Residential exposure to air pollution, access to neighborhood greenspace and their association with hair cortisol concentrations in the second and third trimester of pregnancy

doi:10.21203/rs.3.rs-33256/v1

Background: Exposure to air pollution during pregnancy has been associated with adverse pregnancy outcomes in studies worldwide, other studies have described beneficial effects of residential greenspace on pregnancy outcomes. The biological mechanisms that underlie these associations are incompletely understood. Recent studies have shown that a biological stress response, with release of cortisol, may underlie associations between air pollution and health effects. The available research on air pollution exposure in relation to biological stress during pregnancy is still scarce.

Methods: We explored associations between residential exposure to air pollution, access to neighborhood greenspace and hair cortisol concentrations in a prospective pregnancy cohort study. We modelled participants’ residential air pollutant concentrations (particulate matter with an aerodynamic diameter ≤ 2.5 μm, nitrogen dioxide (NO₂), black carbon (BC)), assessed residential distance to a major road and access to a neighborhood greenspace. Hair cortisol concentrations, reflecting cortisol secretion over a period of 3 months prior to sampling, were determined at the end of the second (n = 133) and third pregnancy trimester (n = 81).

Results: Three-month mean residential NO₂ and BC concentrations were positively associated with third pregnancy trimester hair cortisol concentrations. The residential distance to a major road was negatively associated with second and third trimester hair cortisol concentrations. Access to a greenspace of 10 hectares or more within 800 meters travel distance significantly moderated the association between residential proximity to a major road and second trimester hair cortisol concentrations. At an average residential distance of 304 meters from a major road, mean second trimester HCC were estimated 22% lower for mothers with access to a neighborhood greenspace (3.71 (95% CI: 3.24, 4.24) pg/mg hair) compared to mothers without access (4.22 (95% CI: 3.26, 5.47) pg/mg hair). The moderation tended towards significance in the third trimester (p < 0.10).

Conclusions: Increased residential exposure to air pollution and closer proximity to a major road are associated with an increased biological stress response in the second and third trimester of pregnancy, access to neighborhood greenspace may moderate the association.

Trial registration: The IPANEMA study is registered under number NCT02592005 at clinicaltrials.gov.

Epidemiology

Toxicology

words: air pollution

proximity to major roads

neighborhood greenspace

pregnancy

longer-term biological stress

hair cortisol concentrations

In the past decade, epidemiological studies throughout the world have linked maternal exposure to road traffic and air pollution to adverse pregnancy outcomes such as low birth weight, preterm birth and intrauterine growth restriction [1–3]. These adverse birth outcomes not only increase perinatal morbidity and mortality, but also increase susceptibility to obesity, diabetes and cardiovascular diseases later in life [4,5]. In terms of maternal health, exposure to air pollution has been linked to hypertensive pregnancy disorders (gestational hypertension, pre-eclampsia) and gestational diabetes mellitus [6–8]. Both conditions amplify young women’s risk of developing cardiovascular diseases later in life [9]. The adverse impact of maternal exposure to air pollution on birth outcomes is of major public health importance, considering the ubiquitous nature of air pollution in urban settings [10]. The biological pathways that relate maternal exposure to air pollution to adverse pregnancy outcomes however, remain incompletely understood. Recent experimental animal research has shown that a neuroendocrine stress response is among the early biological responses triggered by exposure to fine particulate matter with an aerodynamic diameter ≤ 2.5 μm (PM_2.5) and exposure to nitrogen dioxide (NO₂) [11]. The biological stress response includes activation of the hypothalamic-pituitary-adrenal (HPA) axis and release of glucocorticoid stress hormones, with the glucocorticoid cortisol as its main downstream effector in humans [12]. The relevance of these experimental observations to humans has been confirmed in a few recent studies [13,14]. To date, most human studies have assessed short-term variations in cortisol levels in relation to air pollution exposure, using blood and saliva as a matrix. Longer-term cortisol concentrations are difficult to evaluate using blood and saliva, due to circadian variations in cortisol secretion and the need for multiple sampling [15]. Repeated sampling increases discomfort for study participants. Hair however, is a suitable matrix for the assessment of longer-term cortisol concentrations [16]. As cortisol is incorporated into growing hair, hair cortisol concentrations (HCC) retrospectively reflect cortisol secretion over a period of several months [17]. Importantly, chronic activation of the maternal HPA axis during pregnancy is associated with gestational hypertensive disorders, intrauterine growth restriction and developmental programming of disease susceptibility [18,19]. With regard to birth outcomes, a significant negative association between maternal HCC in the 2^nd pregnancy trimester and gestational age at delivery has been reported [20].

Interestingly, a growing body of research suggests a beneficial relationship between residential access to greenspaces (publicly accessible vegetation, urban parks, forests) and pregnancy outcomes for both mothers and babies [21]. Residential exposure to air pollution and greenspace occur simultaneously, therefore, the adverse effects of air pollution may, to some degree, be moderated by the beneficial effects of residential access to greenspace [22].

To our knowledge, no research is available on residential air pollution exposure or access to neighborhood greenspace in relation to longer-term biological stress during pregnancy. Accordingly, the main aim of this study was to explore associations between residential exposure to air pollution and road traffic and maternal hair cortisol concentrations in a pregnancy cohort. In addition, we aimed to explore whether residential access to neighborhood greenspace could moderate associations between residential air pollution exposure and maternal biological stress during pregnancy.

