Prenatal Oxidative Balance and Risk of Asthma and Allergic Disease in Adolescence

Abstract

Background:

Fetal oxidative balance (achieved when protective prenatal factors counteract sources of oxidative stress), may be critical for preventing asthma and allergic disease.

Objective:

We examined prenatal intakes of hypothesized protective nutrients (including antioxidants) in conjunction with potential sources of oxidative stress, in models for adolescent asthma and allergic disease.

Methods:

We used data from 996 mother-child pairs in Project Viva. Exposures of interest were maternal pre-pregnancy body mass index and prenatal nutrients (energy-adjusted intakes of vitamins D, C, and E, β-carotene, folate, choline, and n-3 and n-6 polyunsaturated fatty acids (PUFAs)), air pollutant exposures (residence-specific 3^rd trimester black carbon or PM_2.5), acetaminophen, and smoking. Outcomes were offspring current asthma, allergic rhinitis, and allergen sensitization at a median age of 12.9 years. We performed logistic regression. Continuous exposures were log-transformed and modeled as z-scores.

Results:

We observed protective associations for Vitamin D (OR = 0.69; 95% CI 0.53 to 0.89 for allergic rhinitis), the sum of n-3 PUFAs EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) (OR=0.81, 95% CI 0.66 to 0.99 for current asthma)), and n-3 PUFA alpha linolenic acid (OR=0.78; 95% CI 0.64 to 0.95 for allergen sensitization). Black carbon and PM_2.5 were associated with ~30% elevated risk for allergen sensitization. No multiplicative interactions were observed for protective nutrient intakes with sources of oxidative stress.

Conclusions:

We identified potential protective prenatal nutrients (Vitamin D, n-3 PUFAs), as well as adverse prenatal pro-oxidant exposures that may alter risk of asthma and allergic disease into adolescence.

Capsule Summary:

In Project Viva, a pre-birth cohort study, we identified potential protective prenatal nutrients (Vitamin D, n-3 PUFAs), as well as adverse prenatal pro-oxidant exposures that may alter risk of asthma and allergic disease into adolescence.

INTRODUCTION

Asthma is the most common chronic disease of childhood, affecting approximately 6 million children in the United States alone.(1) Susceptibility to asthma and other allergic diseases likely begins in fetal life, when the developing immune system is especially vulnerable.(2) Exposures to sources oxidative stress in fetal life may be particularly influential, given their ability to enhance inflammatory responses and their potential to alter epigenetic patterns with downstream consequences for gene expression. (3) Asthma, allergic rhinitis and wheeze symptoms may not only have consequences during childhood resulting from enhanced IgE-mediated immune response, but also may not remit if prenatal oxidative stressors permanently shift innate and other non-allergic pathways to amplify airway inflammation and overwhelm homeostatic mechanisms.

Epidemiological studies and animal models of individual exposures encountered in utero provide supportive evidence for the protective role of nutrients, many of which function as antioxidants that may be anti-inflammatory.(4–6) Other observational studies in humans and experimental data in animals demonstrate the potential adverse effects of oxidative stressors, including traffic pollution and adiposity in allergic disease development.(7–8) The hypothesized role of oxidative balance in fetal life (the adverse effects of oxidative stressors, and the mitigation of these effects by antioxidants such as protective nutrients) can be considered one facet of the Developmental Origins of Health and Disease (DOHaD) hypothesis.

The roles of protective prenatal factors (nutrient intakes of Vitamin D, n-3 fatty acids, Vitamin C and others) and those of potential adverse exposures (prenatal exposure to air pollution, increased maternal adiposity, smoking and acetaminophen intake), are often considered individually in epidemiological studies, but are rarely modeled simultaneously. Furthermore, associations between these prenatal exposures and allergic diseases are typically reported for early (preschool age) and mid-childhood (~ages 6–7 years) outcomes, but little is known about the persistence of these associations into adolescence.

In this work, we used data from mother-child pairs in Project Viva, a Massachusetts-based prenatal birth cohort study with follow-up data through early adolescence (median age 12.9 years). We examined associations of prenatal intakes of maternal antioxidant nutrients and hypothesized oxidative stress exposures encountered in utero would be associated with current allergic disease outcomes (current asthma, current rhinitis, and allergen sensitization) in adolescence. We tested all protective and adverse exposures with statistically significant or suggestive main effects for interactions with one another. We hypothesized that associations for individual nutrients previously identified (Vitamin D and n-3 PUFAs) as protective against allergic disease risk at the mid-childhood time point (9–10) in our cohort would carry forward into adolescence, and that the combined exposure to multiple hypothesized protective nutrients from diet (Vitamin C, Vitamin E, Vitamin D, beta-carotene, folate) would have the largest impact on reducing risk.

