’s birth. Questionnaires were designed to assess maternal health problems

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152 including allergic diseases (atopic dermatitis, allergic rhinitis, or asthma); socioeconomic status (income and educational levels); and prenatal environmental factors such as tobacco

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154 smoking exposure at 36 weeks of gestation of pregnant women. Newborn’s sex and weight, gestational age, season of birth, and health conditions were collected by a questionnaire after

155 birth.

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Written informed consent was obtained from all women and the study was approved by Asan

Medical Center (IRB No. 2008-0616), Samsun Medical Center (IRB No. 2009-02-021),

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Yonsei Univerisity (IRB No. 4-2008-0588), and CHA Medical Center (IRB No. 2010-010).

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Anxiety assessment

Data on perinatal maternal anxiety were obtained by self-report at the 36th week of pregnancy. The Korean version of STAI (K-STAI) was used which consists of two subscales;

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State Anxiety (anxiety about an event) and Trait Anxiety (anxiety levels as a personal characteristic). Among the two subscales, Trait Anxiety subscale (STAI-T) was used in this study, since it reflects the baseline anxiety levels of the subjects rather than a transient state.


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STAI-T is a 20 item questionnaire scored on a four-point Likert-type scale that measures a

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general tendency to be anxious [20]. The score ranges from 20 to 80 with a higher score

indicating a more severe anxiety level. K-STAI has been shown to exhibit excellent

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psychometric properties[21] and its internal consistency has been reported as having a

Cronbach’s α coefficient of 0.91 [22]. In this study, the reliability coefficient (Cronbach’s α)

of STAI-T was 0.92.

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Assessment of 1 year outcome variables

The infants were examined at 1 year of age with at least one parent present at follow-up visits.

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RTI was assessed by parent-reported doctor diagnosis of RTIs during the first year of life.

RTIs were further categorized as upper (URTIs) and lower (LRTIs) RTIs. URTIs included

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179 common colds, sinusitis, otitis media, and croup; LRTIs included pneumonia, tracheobronchitis, and bronchiolitis.

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DNA isolation and genotyping

The genomic DNA was extracted from the buffy coat of the cord blood at delivery using the

Gentra® Puregene® Blood kit (Qiagen, Maryland, USA) as recommended by the

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184 manufacturer. The genotyping of CD14 (rs2569190) and GSTP1 (rs1695) were conducted using a TaqMan assay (ABI, Foster City, CA, USA). Assay identification numbers for CD14

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186 and GSTP1 were C_16043997_10 and C_3237198_20, respectively. Copy number variations

(CNV) of GSTM1 and GSTT1 were measured by real-time polymerase chain reaction. The

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189 primers and probe for GSTM1 and GSTT1 were synthesized according to Brasch-Andersen C,

et al..[23] Reactions were performed as a triplex, with RNAse P as the reference gene. The

allele frequencies of the polymorphisms in GSTP1 (rs1695), GSTM1 , GSTT1 , and CD14

190 (rs2569190) were in Hardy-Weinberg equilibrium ( P > 0.1).


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Statistical analysis

We examined the association between perinatal maternal anxiety levels and RTIs (URTIs,

LRTIs, and bronchiolitis) during the first year of life, using logistic regression. Adjustments

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tobacco smoking during the first year, presence of maternal and paternal allergic diseases [24,

25]. The parental history of allergic diseases was adjusted for in this study since infants with allergic parents are shown to have a higher risk of developing LRTIs [26].

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The unit of STAI score used in the regression analyses was standard deviation (SD) of samples, which was 8.47. Parameter estimates were multiplied by the SD of the STAI score,

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204 and for 95% confidence intervals (CI), the following equation was used: BETA*(SD of STAI score) ± 1.96*SE*(SD of STAI score). In logistic regression analysis, the estimates were exponentiated to obtain the estimate and 95% CI of the odds ratio.

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The combined effects of perinatal anxiety and genetic polymorphisms on RTI risk were investigated by multivariate logistic regression analysis. To analyze the combined effect of the two factors, STAI scores were divided into two categories on the basis of mean levels and

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209 genetic polymorphisms were classified according to: GSTT1 null or present, GSTM1 null or present, and polymorphisms of GSTP1 (rs1695) and CD14 (rs2569190). All statistical

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211 analyses were performed using SAS for Windows (version 9.2).