Study population and design

This study was conducted in the framework of IPANEMA (Impact of Particulate Matter on Mothers and Babies in Antwerp), a prospective pregnancy cohort study of the Antwerp University Hospital (UZA) in collaboration with the Flemish Institute for Technological Research (VITO) and the University of Antwerp (UA). Pregnant women were recruited between April 2015 and January 2018 at the UZA prenatal clinic by a midwife or obstetrician at a gestational age of 12 to 14 weeks. The inclusion criteria were: a singleton pregnancy; the ability to fill out extensive Dutch questionnaires; delivery planned in the Antwerp University Hospital. All participating mothers gave written informed consent. The study protocol was approved by the ethical committee of the University of Antwerp (14/40/411) and registered under number NCT02592005 at clinicaltrials.gov. Health-related information on mothers and babies was extracted from the hospital records and questionnaires that participants completed at enrolment, during pregnancy and after delivery. These questionnaires provided detailed information on participants’ socio-demographic and lifestyle characteristics. A detailed protocol of the IPANEMA study can be found elsewhere [23].

Hair sample collection and cortisol measurement

At the end of second trimester of pregnancy in-hospital consultation and shortly after delivery, a strand of hair of at least 2mm thick was bound together with a cotton thread and cut close to the scalp from the posterior vertex region of the head. This area of the scalp exhibits the lowest intra-individual variability in HCC [24]. Hair samples were stored in paper envelopes at room temperature until analysis. When protected from ultraviolet light, cortisol concentrations in hair samples remain stable at room temperature for several years [25]. Cortisol concentrations were determined from the 3 cm of hair closest to the scalp. Based on an average hair growth of 1 cm per month, this length represents cortisol secretion in a 3-month period, a trimester, prior to sampling [26]. There is a wide consensus that the first 5–6 cm of hair nearest to a person's scalp can reliably reflect HPA activity [27]. Analysis was performed at the Institute of Public Health, Department of Environmental Medicine of the University of Southern Denmark (SDU), using liquid chromatography and tandem mass spectrometry (LC-MS/MS) as described by Chen et al. [28], after minor modifications. Hair samples were washed with methanol and dried at room temperature. The 3 cm of hair closest to the scalp was cut into segments of 2-3 mm. A typical amount of hair weighed 20-30 mg. Aliquots of 100 mL 20 ng/mL isotope labeled cortisol (cortisol-D₄) were added as internal standard, together with 0.9 mL methanol. Samples were incubated in the dark at 25°C while whirl mixed at 2000 revolutions per minute for 5 days and subsequently centrifuged at 3000g for 5 minutes. 20 μL of the supernatant was injected onto a High-Performance Liquid Chromatography (HPLC) column. HPLC was performed using an Accella 1250 pump (Thermo Scientific, San Jose, CA) and a PAL autosampler (CTC analytics, Zwingen, Switzerland). The analytical column was a Kinetex C18 column, 100 x 4.6 mm (2.6 μm) equipped with a 2 x 4 mm C18 SecurityGuard column (Phenomenex, Torrance, CA). Isocratic elution was performed with a mobile phase system consisting of methanol and 0.1 M formic acid (80:20) at a flow rate of 400 μL/min for 6 min. After the peaks were eluted, a wash procedure was performed before the next samples was injected onto the column. The triple quadrupole mass spectrometer utilized was a TSQ Vantage (Thermo Scientific, San Jose, CA). The calibration curve and calculation of the sample concentration were based on the area ratio of the analyte/isotope labeled internal standard. Quality control samples were included in each series of samples. The limit of quantification (LOQ) for cortisol was 1.0 pg/mg hair. The intra-day repeatability coefficient of variation was 8.7% and the inter-day reproducibility coefficient of variation was 9.5%

Residential exposure assessment

Assessment of all residential exposure variables was based on the participants’ geocoded home address. Geographical Information System (GIS) analyses were carried out using ESRI ArcGIS software version 10.4 (Environmental Systems Research Institute, Redlands, California, USA). The residential degree of urbanization was assessed according to the Eurostat definition that classifies local administrative units as cities, towns, suburbs or rural areas based on a combination of geographical contiguity and population density, applied to 1 km² population grid cells [29]. We assessed residential exposure to fine particulate matter (PM_2.5), nitrogen dioxide (NO₂) and black carbon (BC), primary constituents of traffic-related air pollution. Residential exposure to PM_2.5, NO₂ and BC was modelled using a spatial temporal interpolation method. In Flanders, atmospheric pollutants are continuously measured by a network of automatic monitoring stations by the Flemish Environment Agency. The Belgian Interregional Environment Agency (IRCEL, Intergewestelijke Cel voor het Leefmilieu) uses these measurements together with information on land cover to interpolate the air pollutant concentrations on a 4x4 km² resolution [30]. These background results are combined with a bi-gaussian dispersion model based on emissions from point sources and line sources, the Immission Frequency Distribution Model (IFDM). The combined RIO-IFDM model chain produces daily averaged pollutant concentrations in Belgium on a high resolution receptor grid [31]. We calculated mean air pollutant concentrations at the residential address over a 3-month period before sampling, similar to the period of cortisol accumulation in the hair samples, and over a 1-year period before sampling. Residential proximity to major roads is often used as a surrogate measure of long-term exposure to traffic-related air pollution [32]. We calculated the straight-line distance from each residence to the nearest major road. Major roads included international motorways (E-roads) and the network of large national and local roads of Belgium (N-roads).