METHODS

This study involved women and their children enrolled in Project Viva, an ongoing longitudinal pre-birth cohort study. The design of this cohort study has been described in detail elsewhere.(11) The study was approved by the Harvard Pilgrim Health Care Institutional Review Board. All methods were performed in accordance with the ethical principles of the Declaration of Helsinki. Mothers provided informed consent at enrollment and for their child during follow-up visits, and children gave verbal assent.

Participants were recruited into Project Viva at 8 offices of a multispecialty group practice (Atrius Harvard Vanguard Medical Associates) in Eastern Massachusetts between 1999 and 2002. Exclusion criteria were multiple gestation, inability to answer questions in English, plans to move out of the study area before delivery of the infant, and gestational age ≥22 weeks at the time of presentation for prenatal care. We saw mothers during the first and second trimesters of pregnancy, and both mothers and children at delivery and periodic postnatal research visits, most recently when children reached adolescence (the “early teen” visit at median 12.9 years, range 11.9 to 16.6 years). At this visit, we collected blood samples.

Of the 2,128 women who delivered a live infant, we excluded 360 who were missing all prenatal exposure data (nutrient intakes and air pollution exposure estimates), and 636 who were missing relevant covariates. Of the 1026 mother-infant pairs with available exposures and covariate measures, a total of 996 participants (97%) had information on current allergic disease status (886 (86%) participants reported information on current asthma status, 712 (69%) participants reported information on current allergic rhinitis, and 572 (56%) had an assessment of allergen sensitization (IgE testing) at the early teen visit.

Comparison of the 1,132 mother-infant pairs excluded from the analysis (due to missing exposure, covariate or outcome data) to the 996 participants in the analysis data set showed some differences (Supplemental table 1). For example, a larger percentage of participants with complete data for analysis were white (69 vs. 66%).

Prenatal Dietary Nutrients and Supplement Intakes

During both the first and second trimesters, we collected data on prenatal nutrient intakes from foods using semi-quantitative food frequency questionnaires (FFQs) validated specifically for use in pregnancy. Use of these questionnaires in the Project Viva cohort have been described previously.(12) For first trimester data, the time referent was “during this pregnancy” (i.e. from the date of the last menstrual period until the assessment at a median of 9.9 weeks gestation at enrollment). The time referent for second trimester intakes collected by FFQ at the second visit (at 26–28 weeks gestation) was “during the past 3 months”. We did not administer a full FFQ that covered diet during late pregnancy, but we have found that diet was fairly stable across the first and second trimesters.(13) We estimated individual nutrient intakes using the Harvard nutrient-composition database, which contains food composition values from the US Department of Agriculture, supplemented by other data sources.(14) We used mean nutrient intakes from food from the first and second trimesters as the exposures in the analyses, adjusted for total energy intake by the nutrient residual method.(15) If a participant completed only one FFQ, we used that value for the exposure variable. For the highly correlated long-chain polyunsaturated n-3 (omega-3) fatty acids EPA (eicosapentaenoic acid) and DHA (docosapentaenoic acid) we used their sum in our models. (We have previously shown that maternal FFQ in pregnancy is a reliable measure of elongated PUFA intake).(16)

Prenatal Air Pollutant Exposures

We estimated third trimester air pollutant exposures of interest, residence-specific 3^rd trimester black carbon (BC) and PM_2.5, using validated spatio-temporal regression models. Methodology for generating daily estimates of these exposures has been published elsewhere.(17) Briefly, daily residence-specific BC estimates were generated from a spatiotemporal regression model that contained land-use predictors (e.g., cumulative traffic-density within 100 m of a given location), meterological terms, and a smooth term of longitude and latitude. This model was applied separately for predictions in the warm (May-October) and cold (November-April) seasons. For BC estimates obtained using this spatiotemporal land-use regression model, the mean “out-of-sample” R was 0.73. For PM_2.5 exposure estimates, we used a spatiotemporal prediction model that uses 10×10 km resolution daily satellite remote sensing data on aerosol optical depth (AOD) from the NASA MODIS satellite, land use terms, and meteorological factors. The models first calibrated the remote sensing AOD data to observed PM_2.5 ground monitoring data while accounting for land use and meteorological factors. A second stage used generalized additive models to fill in missing AOD data, due to cloud cover, snow, or other factors, using regional measured PM_2.5, AOD values in neighboring cells, and land use terms. A final third stage used fine-scale spatial information at 100m resolution to account for very local traffic particle emissions. Daily PM_2.5 exposure estimates at each residence yielded mean “out-of-sample” R² of 0.83 for days having AOD remote sensing data and 0.81 for days without.(18) Participants reported their residential address at enrollment and updated it at the end of the second trimester and shortly after birth. Third trimester exposure estimates were calculated by averaging daily exposures from the 188^th day after LMP to the day before birth. We chose to model 3^rd trimester air pollutant exposures, given that the third trimester is a very active period of lung development and therefore a biologically plausible window of vulnerability for airway disease outcomes, and also because satellite model data for PM_2.5 was available starting in the year 2000 (and therefore the 3^rd trimester exposure data were most complete for mothers who enrolled at the beginning of the study, which began in 1999).