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Demographics

Results

Table I summarizes the descriptive statistics across the total sample. Overall, mean maternal

215 age was 32.5 years. Exposure to tobacco smoking during pregnancy was reported by 60.8%


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219 was 26.1%. STAI score demonstrated a normal distribution of 41.0 ± 8.47 (mean ± SD).

About half (49.3%) of the offspring were girls, with a mean gestational age of 39.2 weeks.

Births were evenly distributed among four seasons (26.6% in spring, 26.6% in summer,

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21.8% in autumn, and 25.0% in winter). About a third (34.4%) were delivered by caesarean section.

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Association between perinatal maternal anxiety and development of RTIs during the

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226 first year of life

Higher levels of perinatal maternal anxiety were associated with an increased risk of LRTI especially bronchiolitis, but not with total RTI (Table II).

The risk of LRTIs was significantly

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229 increased in infants exposed to higher levels of perinatal anxiety. For a 1 SD increase in STAI score, the odds of LRTIs risk during the first year of life were 1.35 times higher (95% CI

1.01–1.78). The crude association between perinatal anxiety levels and development of

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232 bronchiolitis was weakly significant (OR 1.35, 95% CI 1.00–1.81; aOR 1.30, 95% CI 1.00–

1.80). However, perinatal anxiety did not increase the risk of URTIs (aOR 1.10, 95% CI

0.87–1.39). In addition, higher levels of perinatal maternal anxiety were not significantly

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234 associated with the occurrence of each type of RTI except bronchiolitis. Figure represents the predicted probability of bronchiolitis at 1 year of age for each observed value of perinatal

235 anxiety score in the multivariate model.

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Combined effect between perinatal maternal anxiety levels and GSTP1 (rs1695), GSTT1,

GSTM1 polymorphisms on the development of RTI during the first year of life

Genetic polymorphisms in GSTP1 (rs1695), GSTT1 or GSTM1 did not increase the risk of


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240 any types of RTIs (data not shown). Exposure to higher levels of perinatal anxiety was

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243 associated with an increased risk for LRTIs especially bronchiolitis in infants with AG+GG genotype of GSTP1 (rs1695), compared with infants with both exposure to lower levels of perinatal anxiety and AA genotype (aOR 2.61, 95% CI, 1.09–6.22; aOR 3.36, 95% CI, 1.25–

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9.03, respectively; Table III). When analyzed by the three genotypes, infants with GG genotype of GSTP1 (rs1695) showed a significantly increased risk for LRTIs especially

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248 bronchiolitis during the first year of life (aOR 22.04, 95% CI, 4.79–101.47; aOR 14.61, 95%

CI, 3.23–66.11, respectively) (Supplementary figure).

In addition, infants with both GSTT1

–null genotype and exposure to higher levels of perinatal

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250 anxiety were associated with an increased risk for LRTIs especially bronchiolitis, compared with infants with both GSTT1

–present genotype and exposure to lower levels of perinatal

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253 anxiety (aOR 2.47, 95% CI 1.04–5.89; aOR 2.79, 95% CI 1.05–7.43; Table IV). In terms of

GSTM1 , exposure to higher levels of perinatal anxiety increased the risk of LRTIs during the first year of life, regardless of the genotypes (present genotype: aOR, 2.95, 95% CI, 1.06–

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8.17; null genotype: aOR, 2.60, 95% CI, 1.01–6.66; Supplementary table I).

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Combined effect between exposure to perinatal maternal anxiety and CD14 (rs2569190) polymorphism on the development of RTI during the first year of life

Infants with TT genotype of CD14 (rs2569190) demonstrated an increased risk of URTIs and

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LRTIs especially bronchiolitis if they were exposed to higher levels of perinatal anxiety, compared with infants exposed to lower levels of perinatal anxiety (aOR 2.51, 95% CI 1.01–

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6.24; aOR 4.60, 95% CI, 1.29–16.41; aOR 4.31, 95% CI, 1.17–15.79, respectively;

Supplementary table II).

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264 Discussion


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Main findings

In this study, we found a positive association between exposure to higher levels of perinatal anxiety and development of RTIs in offspring during the first year of life. The risk of LRTIs, especially bronchiolitis during early infancy was modified by additive effects between

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270 exposure to perinatal anxiety and polymorphisms in antioxidant related genes. To the best of our knowledge, this is the first study examining the gene-environment interactions with

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273 respect to perinatal anxiety on the development of LRTIs especially bronchiolitis during early life.