Residential access to a neighborhood greenspace was based on the 2016 version of the land-use map of Flanders, which maps land cover types, i.e. natural vegetated land cover and urban greenery, in 10x10 m² raster cells [33]. Green cells were clustered to assess the area and public accessibility of greenspace in the maternal residential surroundings. Access to a small neighborhood greenspace (NHGS) was defined as access to at least 0.2 hectares (ha) of greenspace within a travel distance of 400 meters (m) from residence, access to a large greenspace was defined as access to at least 10 ha of greenspace within a travel distance of 800 m from residence. In the large greenspace typology, small water bodies are included when surrounded by > 50% greenspace, agricultural land is included when surrounded by > 30% greenspace. More technical background information on the green typology indicators can be found elsewhere [34].

Potential covariates of hair cortisol concentrations

Possible covariates of HCC were identified based on available data within the IPANEMA cohort and on existing literature [35]. Tested covariates included maternal age, parity, maternal socioeconomic status (SES) defined as the highest educational attainment of the mother and categorized as low/intermediate/high, pre-existing chronic diseases (diabetes, asthma, cardiovascular disease), pre-pregnancy body mass index (BMI), gestational week at sampling, season of sampling, smoking and alcohol consumption before pregnancy, systemic use of glucocorticoids and daily hair washing. We assessed maternal ethnic background as European/non-European country of birth since hair growth rate may be influenced by ethnicity [36]. We additionally tested variables that may have a link with both residential environment and biological stress, i.e. neighborhood SES and residential exposure to noise. A systematic review in the World Health Organization (WHO) European Region showed that lower neighborhood SES is usually linked with higher levels of air pollutants [37]. Independent from higher levels of exposure, deprived mothers may have a higher vulnerability, leading to more pronounced adverse health effects of a given environmental exposure [38]. The Area Deprivation Index (ADI) is a yearly calculated indicator for neighborhood SES on a sub-municipality level in Flanders (Statistics Flanders, n.d.). Deprivation is recorded by the Flemish Child and Family Government Agency (www.kindengezin.be). Selection criteria for deprivation are the family’s monthly income, the parents’ educational attainment, the children’s development, the parents’ employment situation, housing and health. If a family fulfils three or more criteria, it is considered to be underprivileged (OECD, 2000). The index of year X (%), i.e. 2017, considers all children born in year X, X-1 and X-2 that live in deprived households in a given area in Flanders, divided by the total number of children born in the area during the same period. The ADI of the participants’ neighborhood was subsequently categorized into tertiles representing low, intermediate and high area deprivation across the range of ADI among all participants.

Residential proximity to major roads may also lead to elevated noise levels [41]. Residential noise exposure levels were assessed using the Flemish strategic noise map of 2016, which includes major road infrastructure as defined in the EU-guideline 2002/49/EG [42]. The strategic noise map expresses noise levels in L_den, the average sound level over a 24 hour period with a penalty of 5 dB added for evening hours and a penalty of 10 dB added for nighttime hours [43]. The WHO guideline for average noise exposure produced by road traffic is set at 53 decibels (dB) L_den, road traffic noise above this level has been associated with adverse health effects, including adverse birth outcomes [44]. Noise exposure was therefore evaluated binary as exposure to a noise level ≥ 53 dB L_den.

Statistical analysis

Statistical analysis was performed using SPSS Statistics (version 25; IBM, Armonk, NY, USA) and R (version 2018; R Foundation for Statistical Computing, Vienna, Austria). Descriptive statistics provide an overview of study population characteristics, residential exposure characteristics and geometric mean HCC concentration with 95% confidence interval. Air pollution variables were logarithmically transformed (ln-scale) because of skewed distributions, distance to major roads was logarithmically transformed to reflect the non-linear distance decay of traffic-related exposure to air pollutants [45].

Spearman rank correlations between residential exposures variables were assessed, since correlations of 0.9 or higher between exposure variables indicate strongly connected exposures that cannot be disentangled [46]. The outcome variable HCC was logarithmically transformed to obtain a normal distribution. For HCC below the LOQ of 1 pg/mg hair, a random imputation from a log-normal probability distribution was performed where the mean was allowed to depend on observed values for hair cortisone concentrations that were measured simultaneously with cortisol, since both glucocorticoids were highly correlated (p < 0.01, Pearson's r = 0.711 for 2^nd trimester cortisol and cortisone, p < 0.01, Pearson's r = 0.758 for 3^rd trimester cortisol and cortisone). Linear regression models were used to analyze associations between 3-month mean air pollutant concentrations (PM_2.5, NO₂, BC) , distance to major roads and access to greenspace as a predictor and 2^nd and 3^rd trimester Hair Cortisol Concentrations as an outcome. Given the limited number of study participants, we decided not to adjust for a set of a priori selected covariates. The final regression models were only adjusted for significant covariates (p < 0.05). All assumptions of linear regression were checked. To quantify the association, the estimated change in HCC (β) with 95% confidence interval (95% CI) is presented for an increase in exposure from the 25^th to the 75^th percentile.

Effect modification by access the neighborhood greenspace was assessed by adding the interaction term of exposure to air pollution or distance to major roads and access to greenspace into the regression model. The level of significance for estimates was set at p < 0.05.

We conducted several sensitivity analyses to evaluate our results. We tested 1-year mean air pollutant concentrations in relation to 2^nd and 3^rd trimester HCC to confirm the robustness of 3-month mean results. We additionally adjusted our 2^nd trimester models for frequency of hair washing, a significant determinant of 2^nd trimester HCC, independent of biological stress.