Outcome Measures

We examined allergic disease outcomes at the early teen visit. Current asthma was defined as mother’s report of a doctor’s diagnosis of asthma since birth plus maternal report of “wheeze (or whistling in the chest)” in the past year or asthma medication use or shortness of breath in the past month each reported at early teen follow-up (comparison group had no asthma diagnosis, no wheeze, no shortness of breath and no asthma medication use). Current allergic rhinitis was defined as mother’s report of a doctor’s diagnosis of hay fever or allergic rhinitis since birth plus report of “sneezing, runny nose or blocked nose without cold or flu” in the past year (comparison group had no hay fever diagnosis and no sneezing, runny nose or blocked nose symptoms without cold or flu).

Of the 996 children in the analysis sample, 706 had blood drawn at early teen for additional studies, of whom 590 had sufficient sample to measure serum total IgE by using ImmunoCAP (Phadia, Uppsala, Sweden); of the 590 with serum IgE measures, 572 had complete information on covariates. A variety of perennial and seasonal environmental allergens common to the northeastern United States were assessed. Allergen sensitization was defined as any specific IgE level of 0.35 IU/mL or greater to common indoor allergens (Dermatophagoides farinae, cat dander, dog dander, and Blattella germanica), mold (Aspergillus fumigatus, Alternaria alternata), food allergens (egg white, milk, wheat, peanut, and soy bean), or outdoor allergens (tree (silver birch, oak), rye grass and ragweed); or a total IgE level of 100 IU/mL or greater.

In secondary analyses, we tested exposures associated with asthma risk with the outcome of lung function at the early teen visit. Trained research assistants measured child height, weight, and lung function,(19) including forced expiratory volume (FEV) and forced vital capacity (FVC), using the EasyOne Spirometer (NDD Medical Technologies, Andover, MA). Spirometric performance was required to meet American Thoracic Society criteria for acceptability and reproducibility, with each subject producing at least 3 acceptable spirograms, 2 of which must have been reproducible.

Maternal BMI, Prenatal Acetaminophen Intakes, and Covariates

At enrollment mothers completed questionnaires reporting their education, history of atopy, history of hay fever, history of maternal and paternal asthma, smoking habits, and pre-pregnancy height and weight, from which we calculated body mass index (BMI). On postpartum questionnaires, mothers reported child race/ethnicity. Mothers reported acetaminophen intake during prenatal interviews conducted in early and mid-pregnancy. We obtained child sex and date of birth from hospital birth records and determined age at outcome using birth date and date of the early teen visit.

Statistical Analysis

Statistical analyses were performed with SAS statistical software, version 9.4 (SAS Institute, Cary,NC). In our primary analyses, we performed logistic regression to investigate the association between each exposure and each of the 3 outcomes. We adjusted for a priori confounders as well as known predictors of asthma and rhinitis. Covariates for all models included child sex, age at outcome, child’s race/ethnicity, sine and cosine of date of birth (to adjust for seasonality),(20) and maternal pre-pregnancy BMI, smoking status (smoked in pregnancy, former smoker, never smoked), acetaminophen intake (never, 1–9 times, 10 or more time in pregnancy), and education level. Models for current asthma were also adjusted for maternal and paternal history of asthma. Models for current allergic rhinitis were adjusted for maternal and paternal history of hay fever. All nutrient and air pollutant exposures were log transformed, and z-scores were calculated based upon log-transformed exposure levels. We report odds ratios for a 1 unit increase in z-score (i.e. a standard deviation increase) in exposure. We conducted separate analyses for long chain n-3 and n-6 fatty acids (DHA + EPA and AA together in the same model) and short chain n-3 and n-6 fatty acids (ALA and LA). ALA and LA intakes were not included in the same model (as these were highly correlated). We chose not to employ classic multiple comparison correction techniques (i.e. Bonferroni), as these would be too conservative, given the correlated nature of the outcomes.(21) We examined 95% confidence intervals as well as effect sizes and were conservative in reporting findings as significant or not, instead highlighting similar associations in related analyses. We tested exposures with main effects (p<0.1) for interactions with each other in models for specific allergic disease outcomes. Statistical model results for black carbon and nutrient prenatal exposures are also shown within the main manuscript, while models for black carbon exposure (a component of PM_2.5) are shown in supplemental tables.

In secondary statistical analyses, we performed linear regression to examine whether those exposures associated with asthma (from our primary analysis) were also associated with measured lung function at the early teen visit. These models were adjusted for all other nutrient and hypothesized “pro-oxidant” exposures.

In addition to examining individual nutrient exposures, we used weighted quantile sum (WQS) regression (implemented using the gWQS package in R statistical software), to determine whether a combination or mixture of prenatal nutrients was associated with our outcomes of interest. For this analysis, we binned maternal nutrient intakes by decile, and derived a composite index of hypothesized protective nutrients (with individual nutrients in the mixture weighted in proportion to their association with the disease outcome). We used 25% of the data to derive weights for the WQS composite nutrient index, and 75% of the data to perform statistical testing using the weighted composite. Results of this analysis are shown in a supplemental file (supplemental tables 4 and 5).

