Residential green space and child intelligence and behavior across urban, suburban, and rural areas in Belgium: A longitudinal birth cohort study of twins

Background Exposure to green space has beneficial effects on several cognitive and behavioral aspects. However, to our knowledge, no study addressed intelligence as outcome. We investigated whether the level of urbanicity can modify the association of residential green space with intelligence and behavior in children. Methods and findings This study includes 620 children and is part of the East Flanders Prospective Twin Survey (EFPTS), a registry of multiple births in the province of East Flanders, Belgium. Intelligence was assessed with the Wechsler Intelligence Scale for Children-Revised (WISC-R) in 620 children (310 twin pairs) between 7 and 15 years old. From a subset of 442 children, behavior was determined based on the Achenbach Child Behavior Checklist (CBCL). Prenatal and childhood residential addresses were geocoded and used to assign green space indicators. Mixed modeling was performed to investigate green space in association with intelligence and behavior while adjusting for potential confounding factors including sex, age, parental education, neighborhood household income, year of assessment, and zygosity and chorionicity. We found that residential green space in association with both intelligence and behavior in children was modified by the degree of urbanicity (p < 0.001). In children living in an urban environment, multivariable adjusted mixed modeling analysis revealed that an IQR increment of residential green space (3,000-m radius) was associated with a 2.6 points (95% CI 1.4–3.9; p < 0.001) higher total intelligence quotient (IQ) and 2.0 points (95% CI −3.5 to −0.4; p = 0.017) lower externalizing behavioral score. In children residing in a rural or suburban environment, no association was found. A limitation of this study is that no information was available on school location and the potential for unmeasured confounding (e.g., time spend outdoors). Conclusions Our results indicate that residential green space may be beneficial for the intellectual and the behavioral development of children living in urban areas. These findings are relevant for policy makers and urban planners to create an optimal environment for children to develop their full potential.


SUMMARY
Surrounding greenness at home, air pollution and maternal stress have been related to reduction of behavioural problems and improvement of cognitive development, but little is known about mechanism(s) underlying such associations. We hypothesize that exposure to residential greenness might affect in utero stress conditions, both social, as maternal perceived stress, as well as biological stress (inflammation) which both might play a role in behavioural and cognitive development in children. Our hypothesis is supported by available animal experiments showing cognitive effects of particulate air pollution exposure due to a pro-inflammatory state. There are very few human studies on cognitive development and residential traffic and greenness surrounding and non in a susceptible population segment such as twins and triplets.
In the first part of this project, neurocognition was assed among 663 twins pairs with a mean age of 10.4 years old by the Wechsler Intelligence Scale for Children-Revised (WISC-R) and among 752 twins pairs (mean age of 8.2 years), the neurobehavioural outcome was examined with the Child Behaviour Checklist (CBCL). Both test were performed by 52 triplet sets (140 children). In addition, placentation and perinatal data were recorded at birth.
In a second prospective part, momentary maternal stress during pregnancy will be assessed using The Experience Sampling Method (ESM), the Perceived Stress Scale and cortisol concentrations measured in hair. The association between maternal stress and residential greenness will be assessed. In addition, the intermediate role of maternal stress will be evaluated in the relationship between residential air pollution and biological marker of oxidative stress/inflammation. Greenness of the immediate home environment will be evaluated using detailed satellite-derived data, exposure to pre-and postnatal exposure will be assessed by use of land use and dispersion models.
The originality of our project include to study in a retrospective fashion, the association between exposure to greenness, traffic, and stress on the one hand and neurodevelopment on the other hand.
In addition, in a prospective fashion, we will study perceived stress as a possible underlying mechanism. The project is relevant for society, indeed, the most recent EU key directive on ambient air quality is Directive 2008/50/EC. It recognizes the need to reduce pollution to levels, which minimize the harmful effects on human health. The limit values adopted for particulate matter are still higher than WHO recommends. For example, the annual average limit value for PM2.5 was set at 25 μg/m 3 , a level 2.5 times higher than recommended by WHO. The current project allows for detailed risk assessments considering interactions with a broad range of environmental exposures.
The results of our study may lead to a better protection of the most vulnerable individuals in society, i.e. newborns, more specifically those with a complex pregnancy such as twins and triplets. We are seeing more twins now than ever before: twinning rates are at a peak, occurring in 1/30 births in western nations, double the 1980 rate of 1/60. The twins further allow to distinguish the impact of the genes and the environment on the neuropsychological tests and IQ. Knowing that genes, as well as environments, affect our abilities, talents and skills is crucial information for everyone to know.
Further, the project might prioritize health related aspects more directly in the future urban planning.