Hair samples for cortisol analysis were provided by 152 participants. Characteristics of the study population are described in Table 1. We excluded 3 participants due to inexplicable high HCC values (> 3 times the interquartile range above the third quartile). As a result, 149 pregnant women were included in this study, of which 133 women donated a sample at the end of the 2^nd trimester (week 26 ± 1.6) and 81 women shortly after delivery (week 39 ± 1.6), 65 women donated a sample twice. Almost half of the 149 mothers (48%) was aged between 26 and 30 years, 61% of participants were primigravid. Most of the study participants were of European origin (75%, 21% data missing), enjoyed higher education (57%, 23% data missing) and were employed prior to their pregnancy (72%, 23.5% data missing).

Residential characteristics are described in Table 2. Study participants lived in cities (38%), towns and suburbs (62%) in Flanders, none of the participants lived in a rural area. The mean ADI of our study population was 16.4% (95% CI: 14.6, 18.1) whereas the mean 2017 ADI for the study region Antwerp was 17.6% (Statistics Flanders, n.d.). We tested the significance of the association between ADI as an area-level SES indicator and maternal educational attainment as a personal SES-indicator. We did not observe a significant association between neighborhood SES and personal SES (Spearman rank r = -0.074, p = 0.404). A small neighborhood greenspace was accessible for 94% of participants, 76% had residential access to a large greenspace. Three-month geometric mean PM_2.5was 11.61 (95% CI: 11.06, 12.21) µg/m³and 11.55 (95% CI: 10.95, 12.18) µg/m³for 2^nd trimester and 3^rd trimester sampling respectively. Geometric mean NO₂ concentrations 3 months before sampling was 23.03 (95% CI: 21.67, 24.47) µg/m³ for the 2^nd trimester and 23.19 (95% CI: 21.5, 24.98) µg/m³for the 3^rd trimester, 3-month geometric mean BC concentration 1.13 (95% CI: 1.05, 1.21) µg/m³ for the 2^nd trimester and 1.17 (95% CI: 1.07, 1.28) µg/m³for the 3^rd trimester. Noise exposure was covered by the Flemish strategic noise map for 144 participants, 31.5% of participating mothers was exposed to noise levels ≥ 53 dB. Geometric mean 2^nd trimester HCC was 3.94 (95% CI: 3.49, 4.45) pg/mg hair, geometric mean 3^rd trimester HCC was 6.12 (95% CI: 4.96, 7.56) pg/mg hair. The coefficient of variance (CV) of 2^nd trim HCC was 51.5%, CV of 3^rd trimester HCC was 51.8%. Second and third trimester cortisol concentrations of participants that donated a hair sample twice were moderately correlated (n = 65, p < 0.01, Pearson's r = 0.571).

Spearman rank correlations of residential exposure characteristics are presented in Table 3 (2^nd trimester study population) and Table 4 (3^rd trimester study population).

We observed strong positive correlations between 3-month mean air pollutant concentrations (r ranged from 0.61 to 0.89). Distance to major roads was negatively correlated with NO₂ and BC concentrations (r ranged from -0.24 and -0.37), but not with PM_2.5concentrations.

Access to a neighborhood greenspace did not significantly correlate with air pollutants and distance to major roads in the 2^nd trimester. In the 3^rd trimester study population, we did find weak negative correlations between access to a large neighborhood greenspace and air pollutants (r ranged from -0.28 to -0.31) and a weak positive association of access to a large neighborhood greenspace with distance to major road (r = 0.26). The ADI was weakly positively correlated with NO₂, BC and noise exposure above the WHO guideline (r ranged from 0.21 to 0.46) and negatively correlated with distance to major roads and access to a large neighborhood greenspace (r ranged from -0.18 to -0.25).

Season of sampling and daily hair washing were identified as significant covariates of 2^nd trimester HCC, no significant covariates were identified for 3^rd trimester HCC (see Table S1 for details). None of the participants reported the systemic use of glucocorticoids. Residential noise exposure above the WHO guideline (≥53 dB L_den) was not significantly associated with 2^nd or 3^rd trimester HCC (p = 0.871, p = 0.190 respectively). Nor did we find significant associations between the ADI and 2^nd or 3^rd trimester HCC (p = 0.661, p = 0.388 resp.).

Results of the associations between air pollution exposure, access to neighborhood greenspace and maternal biological stress are presented in Table 5. We found a significant negative association between 3-month mean PM_2.5 concentrations and 2^nd trimester HCC in the unadjusted model (p = 0.009), the association did not remain significant after adjustment for season of sampling (p = 0.357). In the 3^rd trimester, 3-month mean PM_2.5 concentrations were not significantly associated with HCC.

We did not observe significant associations between 3-month mean NO₂ and BC concentrations and 2^nd trimester HCC. We did observe a significant positive association between 3-month mean NO₂ concentrations and 3^rd trimester HCC (p = 0.016). For an increase of 3-month mean residential NO₂ concentrations from 18.35 µg/m³ (p25) to 30 µg/m³ (p75), an increase of 3^rd trimester HCC with a factor 1.42 (95% CI: 1.07, 1.88) was estimated. We also observed a significant positive association between 3-month mean BC concentrations and 3^rd trimester HCC (p = 0.032). For an increase of 3-month mean residential BC concentrations from 0.84 µg/m³ (p25) to 1.48 µg/m³ (p75), an increase of 3^rd trimester HCC with a factor 1.37 (95% CI: 1.03, 1.82) was estimated. Residential 3-month mean NO₂ concentrations explained 6.2% of the variations in 3^rd trimester HCC, 3-month mean BC concentrations explained 4.6%.