RESULTS

Sociodemographic and perinatal characteristics of the mothers and children included in this analysis are shown in Table 1. Teen current allergic disease status was associated with maternal history of allergic disease. Children with current asthma were more likely to be of black or other race/ethnicity. Characteristics of the mother-infant pairs with prenatal exposure assessment were similar to the characteristics of those with asthma, allergic rhinitis and allergen sensitization at the early teen visit (Supplemental table 1). Rates of maternal asthma and hay fever were similar to those in the general population. As expected, allergen sensitization at the early teen time point showed the highest prevalence out of all the allergic disease outcomes (62%), followed by current allergic rhinitis (22%) and current asthma (14%). Overall, participants with current asthma had mild disease. Maternal report of disease severity showed that the vast majority (89%) of participants with asthma were well-controlled, and rarely missed school because of their asthma (72% did not miss any school over the past year due to asthma, 16% missed only once, and 11% missed school more than twice due to asthma symptoms). Emergency room visits for asthma over the past year were rare (6% of participants with current asthma) and very few participants (4% of all current asthmatics) reported hospitalizations for asthma.

Table 1.

Characteristics of Parents and Children in the Project Viva Pre-birth cohort

Characteristic	Participants with Current Asthma Outcome Data (Total N=886)		Participants with Current Allergic Rhinitis Outcome Data (Total N=712)		Participants with Allergen Sensitization Outcome Data (Total N=572)
	Current Asthma= Yes (N=134)	Current Asthma= No (N=752)	Current Allergic Rhinitis=Yes (N=159)	Current Allergic Rhinitis= No (N=553)	Atopy=Yes (N=357)	Atopy=No (N=215)
Smoking in Pregnancy
Never	68%	71%	65%	75%	69%	70%
Smoked during Pregnancy	10%	8%	11%	7%	12%	7%
Former	22%	21%	25%	18%	19%	23%
Maternal Allergic rhinitis
Yes	43%*	29%	50%*	23%	35%*	24%
No	58%	71%	50%	77%	65%	76%
Maternal Asthma
Yes	24%*	9%	20%*	8%	14%*	7%
No	76%	91%	80%	92%	86%	93%
Child’s Race/Ethnicity
Black	20%	11%	13%	12%	17%	13%
White	57%	73%	68%	67%	62%	68%
Hispanic	3%	2%	5%	1%	3%*	6%
Other	20%	14%	14%	20%	18%	13%
Comparison across all groups	**
Child’s sex
Female	47%	51%	44%	49%	45%	52%
Male	53%	49%	56%	51%	55%	48%

^*p<0.05 for χ² test statistic comparing frequency of a characteristic vs. outcome

^**p<0.05 for χ² test statistic comparing frequency of a given outcome across groups

The distributions of nutrient intakes and air pollutants are shown in table 2. Overall, associations of prenatal exposures with early teen outcomes were different depending upon the type of allergic disease outcome assessed (Tables 3 and 4). Prenatal vitamin D intake showed protective associations with current allergic rhinitis, (aOR= 0.69, 95% CI 0.53 to 0.89) but not with either current asthma or allergen sensitization. The sum of n-3 fatty acids DHA and EPA showed a protective association for current asthma (aOR=0.81, 95% CI 0.66 to 0.99) and a trend toward potential protective effect for allergen sensitization although the confidence interval crossed the null (aOR=0.85, 95% CI 0.68 to 1.07). Alpha-linolenic acid showed protective associations for both allergen sensitization (aOR=0.78, 95% CI 0.64 to 0.95) and current asthma (aOR=0.79, 95% CI 0.64 to 0.99), but not for allergic rhinitis. Linoleic acid showed a protective association with current asthma (aOR=0.79, 95% CI 0.63 to 0.99), but not allergic rhinitis (aOR=0.89, 95% CI 0.70 to 1.14) or allergen sensitization (OR=0.91, 95% CI 0.75 to 1.12) (results reported here in text only). We should note that our results for asthma were somewhat sensitive to disease definition; including “shortness of breath” in addition to wheeze symptoms and asthma medication use shifted our risk estimates towards slightly stronger estimates (data not shown).

Table 2.