Greenness
Recently home surrounding greenness has been associated with a protective impact on emotional and behavioural problems in schoolchildren in Barcelona 1 which was also observed in a German birth cohort. 2 Both studies reported a larger decrease on hyperactivity and in attention symptoms. These findings were consistent with the effects of 'nature restoration' observed in a small sample of 17 children who improved their cognitive function after relocation to residences with higher 'naturalness'. 3 Accordingly, four trials have shown an improvement of attention deficit and hyperactivity disorder (ADHD) symptomatology after exposures to the natural environment. [4][5][6] Consistently, a recent US cross-sectional study has reported an improved school performance associated with school greenness based on data from all the schools in Massachusetts. 7 The mechanisms underlying observed associations between exposure to greenness and mental and psychological status are not well-understood.

Air pollution
Solid and liquid phase material suspended in the atmosphere is referred to as 'fine particulate matter' (PM). Cardiorespiratory effects and mechanism of particulate air pollution have been largely investigated. 8,9 In contrast, little is known about neurological effects of fine particulate air pollution.
There exist several pathways by which air pollution may exert neurotoxic effects. The direct ones results in an accumulation of ultrafine particles in het human brain. 10,11 In rats, inhaled ultrafine particles translocate from the nose along the olfactory nerve to the olfactory bulb and other regions of the brain. [12][13][14][15] Additionally, studies in rodents have demonstrated that ultrafine particles translocate from the lungs into the blood, from which they can reach extrapulmonary organs including the brain. 16,17 The indirect pathways involve a pro-inflammatory response in the lung when exposed to air pollution leading to increased systemic inflammation, which in turn, may adversely affect the brain. [18][19][20][21][22] Inflammatory mediators produced in the lung are able to translocate into the circulation 23 and trigger a sequel of pro-inflammatory events. 19,[24][25][26] Exposure to airborne polycyclic aromatic hydrocarbons (PAH) during the third trimester of pregnancy in women residing in New York. 27 Children with a prenatal PAHs exposure above the median had a 4.7 points (95% CI: -7.7 to -1.6) lower verbal IQ and 4.3 points (95% CI: -7.4 to -1.2) lower full scale IQ than those with exposure below the median. 27 The impact of PAHs on mental development at 3 years of age does not appear to be mediated by birth weight or head circumference, fetal growth parameters previously shown to be associated with prenatal PAH exposure in this cohort. 27 Studies observed a significant association between neighborhood green space and lower levels of symptomology for depression, anxiety and stress. 33,34 In addition maternal stress during pregnancy is associated with adverse neurodevelopmental outcomes in the child. These include emotional or cognitive problems, such as increased risk of attentional deficit/hyperactivity, anxiety, and language delay. [35][36][37] However many questions remain about the underlying mechanism. A proposed mechanism by which prenatal stress may affect the fetus is cortisol. 38 During pregnancy, women have naturally elevated levels however under stressful conditions maternal cortisol concentrations can reach high levels. Consequently high concentrations of cortisol may affect fetal development and growth 39 and according to animal studies this may even alter the development of neurons in the brain. [40][41][42] The emotional state of the mother may also alter the function of the placenta in other ways, independent of cortisol. There is some evidence for effects on uterine blood supply, which could affect the neurodevelopment of the fetus. 43 Besides maternal stress also biological stress, such as inflammation and oxidative stress, may serve as a mediator between environmental exposure and neurocognitive development. Exposure to particulate air pollution can result in oxidative stress and inflammation. 44,45 This is supported by studies showing an association between in utero traffic exposure and shorter telomere length and decreased mitochondrial DNA content in placental tissue, both sensitive to the effects of oxidative stress. 46,47 However the effects of biological stress during pregnancy on the development of the fetal brain and neurocognition in childhood are currently unknown.