In addition, we found that exposure to higher levels of perinatal anxiety affected the

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275 development of RTIs especially bronchiolitis during infancy differently, according to the polymorphisms of reactive oxidative stress (ROS) detoxification genes such as GSTP1

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(rs1695) and GSTT1 . The combined effect between exposure to perinatal maternal stress and polymorphisms of ROS detoxification genes on the occurrence of LRTI especially bronchiolitis may be partially attributable to the increased generation of ROS with exposure

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281 to higher levels of perinatal maternal anxiety and LRTI especially bronchiolitis. Perinatal anxiety increases glucocorticoid levels in both mother and fetus, which induces superoxide

production [27]. The capacity to effectively detoxify ROS is affected by polymorphisms of

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these genes [28, 29]. The reason for the increased risk of LRTIs, especially bronchiolitis in

infants with AG+GG genotype of GSTP1 (rs1695) or GSTT1

–null genotype in response to

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285 exposure to higher levels of perinatal anxiety may be explained by partially reduced enzymatic activity in AG+GG genotype of GSTP1 (rs1695) and by the loss of enzymatic activity in GSTT1

–null genotype [28, 29]. Since GST subfamily genes are expressed mainly

in the respiratory tract and oxidative stress is increased during LRTIs including bronchiolitis

[30], genetic variants of the GST subfamily may modify the risk of LRTIs including

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Possible underlying mechanisms

Psychological stress may affect the imbalance of oxidant and antioxidant states and thereby

influence an individual’s health [31, 32]. In response to stress, serum glucocorticoid levels

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rise to modulate the stress response [33]. Glucocorticoid levels affect antioxidant enzyme levels and antioxidant enzyme activity in the lungs begins in late gestation [34, 35].

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Considering the impact of ROS in fetal programming and variants of ROS-related genes on

the capacity to detoxify ROS [11, 12], the increased susceptibility to RTIs in offspring

exposed to perinatal anxiety might be explained by their decreased ability to mitigate the

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oxidative stress generated during RTIs [36].

An experimental study showed that prenatal maternal stress influences epigenetic re-

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303 programming via altered expression of microRNA involved in the stress response, oxidative

stress, and metabolism [6]. In addition, prenatal anxiety-related immune and oxidative

responses can cause telomere erosion, possibly increasing the risk for infection [37, 38]. On

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306 the basis of these findings, we suggest that perinatal anxiety influences susceptibility to RTIs in offspring during critical periods in combination with genetic variants of ROS-related genes or through epigenetic mechanisms. Additional studies are needed to clarify the underlying

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Previous studies showed that T allele of CD14159C/T is associated with enhanced

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310 transcriptional activity and increased soluble CD14 levels, which are markers of monocyte

and macrophage activation [39, 40]. The positive relationship between perinatal anxiety

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313 levels and risk of RTIs in infants with TT genotype of CD14 (rs2569190) might be associated with the essential role of CD14 as an initiator of innate immunity against viral infections in

the respiratory tract [14]. Alterations in cellular and humoral immune responses caused by the

314 interactions between exposure to higher levels of perinatal maternal stress and genetic


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might partially explain these findings [41, 42].

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In this study, we proposed that perinatal maternal anxiety differentially affects the risk of

LRTIs, especially bronchiolitis depending on their anatomic location. Increased glucocorticoid levels during fetal periods may affect the development and function of lung

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during early life [43]. In addition, differences in defense mechanisms, depending on the

anatomy, and more severe characteristics in LRTIs than URTIs may account for the increased

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risk of LRTIs [44, 45]. In addition, functional differences attributable to the genetic variants

in ROS–related genes may add an increased susceptibility to LRTIs, especially bronchiolitis

in affected infants [28, 42].

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Potential limitations

This study has a few limitations. First, the sample size in this study is relatively small because this study is an ongoing prospective birth cohort study. Although the statistical power was weak to analyze the associations with respect to each type of LRTI except bronchiolitis due

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331 to small sample size, this study implicates the importance of the effect of perinatal maternal anxiety on the occurrence of RTI during early life. Further large scale studies are needed to validate the results of this study.

Second, mechanisms underlying the additive effects between

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333 perinatal anxiety and variants of antioxidant defense genes on the occurrence of LRTI, especially bronchiolitis were not explored. However, we tried to explain the underlying

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335 pathophysiology based on the previously reported findings. Also, we analyzed only one single nucleotide polymorphism (SNP) in each gene; however, this selection was based on

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338 previous studies demonstrating an association between SNPs and development of respiratory

illness [30, 35, 39].