Residential distance to a major road was negatively associated with second trimester HCC (p = 0.016 unadjusted model, p = 0.11 adjusted model). For an increase in distance to a major road from 143 m (p25) to 642 m (p75), a change of 2^nd trimester HCC with a factor 0.82 (95% CI: 0.70, 0.95) was estimated in the adjusted model. The model explained 7.8% of the variation in 2^nd trimester HCC. Distance to a major road was also negatively associated with 3^rd trimester HCC (p = 0.04). For an increase of distance to a major road from 114 m (p25) to 598 m (p75), a change of 3^rd trimester HCC with a factor 0.74 (95% CI: 0.55, 0.99) was estimated. Distance to a major road explained 4% of the variations in 3^rd trimester HCC. Access to a small neighborhood greenspace tended towards a significant negative association with 2^nd trimester HCC (p = 0.117 unadjusted model, p = 0.061 adjusted model) and was not significantly associated with 3^rd trimester HCC (p = 0.354). Access to a large greenspace tended towards a significant negative association with 2^nd trimester HCC (p = 0.073 unadjusted model, p = 0.095 adjusted) and with 3^rd trimester HCC (p = 0.062).

We tested whether access to a neighborhood greenspace moderated the associations between air pollution exposure, proximity to major roads and maternal biological stress. We found no significant interaction between access to a small or large neighborhood greenspace and air pollution constituents in relation to 2^nd or 3^rd trimester HCC (see Table S2 for details). We did observe a significant interaction between access to a large neighborhood greenspace (NHGS) and distance to major roads in relation to 2^nd trimester HCC in both the unadjusted model, as presented in Figure 1, and the model adjusted for season of sampling (p = 0.021, p = 0.034 resp.). At an average residential distance of 304 meters from a major road, estimated mean 2^nd trimester HCC were 22% lower for mothers with access to a large neighborhood greenspace (3.71 (95% CI: 3.24, 4.24) pg/mg hair) compared to mothers without access (4.22 (95% CI: 3.26, 5.47) pg/mg hair). The adjusted interaction model explained 10.8% of the variations in 2^nd trimester HCC.

Figure 1 Interaction between distance to a major road and access to a large neighborhood greenspace in relation to 2nd trimester HCC

The interaction between access to a large neighborhood greenspace (NHGS) and distance to major roads in relation to 3^rd trimester HCC tended towards significance (p = 0.073), as presented in Figure 2. At an average residential distance of 264 m from a major road, the model estimated 19% lower mean 3^rd trimester HCC for mothers with access to a large neighborhood greenspace (5.46 (95% CI: 4.30, 6.95) pg/mg hair) compared to mothers without access (6.75 (95%CI: 4.46, 10.22) pg/mg hair). The interaction model explained 7.9% of the variations in 3^rd trimester HCC.

Figure 2 Interaction between distance to a major road and access to a large neighborhood greenspace in relation to 3rd trimester HCC

In a sensitivity analysis, we evaluated the significance of associations between 1-year mean PM_2.5, NO₂ and BC concentrations and HCC to reflect the participants’ longer-term residential exposure to air pollution. Results are presented in Table S3. Extending the exposure period did not change our results, we found significant positive associations between 1-year mean NO₂ concentrations and 3^rd trimester HCC ( p = 0.013) and between 1-year mean BC concentrations and 3^rd trimester HCC (p = 0.046). The robustness of our results was also evaluated by additional adjustment of our 2^nd trimester model with daily hair washing, a significant but external, non-biological determinant of HCC. Our results, presented in Table S4 and S5, remained robust. Residential distance to a major road, adjusted for season of sampling and daily hair washing was significantly associated with 2^nd trimester HCC (n = 103, p = 0.006, R² = 17.4% ). In accordance, access to a large greenspace significantly moderated the association between distance to major roads and 2^nd trimester HCC in the model adjusted for season of sampling and daily hair washing (n = 103, p = 0.001, R²= 22.1%). The model estimated 17.66% lower mean 2^ndtrimester HCC for participants with access to large NH greenspace compared to participants without access to large NH greenspace (2.89 (95 %CI: 2.34, 3.57) versus 3.51 (95%CI: 2.54, 4.85) pg/mg hair).