Epigenetics to link the role of early life exposure with brain development
The placenta, aside from its role as exchange barrier, also contributes to neurodevelopmental processes through adaptive responses to the maternal environment. These critical developmental processes in the placenta and fetal brain are shaped by the same molecular signals. 48 Transcriptional changes during prenatal development are associated with morphological and functional development of the fetal brain. 49 In mice, a strong correlation was observed between a set of genes, co-expressed in the hypothalamus and placenta at mid-gestation (embryonic day [11][12][13], an important period of neuronal proliferation and differentiation. 50 Bonnin et al. 51 showed in an ex vivo model that the placenta can convert maternal tryptophan into the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) and functions as primary source of 5-HT for the developing mouse forebrain at mid-gestation.
Besides the serotenergic pathway, other pathways in the placenta such as the dopaminergic, the glucocorticoid and the brain-derived neurotrophic factor (BDNF) signalling pathway may also be involved in fetal neurodevelopment. A reduction in gene expression of the brain-derived neurotrophic factor (BDNF) pathway in human placental tissue and cord blood has been found to negatively affect neurodevelopment during childhood. 52 This pathway is involved in critical developmental processes, including embryo implantation, placental development, and fetal growth. Single nucleotide polymorphisms (SNPs) of genes of the serotonergic and dopaminergic pathways 53,54 have been linked to neurological disorders, such as psychosis and schizophrenia. 55,56 These pathways have also been characterized in relation to the placenta and neurological development. 57,58 The concept of the placental regulation in brain development is relatively new and is in line with the observations of fetal programming and disease susceptibility later in life. 59 Epigenetic mechanisms, including promoter DNA methylation, are believed to play an essential role in this process. Indeed, recent studies have shown that epigenetic regulation through DNA methylation in the placenta is associated with infant neurobehavioural outcomes. 60,61 Groundbreaking nature and implications for twins The intelligence score of twins and triplets is lower than the score of a singleton. 62 This difference in intelligence scores is partly attributed to differences in intrauterine growth. 62 However, to the best of our knowledge, the association between neurodevelopment and residential greenness, air pollution, traffic, noise and stress exposure in utero and during childhood has not been investigated in twins and triplets.
The originality lays in: I) studying the association between exposure to greenness, air pollution and stress and neurodevelopment using validated and objective neuropsycholocial tests and IQ together with high quality satellite data on greenness and conducting a follow-up design capable of linking exposures early in life in relation to the brain effects latter during the school period, II) assessing intermediate pathways such as inflammation in this association in a group of school age twins, III) the use of placental tissue as surrogate to study epigenetic targets of brain development and IV) the twin design to unravel genetic versus environmental effects.

Objectives and aim
Overall Aim: To study the association between neurodevelopment and residential surrounding greenness, air pollution and stress conditions, both social as maternal perceived stress, as well as biological stress (inflammation).

Maternal stress perception and assessment
Mothers will complete questionnaires retrospective about stress perception during pregnancy. The Perceived Stress Scale (PSS) is the most widely used psychological instrument for measuring the perception of stress. It is a measure of the degree to which situations in one's life are appraised as stressful.

Neurodevelopment and behaviour
The neurodevelopment outcome was assessed by the Wechsler Intelligence Scale for Children-Revised (WISC-R). The WISC-R consists of six verbal and six performance subscales and has been validated for use in this population. 68  The neurobehavioural outcome was assessed by the Achenbach Child Behaviour Checklist (CBCL).
The CBCL was developed by Achenbach (1991) to examine the extent to which children have behavioural and emotional problems as perceived by their parents. Although the CBCL allows for the calculation of separate scores corresponding to several behavioural dimensions based on exploratory factor analysis, in this study the total amount of psychopathology, as measured by the total problem score, was examined.

Underlying mechanism: retrospective study
Epigenetic mechanisms, including promoter DNA methylation, are believed to play an essential role in the process of placental regulation in brain development. We will investigate methylation of CpG sites within promoter regions of the most important genes involved in early neurodevelopment based on literature. Genes within several pathways will be studied since the serotenergic pathway, the dopaminergic, the glucocorticoid and the brain-derived neurotrophic factor (BDNF) signalling pathway may be involved in fetal neurodevelopment.
To investigate molecular effects of oxidative stress, biomarkers in processes associated with oxidative stress and inflammation will be measured. Postnatal measures of these biomarkers, telomere length and mitochondrial DNA content, will be applied in placental tissue.

Subject recruitment
We will recruit mothers pregnant of twins in East-Flanders (Belgium). At birth besides placental tissue and cord blood, also maternal blood and hair will be collected. In addition, we asses stress during pregnancy and at time of birth.