Finally, we cannot exclude the effects of postnatal maternal anxiety on the

339 offspring’s health. However, after adjustment for the exposure to the postnatal maternal anxiety during their infants’ 1 st

year of life, exposure to the prenatal maternal stress was


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340 associated with the increased risks of LRTIs. Finally, about 30% of the participants did not

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342 report their anxiety status and was thus excluded from the analysis.

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Conclusion

We suggest that exposure to perinatal maternal anxiety is a significant risk factor for the development of bronchiolitis in the first year of life. Also, this study implicates that variants

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348 in antioxidant defense genes modulate the effect of in utero exposure to perinatal anxiety on infant susceptibility to bronchiolitis during critical periods. In view of the fact that bronchiolitis during early infancy are an economic and medical burden and are associated

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350 with an increased risk of recurrent wheezing and asthma in later life, efforts to decrease perinatal anxiety may be helpful in preventing bronchiolitis during infancy, especially in

351 genetically susceptible infants.


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Abbreviations:

CB : cord blood

CI : confidence intervals

COCOA : COhort for Childhood Origin of Asthma and allergic disease

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GSTM1: glutathione S -transferase M1

GSTP1 : glutathione S -transferase P1

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GSTT1 : glutathione S -transferase T1

LRTIs : lower respiratory tract infections

PCR : polymerase chain reaction

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ROS : reactive oxygen species

RSV : respiratory syncytial virus

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RTI : respiratory tract infection

SD : standard deviation

STAI : State-trait anxiety inventory

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URTIs : upper respiratory tract infections

POTENTIAL CONFLICT OF INTERESTS TO COMPETING INTERESTS: The

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370 authors have no conflicts of interest relevant to this article to disclose.

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FINANCIAL DISCLOSURE : The authors have indicated that they have no financial relationships relevant to this article to disclose.

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FUNDING: This research was supported by funds (2008-E33030-00, 2009-E33033-00.

2011-E33021-00, 2012-E33012-00) from Research of Korea Centers for Disease Control and

Prevention.

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Role allocations

Eun Lee and Hyoung Yoon Chang contributed to the analysis and interpretation of data and drafting the manuscript.

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Kyung-Sook Lee, Jinah Park, and In Ae Choi contributed to collection of data on perinatal maternal stress.

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Dong In Suh, Kyung Won Kim, Youn Ho Shin, Kang Mo Ahn, Eun-Jin Kim, and Joo-Shil

Lee contributed to the construction of this cohort study and conducted the follow-up.

Ja-Young Kwon, Suk-Joo Choi, Kyung-Ju Lee, and Hye-Sung Won contributed to the

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387 enrolment of pregnant women in the birth cohort study.

Mi-Jin Kang and Ho-Sung Yu performed the genetic studies and analyzed the data.

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Song I Yang, Young-ho Jung, Hyung Young Kim, Ju-Hee Seo, Ji-Won Kwon, Byoung-Ju

Kim, Hyo-Bin Kim, So-Yeon Lee, and Katherine M. Keyes contributed to the thorough review of this manuscript and the acquisition of data.

Soo-Jong Hong and Yee-Jin Shin contributed to the conception of this study, its design, and interpretation of the data as co-corresponding authors.

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ACKNOWLEDGMENTS

The authors thank Jung Yeon Shim, Woo Kyung Kim, Gwang Chen Jang, and Hyeon Jong

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Yang for their valuable support during this study. In addition, the authors are grateful to the research nurses for their continuous collaboration over the years. We thank to Sung-Cheol

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Yun for statistics advice.

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10

Table I. Characteristics of study population and subjects not included in the current analysis

Covariates (mother)

Maternal age (years)

BMI (kg/m 2 )

Maternal educational level

Secondary school

College or University

Graduate school

Environmental tobacco exposure

History of any allergic disorder (AD, AR, Asthma)

Covariates (father)

History of any allergic disorder (AD, AR, Asthma)

Covariates (child)

Sex: Girl

Birth weight (g)

Season of birth

Spring

Summer

Autumn

Winter

Mode of delivery: Caesarean section

Gestational age (months)

Presence of siblings

Daycare attendance during the first year of life

Women included in this study n Mean ± SD or n (%)

440

32.5 ± 3.55

20.7 ± 2.67

31 (7.0%)

289 (65.7%)

120 (27.3%)

265 (60.8%)

124 (28.2%)

410

440

115 (26.1%)

217 (49.3%)