Distribution of Prenatal Exposure Variables (Nutrient Intakes and Air Pollutant Exposures) in Children with Covariates and Allergic Disease Outcome Data at the Early Teen Visit

Prenatal Exposure Variable	N	25th Percentile	Median	75th Percentile
Vitamin C, mg/d	996	133.7	170.0	211.6
Vitamin E, mg/d	996	5.4	6.4	8.0
Beta Carotene, μg/d	996	2435.4	3538.4	4954.5
Folate, μg/d	996	289.8	352.1	425.8
Choline mg/d	996	290.3	322.6	360.3
Vitamin D, IU/d	996	149.2	209.2	282.1
DHA + EPA, gm/d	996	0.07	0.14	0.23
Arachidonic Acid, gm/d	996	0.07	0.09	0.11
Alpha-linolenic Acid, gm/d	996	0.70	0.88	1.13
Linoleic gm/d	996	9.90	11.6	13.5
Black Carbon μg/m3	996	0.5	0.7	0.8
PM2.5 μg/m3	878	10.6	11.7	12.9

Table 3.

Prenatal Exposures (Nutrient intakes, Long Chain n-3 and n-6 PUFA intakes, Air Pollution (Black Carbon)) and Allergic Disease Outcomes in Early Teen

Exposure Group	Prenatal Exposure (z-score)	Current Asthma No=752 (85%), Yes=134 (15%)	Current Allergic Rhinitis No=553 (78%), Yes=159(22%)	Allergen Sensitization No=215(38%), Yes=357(62%)
		aOR (95% CI)a,b	aOR (95% CI)a,c	aOR(95% CI) a
Hypothesized Antioxidants/Protective Nutrients	DHA+ EPA	0.81 (0.66 to 0.99)	1.24 (0.93 to 1.63)	0.85 (0.68 to 1.07)
	AA	1.06 (0.83 to 1.37)	0.93 (0.71 to 1.21)	0.87 (0.68 to 1.11)
	Vitamin E	1.05 (0.83 to 1.33)	1.12 (0.88 to 1.44)	1.00 (0.80 to 1.25)
	Vitamin C	1.12 (0.87 to 1.43)	1.09 (0.83 to 1.42)	1.12 (0.89 to 1.41)
	Beta Carotene	0.93 (0.74 to 1.16)	0.83 (0.66 to 1.05)	1.10 (0.89 to 1.37)
	Folate	0.97 (0.71 to 1.31)	0.91 (0.66 to 1.24)	0.78 (0.58 to 1.03)
	Choline	0.86 (0.66 to 1.12)	1.09 (0.82 to 1.44)	1.33 (1.03 to 1.70)
	Vitamin D	1.09 (0.85 to 1.38)	0.69 (0.53 to 0.89)	0.92 (0.73 to 1.17)
Hypothesized Pro-oxidants	Black Carbon	0.99 (0.79 to 1.25)	0.99 (0.79 to 1.23)	1.35 (1.10 to 1.67)
	Acetaminophen*	1.29 (0.98 to 1.70)	1.21 (0.92 to 1.60)	0.89 (0.70 to 1.15)
	Maternal BMI	1.09 (0.90 to 1.32)	1.16 (0.95 to 1.41)	1.09 (0.91 to 1.32)
	Smoking in Pregnancy**	1.09 (0.55 to 2.15)	1.31 (0.66 to 2.60)	1.82 (0.95 to 3.50)
	Former Smoker**	1.12 (0.68 to 1.84)	1.59 (0.98 to 2.57)	0.93 (0.60 to 1.45)

^*Maternal acetaminophen intake (never, 1–9 times, 10 or more time in pregnancy)

^**Categorical variable; reference group never smoker

^aModel adjusted for sex, age at outcome, sine and cosine of date of birth, child’s race, and maternal education level

^bAdditional adjustment for maternal and paternal asthma

^cAdditional adjustment for maternal and paternal allergic rhinitis

Table 4.

Prenatal Exposures (Nutrient intake, Short Chain n-3 PUFA intake, and Air Pollution (Black Carbon)) and Allergic Disease Outcomes in Early Teen

Exposure Group	Prenatal Exposure (z-score)	Current Asthma No=752 (85%), Yes=134 (15%)	Current Allergic Rhinitis No=553 (78%), Yes=159(22%)	Allergen Sensitization No=215(38%), Yes=357(62%)
		aOR (95% CI)a,b	aOR (95% CI)a,c	aOR(95% CI)a
Hypothesized Antioxidants/Protective Nutrients	Alpha-linolenic	0.80 (0.65 to 0.99)	0.93 (0.74 to 1.17)	0.78 (0.64 to 0.95)
	Vitamin E	1.05 (0.83 to 1.33)	1.17 (0.91 to 1.50)	1.00 (0.80 to 1.24)
	Vitamin C	1.05 (0.82 to 1.34)	1.10 (0.85 to 1.44)	1.08 (0.85 to 1.35)
	Beta Carotene	0.88 (0.71 to 1.09)	0.86 (0.68 to 1.08)	1.10 (0.90 to 1.35)
	Folate	0.95 (0.71 to 1.27)	0.86 (0.63 to 1.16)	0.80 (0.60 to 1.05)
	Choline	0.90 (0.71 to 1.14)	1.06 (0.84 to 1.35)	1.26 (1.01 to 1.56)
	Vitamin D	0.97 (0.76 to 1.23)	0.73 (0.57 to 0.93)	0.85 (0.68 to 1.06)
Hypothesized Pro-oxidants	Black Carbon	0.99 (0.79 to 1.24)	1.00 (0.80 to 1.24)	1.34 (1.09 to 1.66)
	Acetaminophen*	1.30 (0.99 to 1.70)	1.22 (0.93 to 1.60)	0.89 (0.70 to 1.14)
	Maternal BMI	1.09 (0.91 to 1.32)	1.17 (0.97 to 1.42)	1.13 (0.94 to 1.37)
	Smoking in Pregnancy**	1.04 (0.53 to 2.05)	1.32 (0.66 to 2.61)	1.69 (0.89 to 3.24)
	Former Smoker**	1.13 (0.69 to 1.84)	1.65 (1.03 to 2.67)	0.90 (0.58 to 1.41)