Exposure assessment
Similar traffic and land-use variables will be measured as described in part I. In addition residential exposure to particulate air pollution will be determined. Regional background levels of PM10 for each mother's residential address will be estimated using a spatial temporal interpolation method (Kriging) that uses land cover data obtained from satellite images (Corine land cover data set) in combination with monitoring stations (n = 34). 69,70 This model provides interpolated daily PM10 values in 4 × 4 km grids from the Belgian telemetric air quality networks.

Maternal stress perception and assessment (Pilot study)
Maternal emotional and daily life stress reactivity will be assessed during one week in the third trimester of pregnancy. The Experience Sampling Method (ESM) will be used. This is a random timesampling self-assessment technique that has been shown to be feasible, valid, and reliable in general and patient populations. 71,72 Subjects will receive a digital wristwatch that emits a signal ten times a day on five consecutive days, at unpredictable moments between 7:30 a.m. and 10:30 p.m.
After each 'beep', subjects will complete ESM selfassessment forms concerning current context, thoughts, emotions, and psychotic experiences. Subjects will be instructed to complete their reports immediately after the beep, thus minimizing memory distortions. 73 In addition, at time of birth mothers will receive a questionnaire; the Perceived Stress Scale (PSS).
Cortisol, which is synthesized from cholesterol, is the main glucocorticoid in the zona fasciculata of human adrenal cortex and its secretion in response to biochemical stress contributes to the wellcharacterized suppression of the hypothalamic-pituitary-adrenal (HPA) axis on health and cognition events. [74][75][76] Since the vast majority of cortisol actions rely on binding to it cytosolic receptors, only the small fraction of unbound, free cortisol is revealed to be biologically active. It comes out of the mitochondrion and moves out of the cell into the extracellular space and into the bloodstream. Due to its low molecular weight and lipophilic nature, unbound cortisol enters cells by passive diffusion that makes it feasible to measure the free cortisol in many body tissues. 77 In this study, maternal cortisol levels during pregnancy will be measured in hair. Measurement of cortisol concentrations in hair is a non-invasive method and a better tool for evaluating chronic stress compared to salivary cortisol. 78 Since hair grows approximately one cm per month, we will collect 9 cm of maternal hair at time of delivery to reflect the 9 months of pregnancy. 79

Inflammation & oxidative stress
Oxidative and nitrosative stress is postulated to be one of the mechanisms by which particulate matter (PM) exerts its effects. 80 Organic chemical components onto the particle surface play an important role in the production of reactive oxygen species (ROS). Mitochondrial DNA (mtDNA) molecules are highly susceptible to oxidative stress. Accumulation of mtDNA mutations leads to alterations of mitochondrial biogenesis and function that might result in decrease of mtDNA content within cells and implies a role of mtDNA content as a potential biomarkers in processes associated with oxidative stress and inflammation. In addition, telomere shortening is a marker of exposure to life time oxidative stress. Postnatal measures of these inflammation biomarkers will be applied in cord blood.

STATISTICAL ANALYSIS PLAN
Mixed modelling will be performed to assess the association between environmental factors and outcomes. The twins will be analyzed as individuals in a multilevel regression analysis by adding a random intercept to the model to adjust for the relatedness between twin members. Covariables and potential confounders will be identified, including newborn's sex, age, parental education, neighbourhood socioeconomic status. We will use a prospective observational design. First, we will investigate the association between exposure (environmental factors) and outcome (cognitive result of this assumption is that a greater within-pair similarity in MZ twins than in DZ twins reflects genetic influences. Using the twin design, the relative influence of genetic and environmental factors on birth outcomes can be estimated (aim 5).

FUTURE PERSPECTIVES
We designed the study in a specific way that after 4 years we not only provided a finished project but also as a stepping stone to new projects. The twins recruited in part II can be invited to a followup study at age 4 year. Measurements of neurodevelopment and micro-circulation at this follow-up can be studied in relation to previous determined parameters; air pollution and stress exposure, molecular markers of inflammation/oxidative stress.

Micro-circulation
Changes in the microcirculation can be explored non-invasively by studying retinal blood vessels that are visualized in fundus images, 81  offers the opportunity to follow up twins from birth to adulthood. Environmental influences on the intrauterine environment may not only effect fetal growth but also may predispose offspring to increased risk of clinical outcomes later in life. Her research shows that prenatal exposure to air pollution and traffic was respectively associated with a suboptimal fetal growth and a change in placental telomere length, a biomarker of ageing. Besides these outcomes at birth, she noted that exposure to traffic early in life was associated with shorter telomere length in young adulthood and that surrounding greenness in early-life is associated with a lower blood pressure in adulthood.