3143.9 ± 422.01

117 (26.6%)

117 (26.6%)

96 (21.8%)

110 (25.0%)

134 (34.4%)

39.2 ± 1.08

198 (45.0%)

105 (23.9%)

Exposure 440

STAI score

Physician diagnosed respiratory tract infection during the first year of life

41.0 ± 8.47 (20 – 71)

Upper respiratory tract infection

Lower respiratory tract infection

337 (76.6%)

65 (14.8%)

Bronchiolitis 48 (10.9%)

AD , atopic dermatitis; AR , allergic rhinitis; N/A, not applicable; STAI , state trait anxiety inventory

N/A

Women not include in this study n Mean ± SD or n (%)

193

32.4±3.45

20.7 ± 2.60

21 (7.3%)

209 (73.1%)

56 (19.6%)

160 (54.6%)

76 (30.4%)

193

65 (26.4%)

129 (44.2%)

3198.3 ± 395.43

42 (14.3%)

40 (13.7%)

139 (47.4%)

72 (24.6%)

100 (35.3%)

39.2±1.09

83 (43.0%)

43 (22.3%)

P value

0.736

0.754

0.060

0.131

0.537

0.651

0.173

0.153

<0.001

0.793

0.968

0.705

0.740


11

Table II. Odds ratio and 95% confidence intervals for perinatal anxiety predicting respiratory tract infection at age 1 year

Exposure Outcome Unadjusted Adjusted

*

OR

†

95% CI OR 95% CI

Upper RTI 1.04

1.31

‡

STAI (*SD) Lower RTI

Bronchiolitis 1.35

‡

(0.84–1.30)

(1.01–1.71)

1.10

1.35

‡

1.30

§

(0.87–1.39)

(1.01–1.78)

(1.00–1.81) (1.00–1.80)

CI , confidence intervals; OR , odds ratio; RTI , respiratory tract infection; SD , standard deviation; STAI , State trait anxiety inventory

* Adjusted for child’s sex, season of birth, maternal age, education level, exposure to tobacco smoking during the first year of life, history of maternal allergic diseases and history of any paternal allergic diseases (atopic dermatitis, allergic rhinitis, or asthma)

†

Logistic regression models were used to calculate OR and 95% CI.

‡ p -value < 0.05

§ p -value < 0.10

diagnosis diagnosis

(-) (+) diagnosis (-) diagnosis (+)


12

STAI

Table III. Interactions between maternal perinatal stress (STAI score) and GSTP1 (rs1695) polymorphism on the development of respiratory tract infection during the first year of life

Upper RTI

*

Lower RTI

†

Bronchiolitis

GSTP1 Upper RTI Upper RTI aOR

‡

Lower RTI Lower RTI aOR

‡

Bronchioli Bronchioliti aOR

‡

(95% CI) (95% CI) tis diagnosis s diagnosis

(+)

95% CI

(-)

138 9 1 low

(≤41) low

(≤41) high

(>41) high

AA

AG+GG

AA

AG+GG

36

17

31

13

111

43

90

53

1

1.10

(0.53-2.27)

1.25

(0.68-2.30)

1.91

133

51

101

52

14

9

20

14

1

1.66

(0.64-4.26)

1.99

(0.92-4.29)

2.61

(>41) (0.86-4.25) aOR , adjusted odds ratio; CI , confidence intervals; GSTP1: glutathione S -transferase P1;

(1.09-6.22)

52

106

54

8

15

12

2.14

(0.74-6.15)

2.07

(0.83-5.16)

3.36

(1.25-9.03)


13

RTI , respiratory tract infection; STAI , State trait anxiety inventory

*

Upper RTI includes common colds, sinusitis, otitis media, and croup.

†

Lower RTI includes pneumonia, tracheobronchitis, and bronchiolitis

‡ Adjusted for child’s sex, season of birth, maternal age, education level, prenatal exposure to tobacco smoking, history of any maternal allergic diseases and history of any paternal allergic diseases (atopic dermatitis, allergic rhinitis, or asthma)

diagnosis (-), diagnosis (+), n n diagnosis (-), diagnosis (+), n n


14

STAI

Table IV. Interactions between maternal perinatal stress (STAI score) and copy number variation of GSTT1 on the development of respiratory tract infection during the first year of life

Upper RTI

*

Lower RTI

†

Bronchiolitis

GSTT1 Upper RTI Upper RTI aOR

‡


‡

Bronchiolitis Bronchiolitis aOR

‡

(95% CI) (95% CI) diagnosis (-), diagnosis (+), n n

(95% CI) low

(≤41)