^*Maternal acetaminophen intake (never, 1–9 times, 10 or more time in pregnancy)

^**Categorical variable; reference group never smoker

^aModel adjusted for sex, age at outcome, sine and cosine of date of birth, child’s race, and maternal education level

^bAdditional adjustment for maternal and paternal asthma

^cAdditional adjustment for maternal and paternal allergic rhinitis

In addition to identifying some protective associations, we also observed adverse associations and trends for prenatal exposures and outcomes at the early teen visit. Maternal report of “former smoker” status was associated with increased odds of allergic rhinitis (OR=1.65, 95% CI 1.03 to 2.67), and maternal acetaminophen intake showed a borderline association with increased current asthma in adolescence (OR=1.30, 95% CI 0.99 to 1.70) (Table 4). Three exposures, including one from diet (choline) and two from air pollution (BC and PM_2.5) were associated with higher allergen sensitization risk. Choline intake was the only nutrient associated with increased allergen sensitization risk (OR=1.33, 95% CI 1.03 to 1.70), and prenatal exposures to either black carbon (OR=1.35, 95% CI 1.10 to 1.67) or PM_2.5 (OR=1.34, 95% CI 1.09 to 1.65) also conferred an increase in risk (Table 3, Supplemental table 2). In addition to 3^rd trimester air pollution, we also tested 1^st and 2^nd trimester black carbon exposures in our models for current asthma, allergic rhinitis, and atopy (data not shown). Our findings mirrored those for 3^rd trimester black carbon exposure (associations were observed for atopy, but not asthma or rhinitis), and the effect sizes found for atopy were strongest for 3^rd trimester exposures.

In secondary statistical analyses, we investigated associations between prenatal n-3 PUFAs and lung function at early teen. We focused specifically on n-3 PUFAs as these nutrients were associated with asthma in our main analyses. We identified a protective association for prenatal DHA + EPA intake and FEV₁/FVC ratio at the early teen visit (table 5).

Table 5.

Prenatal Intakes of n-3 PUFAs and Lung Function in Early Teen

	Prenatal Exposure (z-score)	FEV1 (% Predicted) (N=903)	FVC (% Predicted) (N= 903)	FEV1/FVC Ratio (N=903)
		Beta (95% CI)a,b	Beta (95% CI)a,c	Beta (95% CI)a
Model 1a	Alpha-linolenic	−0.33 (−1.29 to 0.59)	−0.42 (−1.28 to 0.44)	0.04 (−0.47 to 0.56)
Model 2b	DHA + EPA	0.66 (−0.34 to 1.67)	−0.18 (−1.12 to 0.76)	0.79 (0.23 to 1.35)

^aAdjusted for additional nutrients (Vitamin C, Vitamin D, Beta Carotene, Folate, Choline), air pollutants (Black carbon), acetaminophen intakes (never, 1–9 times, 10 or more time in pregnancy), maternal BMI, smoking in pregnancy, former smoker status, for sex, age at outcome, sine and cosine of date of birth, child’s race, and maternal education level

^bAdditional adjustment for arachidonic acid

As many reports have noted interactions between prenatal exposure to air pollutants and child’s sex, we tested our models for sex by PM_2.5 and sex by BC exposure interactions but we did not see strong evidence for sex-specific associations (P values for all interaction terms were >0.05). We tested exposures with main effects (p<0.1) for interactions with each other in models for specific allergic disease outcomes, but detected no interactions at the p <0.05 level.

Our WQS analysis of all hypothesized protective nutrients as a composite mixture did not show any clear associations of this nutrient mixture measure with asthma or allergic disease outcomes in adolescence (Supplemental Tables 4 and 5). In one model (with the nutrient mixture including ALA and LA PUFAs) for the weighted nutrient composite index and current asthma we detected an association suggestive of protection (aOR=0.75, 95% CI 0.55 to 1.01 for an interquartile range increase); however confidence intervals included the null.