This study provides new insights in the relation between residential exposure to air pollution and road traffic and hair cortisol as a biomarker for longer-term biological stress during pregnancy. We observed significant positive associations between residential 3-month mean NO₂ and BC concentrations and maternal biological stress in the 3^rd pregnancy trimester. It should be noted that NO₂exposure levels were strongly correlated with BC exposure levels (r=0.89), making it impossible to disentangle the effects of both pollutants. In urban settings, road traffic is the principal source of NO₂ and BC in ambient air [47]. We also observed significant associations between residential proximity to major roads and maternal biological stress in the 3^rd pregnancy trimester. As previously reported, residential proximity to major roads and maternal biological stress in the 2^nd pregnancy trimester were significantly associated [48]. The difference in significant associations between traffic-related exposures and 2^nd and 3^rd trimester HCC may be due to the difference in study population between both trimesters or to the increase in circulating cortisol concentrations towards the end of pregnancy, which is a normal biological process [20]. Our observations are in line with recent human studies that reported associations between air pollutants and short-term variations in cortisol secretion. In a panel study among 43 students in Shanghai, residential exposure to PM_2.5was associated with higher serum cortisol levels [13]. In a cross-sectional analysis of 1793 adults, residential NO₂exposure was associated with higher wake-up salivary cortisol [14]. To our knowledge, only one epidemiological study has examined the association between personal air pollution exposure and HCC; the study, including Belgian schoolchildren and adolescents, did not find a significant relationship [49]. Pregnancy however, is a vulnerable period for both mother and fetus [50]. Several mechanisms potentially underlie the association between air pollution and biological stress during pregnancy. Air pollutants may induce oxidative stress and low grade inflammation [51]. Depending on size and chemical composition, inhaled air pollution constituents may translocate from the lungs to the systemic circulation or migrate via olfactory transport to the brain and directly interact with brain tissues including the hypothalamus [11]. During pregnancy, oxidative stress is known to be higher than in the non-pregnant state; residential exposure to air pollutants and road traffic may further amplify the level of maternal oxidative stress [52]. Oxidative stress may in turn lead to low grade inflammation and HPA axis activation, resulting in a marked increase in the secretion of cortisol into the circulation [53]. In addition to indirect activation of the HPA axis by systemic low grade inflammation, low grade inflammation in the brain may directly activate the hypothalamus [54]. Flanders, the IPANEMA study region, is characterized by a dense road network and high emissions from traffic [55]. The fraction of the Flemish population, living and working in close proximity to traffic, is high and access to neighborhood greenspace is typically limited. Interestingly, n our urban and suburban pregnancy cohort, access to significantly moderated the association between residential proximity to traffic and maternal biological stress in the 2^nd pregnancy trimester. Beneficial relationships between residential access to greenspace and hair cortisol concentrations have been described in previous studies [56,57]; whereas other studies have reported a beneficial impact of surrounding greenness on fetal growth and birth weight [58–61]. Neighborhood greenspace may improve health by relieving psychophysiological stress, supporting physical activity, increasing social contacts and by reducing exposure to air pollution, noise and excessive heat [62–65]. In our study, we found a weak inverse correlation between access to a large greenspace and residential air pollutant concentrations in the 3^rd pregnancy trimester, but not in the 2^nd trimester. This suggest a moderating effect of residential access to a large greenspace, independent of the effect on air pollution exposure levels.

The added value of prospective cohort studies such as IPANEMA, is the possibility to provide more insight into early pathophysiological mechanisms, triggered by air pollution exposure, in real-world settings. The urban and suburban character of the IPANEMA cohort made it possible to go deeper into traffic-related air pollution exposure, notwithstanding the low number of participants. Residential exposure to air pollution was estimated using a high spatial resolution model, residential mobility of participants was considered. In addition to maternal traffic and air pollution exposure, we took access to neighborhood greenspaces into account. Evaluation of only one residential environmental exposure i.e. air pollution, ignoring potential interaction with other jointly occurring exposures i.e. access to greenspaces, could lead to an inaccurate estimate of the true effect of exposures [66]. We measured hair cortisol concentrations, a novel method in epidemiological studies to retrospectively determine longer-term biological stress in a non-invasive and reliable way. Hair samples were collected according to a strict protocol by trained midwives at the in-hospital consultation to avoid interindividual differences in hair collection. Some limitations of the study need to be addressed. In this study, we had a limited number of participants and did not have the same study population in the second and third pregnancy trimester, leading to differences in residential exposures. We had a considerable percentage of missing questionnaire-based data. Future prospective cohort studies, ideally including a larger number of participants from pre-conception onwards, should enhance efforts to collect questionnaires from all participants, including relevant information on time spent in residential greenspaces, physical activity, wellbeing and health. The exposure assessment was limited to residential surroundings, we did not consider air pollution exposure while commuting and working. IPANEMA participants were mostly of a higher socioeconomic status, it was therefore not possible to explore increased vulnerability to environmental exposure in participants with lower SES. Moreover, the neighborhood SES indicator in this study did not reflect participants with lower socio-economic status. In literature, the pattern of air pollution is often described as U-shaped , although the most deprived areas have the highest levels of poor air quality, the least deprived areas also experience higher levels of air pollutants than some other social groups [37]. In our cohort, the mean ADI was 18.1% for lower educated mothers, 15.3% for medium education mothers and, as described in literature, we observed a slightly higher ADI of 15.6% for higher educated women. Future studies should enhance efforts to include participants of all SES.

This study observed significant positive associations between residential exposure to traffic-related air pollution during pregnancy and longer-term biological stress in the 2^nd and 3^rd trimester of pregnancy. In the 2^nd trimester of pregnancy, the association was significantly moderated by residential access to a large neighborhood greenspace. Air pollution and urban spatial planning are in the center of public debate in Flanders. Because of the ubiquitous nature of traffic-related air pollution and the adverse pregnancy outcomes that have been associated with elevated maternal biological stress for both mothers and babies, even a small increase in maternal biological stress may be of public health interest. Our research, if confirmed in future studies, may provide guidance towards a more sustainable urban planning and support environmental health protection for both pregnant women and their babies.

ADI:	Area deprivation index
BC:	Black carbon
BMI:	Body mass index
HCC:	Hair cortisol concentrations
HPA axis:	Hypothalamic-pituitary-adrenal
HPLC:	High-Performance Liquid Chromatography
IPANEMA:	Impact of Particulate Matter on Mothers and Babies in Antwerp
IRCEL:	Belgian Interregional Environment Agency
LC-MS/MS:	Liquid chromatography and tandem mass spectrometry
Lden:	Day–evening–night noise level
LOQ:	Limit of quantification
NHGS:	Neighborhood greenspace
NO₂:	Nitrogen dioxide
PM_2.5:	Particulate matter with an aerodynamic diameter ≤ 2.5 μm
RIO-IFDM:	Air quality interpolation model combined with Immission Frequency Distribution Model
SES:	Socioeconomic status
WHO:	World Health Organization

Ethics approval and consent to participate

The study protocol was approved by the ethical committee of the University of Antwerp (14/40/411). All participants gave written informed consent.