CNV≥1 low

(≤41)

CNV=0 high

(>41)

CNV≥1 high

(>41)

CNV=0

16

37

22

22

76

76

65

77

1

0.43

(0.21-0.88)

0.66

(0.30-1.45)

1.02

(0.46-2.26)

82

102

75

77

10

11

12

22

1

0.86

(0.33-2.21)

1.29

(0.50-3.33)

2.47

(1.04-5.89)

85

104

79

80 aOR , adjusted odds ratio; CI , confidence intervals; CNV , copy number variation; GSTT1, glutathione S -transferase T1;

RTI , respiratory tract infection; STAI , State trait anxiety inventory

7

9

8

19

1

0.97

(0.33-2.83)

1.13

(0.37-3.34)

2.79

(1.05-7.43)


15

*


†




16

Figure Legends

Figure.

Relationship between perinatal maternal anxiety levels and predicted probability of bronchiolitis at 1 year of age.


17

Supplementary figure.

Effect of GSTP1 (rs1695) polymorphisms on respiratory tract infection according to perinatal maternal anxiety levels. (A) The risk of upper RTIs during the first year of life. (B) The risk of lower RTIs during the first year of life. (C) The risk of bronchiolitis during the first year of life.

STAI, state trait anxiety inventory


18

Supplementary table I . Interactions between maternal perinatal stress (STAI score) and copy number variations of GSTM1 on the development of respiratory tract infection during the first year of life

Upper RTI *

STAI

Upper RTI Upper RTI

GSTM1 diagnosis (-) diagnosis (+) low (≤41)

CNV≥1 21 77 low (≤41) CNV=0 32 76 aOR

‡

(95% CI)

1

0.68

(0.35-1.34) high (>41) high (>41)

CNV≥1

CNV=0

24

20

55

87

0.81

(0.38-1.73)

1.43

(0.68-3.01)

Lower RTI†

Lower RTI Lower RTI diagnosis (-) diagnosis (+)

91 7

93

65

87

15

14

20 aOR

‡

(95% CI)

1

1.75

(0.66-4.66)

2.95

(1.06-8.17)

2.60

(1.01-6.66)

Bronchiolitis

Bronchiolitis Bronchiolitis diagnosis (-) diagnosis (+)

92 6

97

69

90 aOR , adjusted odds ratio; CI , confidence intervals; CNV , copy number variation; GSTM1, glutathione S -transferase M1;

11

10

17

RTI , respiratory tract infection; STAI , State trait anxiety inventory aOR

‡

(95% CI)

1

1.35

(0.46-3.97)

2.02

(0.65-6.25)

2.35

(0.86-6.47)

*Upper RTI includes common colds, sinusitis, otitis media, and croup.

†


† Lower RTI includes pneumonia, tracheobronchitis, and bronchiolitis


19

‡

Adjusted for child’s sex, season of birth, maternal age, education level, prenatal exposure to tobacco smoking, history of any maternal allergic diseases and history of any paternal allergic diseases (atopic dermatitis, allergic rhinitis, or asthma)

STAI CD14 diagnosis

(-), n

Diagnosis

(+), n diagnosis

(-), n diagnosis

(+), n


20

Supplementary table II.

Interactions between STAI score and CD14 (rs2569190) polymorphism on the development of respiratory tract infection during the first year of life

Upper RTI

*

Lower RTI

†

Bronchiolitis

Upper RTI Upper RTI aOR

‡


‡

Bronchiolitis Bronchiolitis aOR

‡

(95% CI) (95% CI) diagnosis

(-), n diagnosis (+), n

(95% CI) low

(≤41)

TT low

(≤41)

TC+CC high

(>41)

TT

21

31

13

43

110

44

1

1.89

(0.93-3.83)

2.51

60

123

46

4

18

11

1

2.22

(0.69-7.14)

4.60

60

128

47 high

(>41)

TC+CC 31 96

(1.01-6.24)

1.90

(0.91-3.94)

105 22

(1.29-16.41)

3.01

(0.94-9.70)

110 aOR , adjusted odds ratio; CI , confidence intervals; RTI , respiratory tract infection; STAI , State trait anxiety inventory

*


4

13

10

17

1

1.58

(0.47-5.31)

4.31

(1.17-15.79)

2.11

(0.63-7.04)


21

†



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