DISCUSSION

In this study, we examined multiple prenatal exposures with the potential to influence oxidative balance in utero, and determined their associations with asthma and allergic disease outcomes in adolescence. Two of the hypothesized pro-oxidant prenatal exposures considered (maternal smoking and prenatal air pollution) showed clear associations for increased risk of at least one outcome. Acetaminophen intake (another potential pro-oxidant exposure), was associated with trends toward increased risk of current asthma. On the other hand, we did not observe any beneficial effects of nutrients that function primarily as antioxidants (Vitamin C, Beta-carotene), nor did we detect any significant associations for the weighted composite index of all nutrients combined. Instead, our findings suggest that a few key prenatal nutrients with immunomodulatory potential (Vitamin D and n-3 PUFAs) may have long-term implications for reducing asthma and allergic disease risk. These findings are consistent with our previous analysis of biomarkers of airway inflammation (FeNO) and allergen sensitization (IgE) in early adolescence, which also showed protective associations for Vitamin D and n-3 PUFAs.(22)

Recent analyses of clinical trial data show that higher levels of prenatal Vitamin D (4000 IU vs. 400 IU in prenatal vitamins)(23) and n-3 PUFAs (2.4g vs. olive oil)(24) reduce asthma and wheeze risk in offspring. These trials were conducted using dietary supplements, and focused on asthma/wheeze outcomes at preschool age. In contrast, our observational study shows the potential benefits of prenatal Vitamin D and n-3 PUFAs (EPA + DHA, alpha linolenic acid) absorbed directly from food intake for childhood allergic disease outcomes at a much later (adolescent) time point. Of note, in Project Viva, we have previously reported protective associations of maternal plasma n-3 PUFAs levels in pregnancy (specifically EPA) with reduced risk of asthma, and of EPA and ALA with allergen sensitization in mid-childhood.(9) We have also demonstrated inverse associations of prenatal Vitamin D intake from diet with reduced allergic rhinitis risk in mid-childhood.(10) Thus findings in the present Project Viva study suggest that protective effects of these nutrients may extend even beyond mid-childhood and into adolescence. Our observation that some nutrients are associated with reduced asthma or allergic rhinitis but not allergen sensitization may seem counter-intuitive. However, it is important to note that allergen sensitization may not necessarily lead to symptoms. In light of this, we may have had more power to detect relationships with active disease (current allergic rhinitis) as opposed to allergen sensitization (which is associated with, but not a direct surrogate for, symptomatic allergic disease). It is not clear to us why the protective associations for Vitamin D were found for upper airway and not lower airway outcomes in our study

Long chain n-3 PUFAs may reduce allergic inflammation by displacing arachidonic acid in cell membranes, inducing a shift in eicosanoid pathways such that the mediators produced are less inflammatory or even anti-inflammatory (resolvins and protectins).(25) Whether or not this mechanism decreases fetal oxidative stress or reduces post-natal susceptibility to allergic disease is unknown. The mechanism of action for Vitamin D is likely mediated through binding of vitamin D receptor expressed on adaptive (T cells and B cells) as well as innate immune cells (dendritic cells,(26) macrophages(27)), with downstream consequences for reduction in allergic immune response pathways.(28) Both vitamin D and n-3 PUFA supplementation in pregnancy have been shown to alter cord blood immune responses; vitamin D supplementation increases innate immune responses to TLR agonists and is related to higher levels of IL-10 production in response to dexamethasone, and prenatal n-3 PUFA supplementation is linked to lower levels of Th2 cytokines IL-4(29) and IL-13.(30) In vitro data in our cohort also suggest that n-3 PUFAs may elicit protective responses in the neonatal immune system.(31) Cord blood EPA and DHA levels in Project Viva infants were associated with reduced cytokine responses to dust mite and cockroach allergens, which supports our current finding that EPA and DHA may be protective against both asthma and allergen sensitization.(31) Conversely, choline was associated with an increased risk of allergen sensitization (Tables 3 and 4). While intake of this nutrient has been associated with increased asthma risk in adults,(32) the contributions of prenatal choline to allergen sensitization are unclear. Integrative genetic and metabolite profiling analysis suggests altered phosphatidylcholine metabolism in asthma.(32)

Hypothesized pro-oxidant exposures considered in our study were multi-faceted, and included maternal smoking, maternal acetaminophen intake, maternal BMI, and prenatal air pollution exposure. In utero exposures to cigarette smoke,(33) air pollutants(34) and acetaminophen intake(35) are all direct sources of toxins or xenobiotic compounds capable of generating free radicals and ROS (reactive oxygen species). Mechanisms for oxidative stress resulting from elevated maternal BMI are also well known,(36) and are driven by production of pro-inflammatory chemokines, adipokines (i.e. leptin) and innate inflammatory cytokines (i.e., TNF-alpha, IL-6) from adipose tissue. Prenatal exposures to maternal smoking, acetaminophen intake and higher BMI have all been linked to altered DNA methylation profiles,(37) suggesting that the influence of these pro-oxidant fetal exposures on disease outcomes may be mediated by alterations in the epigenome.