All co-authors have read the manuscript and consented with publication.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

Veerle Verheyen was supported by a VITO PhD scholarship. The IPANEMA study was sponsored by the Antwerp University. This research did not receive a specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Authors' contributions

VV contributed to the study design, data acquisition, analysis and interpretation and was the first author of the manuscript draft. SR contributed to data interpretation, data visualization and revision of the manuscript. NL contributed to the study concept and design and revision of the manuscript. EG and AC contributed to the data analysis and revision of the manuscript. LP, EV, WL and CV contributed to the acquisition of environmental data. PM contributed to the environmental data analysis. FN conducted the analysis of hair cortisol and therefore contributed to the biomarker data acquisition. LVdE contributed to the in-hospital data acquisition. YJ supervised the IPANEMA study and contributed to the study concept and acquisition of data. GS contributed to the study concept, study design and revision of the manuscript and provided general guidance.

Acknowledgements

The authors wish to thank the IPANEMA study participants and the nurses and obstetricians at the Antwerp University Hospital that recruited the participants. Exposure data was provided by the Flemish Department of Environment and Spatial Development, the Flemish Institute for Technological Research, the Belgian Interregional Environment Agency and the Belgian Royal Meteorological Institute (KMI). Erik Fransen (Antwerp University Doctoral School) provided relevant insights in statistical data-analysis.

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Table 1. Basic characteristics of the study participants

Characteristic	n (%)
Age
≤ 25	17 (11.4)
26-30	71 (47.7)
31-35	44 (29.5)
>35	17 (11.4)
Missing	0
Parity
0	91 (61.1)
1	43 (28.9)
≥ 2	15 (10)
Missing	0
Pre-existing chronic diseases
No	135 (90.6)
Yes	12 (8.1)
Missing	2 (1.3)
Pre-pregnancy Body Mass Index (kg/m²)
Underweight (<18.5)	8 (5.4)
Normal (18.5-24.9)	84 (56.4)
Overweight (25-29.9)	20 (13.4)
Obese (≥30)	12 (8.1)
Missing	25 (16.7)
Smoking before pregnancy
Never	94 (63.1)
Former smoker	21 (14.1)
Missing	34 (22.8)
Alcohol consumption before pregnancy
No	16 (10.7)
Yes	99 (66.4)
Missing	34 (22.8)
Ethnic background
European	112 (75.2)
Non-European	4 (2.7)
Missing	33 (21.1)
Educational attainment
Low (Basic level)	15 (10.1)
Intermediate (Secondary school)	15 (10.1)
High (Higher education)	85 (57.0)
Missing	34 (22.8)
Pre-pregnancy employment
No	7 (4.7)
Yes	107 (71.8)
Missing	35 (23.5)
Daily hair washing
No	97 (65.1)
Yes	18 (12.1)
Missing	34 (22.8)
Season of 2^nd trimester sampling (n=133)
Autumn	28 (21.2)
Winter	23 (17.3)
Spring	38 (28.6)
Summer	44 (33.1)
Missing	0
Season of 3^rd trimester sampling (n=81)
Autumn	23 (28.4)
Winter	21 (25.9)
Spring	12 (14.8)
Summer	25 (30.9)
Missing	0

Note: pre-existing chronic diseases include diabetes, asthma, cardiovascular diseases

Table 2. Residential characteristics of the study participants

Variable
Categorical variables (n=149)	n (%)
Neighborhood greenspace
Access to small neighborhood greenspace
No	9 (6.0)
Yes	140 (94.0)
Access to large neighborhood greenspace
No	36 (24.2)
Yes	113 (75.8)
Eurostat urbanization
Cities	56 (37.6)
Towns and suburbs	93 (62.4)
Rural	0
Noise levels
L_den < 53 dB	97 (65.1)
L_den ≥ 53 dB	47 (31.5)
Missing	5 (3.4)
Continuous variables	Geometric mean (95% CI)
Distance to major roads (m) (n=149)	290 (240, 349)
2^nd trimester air pollution ( µg/m³) (n=133)
NO₂-90 days prior to sampling	23.03 (21.67, 24.47)
NO₂ - 1 year prior to sampling	24.55 (23.39, 25.76)
PM_2.5-90 days prior to sampling	11.61 (11.06, 12.21)
PM_2.5- 1 year prior to sampling	13.09 (12.81, 13.37)
BC-90 days prior to sampling	1.13 (1.05, 1.21)
BC - 1 year prior to sampling	1.29 (1.24, 1.36)
3^rd trimester air pollution (µg/m³) (n=78)
NO_{2 -}90 days prior to sampling	23.19 (21.53, 24.98)
NO₂ - 1 year prior to sampling	24.70 (23.27, 26.23)
PM_2.5-90 days prior to sampling	11.55 (10.95, 12.18)
PM_2.5-1 year prior to sampling	12.70 (12.42, 12.99)
BC-90 days prior to sampling	1.17 (1.07, 1.28)
BC - 1 year prior to sampling	1.29 (1.21, 1.37)
Ambient temperature (°Celsius)
90 days prior to 2^nd trimester sampling (n=133)	10.5 (9.7, 11.4)
90 days prior to 3^rd trimester sampling (n=81)	12.6 (11.7, 13.3)
Area deprivation index (%) (n=149)	16.4 (14.6, 18.1)

Note:Categorical data is described as frequencies (%), continuous data is described by geometric mean with 95% confidence interval (95% CI). All data is based on the maternal residential address. Major roads include E- or N-roads. Access to small neighborhood greenspace is defined as access to > 0.2 hectares (ha) of greenspace within a travel distance of 400 meters (m) from residence, access to large neighborhood greenspace is defined as access to > 10 ha of greenspace within a travel distance of 800 m from residence. L_den, day–evening–night noise level_;NO₂, nitrogen dioxide; PM_2.5, fine particulate matter with an aerodynamic diameter ≤ 2.5 μm; BC, black carbon.

Table 3. Spearman rank correlations between residential exposures in the 2nd trimester study population (n=133)

	BC	NO₂	Distance to major road	Small greenspace	Large greenspace	Noise	ADI
PM2.5	0.68^*	0.68^*	-0.15	-0.02	-0.04	0.08	0.15
	BC	0.89^*	-0.29^*	-0.09	-0.14	0.28^*	0.38^*
		NO₂	-0.37^*	-0.08	-0.12	0.31^*	0.46^*
			Distance to major road	0.01	0.17	-0.32^*	-0.19^*
				Small greenspace	0.43^*	-0.14	-0.11
					Large greenspace	-0.20^*	-0.18^*
						Noise	0.21

^*Significant correlations (p < 0.05)

Note: Air pollutants were modelled at the maternal home address, 3-mont mean concentrations were calculated. Major roads include E- or N-roads. Access to small neighborhood greenspace is defined as access to > 0.2 hectares (ha) of greenspace within a travel distance of 400 meters (m) from residence, access to large neighborhood greenspace is defined as access to > 10 ha of greenspace within a travel distance of 800 m from residence. Noise exposure is evaluated as exposure above the WHO health-based guideline of 53 dB Lden (day–evening–night noise level). ADI, area deprivation index; NO₂, nitrogen dioxide; PM_2.5, fine particulate matter with an aerodynamic diameter ≤ 2.5 μm; BC, black carbon.

Table 4. Spearman rank correlations between residential exposure in the 3rd trimester study population (n=81)

	BC	NO₂	Distance to major road	Small greenspace	Large greenspace	Noise	ADI
PM2.5	0.61^*	0.59^*	-0.06	-0.19^*	-0.31^*	0.01	0.04
	BC	0.89^*	-0.24^*	-0.22^*	-0.3^*	0.18	0.31^*
		NO₂	-0.32^*	-0.21^*	-0.28^*	0.24^*	0.39^*
			Distance to major road	0.13	0.26^*	-0.31^*	-0.25^*
				Small greenspace	0.41^*	-0.37^*	-0.08
					Large greenspace	-0.15^*	-0.19
						Noise	0.25^*

^*Significant correlations (p < 0.05)

Note: Air pollutants were modelled at the maternal home address, 3-mont mean concentrations were calculated. Major roads include E- or N-roads. Access to small neighborhood greenspace is defined as access to > 0.2 hectares (ha) of greenspace within a travel distance of 400 meters (m) from residence, access to large neighborhood greenspace is defined as access to > 10 ha of greenspace within a travel distance of 800 m from residence. Noise exposure is evaluated as exposure above the WHO health-based guideline of 53 dB Lden (day–evening–night noise level). ADI, area deprivation index; NO₂, nitrogen dioxide; PM_2.5, fine particulate matter with an aerodynamic diameter ≤ 2.5 μm; BC, black carbon.

Table 5. Associations between residential exposures and hair cortisol concentrations in the second and third pregnancy trimester

	2nd trimester unadjusted model (n=133)		2nd trim HCC adjusted model^a (n=133)		3rd trimester unadjusted model (n=81)
Exposure	p-value	β (95% CI)	p-value	β (95% CI)	p-value	β (95% CI)
3-month mean PM_2.5	0.009	0.81 (0.70, 0.95)	0.200	0.87 (0.70, 1.08)	0.227*	1.20 (0.89, 1.62)
3-month mean NO₂	0.934	0.99 (0.83, 1.18)	0.287	1.10 (0.92, 1.34)	0.016*	1.42 (1.07, 1.88)
3-month mean BC	0.551	0.94 (0.79, 1.13)	0.775	1.03 (0.84, 1.27)	0.032*	1.37 (1.03, 1.82)
Dist. to major roads	0.016	0.82 (0.70, 0.96)	0.011	0.82 (0.70, 0.95)	0.040	0.74 (0.55, 0.99)
Small greenspace	0.117	0.68 (0.42, 1.10)	0.061	0.63 (0.38, 1.02)	0.354	0.67 (0.28, 1.05)
Large greenspace	0.073	0.77 (0.57, 1.03)	0.095	0.78 (0.59, 1.04)	0.062	0.65 (0.41, 1.02)

Note: ^aadjusted for season of sampling. Estimates are presented for an increase of exposure from the 25^th to the 75^th percentile. *Data available of 78 participants. Air pollutants were modelled at the maternal home address, 3-mont mean concentrations were calculated. Major roads include E- or N-roads. Access to small neighborhood greenspace is defined as access to > 0.2 hectares (ha) of greenspace within a travel distance of 400 meters (m) from residence, access to large neighborhood greenspace is defined as access to > 10 ha of greenspace within a travel distance of 800 m from residence. CI, confidence interval; NO₂, nitrogen dioxide; PM_2.5, fine particulate matter with an aerodynamic diameter ≤ 2.5 μm; BC, black carbon

supplement6.docx

Residential exposure to air pollution, access to neighborhood greenspace and their association with hair cortisol concentrations in the second and third trimester of pregnancy

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Abstract

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Background

Methods

Results

Discussion

Conclusions

Abbreviations

Declarations