In our analyses, maternal smoking (either smoking during pregnancy or status as a former smoker) showed the largest effect sizes overall, followed by acetaminophen intake and air pollutant exposures.. While the literature linking maternal smoking to allergic sensitization in children is not entirely consistent,(38) (39) identification of adverse associations with maternal smoking are in accordance with multiple published reports that identify in utero smoke exposure as a risk factor for asthma and allergen sensitization in offspring, particularly for early life outcomes.(40) Our finding of increased risk of allergen sensitization in early adolescence with maternal smoking in pregnancy is in contrast to Swedish cohort study that did not detect an association between maternal smoking and allergen sensitization up to 16 years of age.(39) However, the authors acknowledge the possibly that maternal smoking rates could have been under-reported in this population, perhaps due to social stigmatization surrounding smoking in the context of children’s health.

In addition to maternal smoking, ambient air pollution exposures, to both PM_2.5 and black carbon, were also related to increased allergen sensitization risk in adolescence. While multiple studies,(7) including our own,(41) have identified potential adverse effects of air pollutant exposures on asthma risk and reduced lung function in children, fewer studies have identified associations with allergen sensitization. In one birth cohort study, perinatal exposure to traffic related air pollution was associated with increased allergen sensitization risk; however sensitization was assessed very early in life (at 1 year of age).(42)

Prenatal acetaminophen is another potential pro-oxidant that has been examined previously in our Project Viva cohort(43) as well as in other studies.(44) In Project Viva, prenatal acetaminophen intake has been associated with increased risk of current asthma in early childhood, and at school age. The findings in the current analysis demonstrate that this adverse association persists into early adolescence. One major challenge in our study and others is the lack of information about indication for acetaminophen intake. A recent study in a large general population sample of over 50,000 mother-infant pairs accounted for the indications for maternal acetaminophen use (respiratory infection, pain or influenza), but found that associations with early childhood current asthma remained even after adjustment for confounding by indication.(45)

In addition to studying potential adverse and protective effects of individual prenatal exposures, we also explored exposure interactions. We hypothesized that protective prenatal nutrients would reduce childhood allergic disease risk in the face of prenatal oxidative stressors, and that these interactions would be identifiable in our statistical models in the form of significant multiplicative interaction terms. We did not detect any significant multiplicative interactions of this type; however, a protective association for prenatal nutrients was identified for each of the allergic disease outcomes tested, suggesting that these nutrients may be beneficial for the fetus when it encounters sources of oxidative stress. Functional data in experimental animal models provide evidence for role of nutrients in modifying the adverse effects of oxidative stressors. In mice, n-3 PUFAs can combat the effects of particulate air pollution, by suppressing IgE production and reducing bronchoconstriction.(46)

Our study had several strengths. We were able to relate multiple sources of prenatal dietary antioxidants and pro-oxidant exposures simultaneously to development of allergic disease, and our extended follow-up period allowed us to demonstrate that many of these associations persist into adolescence. Some study weaknesses deserve mention. In some instances, we may have had limited statistical power to detect associations within our cohort. Follow-up testing of hypothesized prenatal anti-oxidant and pro-oxidant exposures in a larger population may enhance our ability to observe potential protective and adverse effects. In our study, prenatal exposure to 3^rd trimester air pollutants did not directly overlap with our nutrient assessments from the 1^st and 2^nd trimesters. It is possible that we may have had greater power to detect interactions between intake of nutrients and “pro-oxidative” exposures if the time windows of assessment had been overlapping (although our measured 1^st and 2^nd trimester intakes are likely reasonable approximations of 3^rd trimester intakes). . Given that the majority of our participants show mild disease, we did not have the statistical power to study associations of prenatal exposures with asthma severity outcomes. We also did not account for cumulative exposure to nutrients and air pollutants up until the assessment of the early adolescent outcomes, as this analysis was beyond the scope of our study.

In conclusion, we have identified potential protective prenatal nutrient exposures (Vitamin D, n-3 PUFAs), as well as prenatal pro-oxidant exposures (maternal smoking, maternal acetaminophen intake, and prenatal air pollutants) that may increase risk of allergic disease outcomes in adolescence. Protective nutrients may counterbalance the adverse effects of pro-oxidant exposures.

Key Messages:

• Prenatal exposures, including those associated with in utero oxidative balance, have the potential to influence risk of asthma and allergic disease in children

• In our study, two of the hypothesized pro-oxidant prenatal exposures considered (maternal smoking and prenatal air pollution) showed clear associations for increased asthma and allergic disease

• We did not observe any beneficial effects of nutrients that function primarily as antioxidants (Vitamin C, Beta-carotene)

• Instead, our findings suggest that a few key prenatal nutrients with immunomodulatory potential (Vitamin D and n-3 PUFAs) may have long-term implications for reducing asthma and allergic disease risk.

Abbreviations:

PUFA

Poly-unsaturated fatty acid