In recent years a growing body of epidemiological research has focused on the potential impact of prenatal exposure to air pollution on birth outcomes. Several outcomes have been related to exposure to air pollution during pregnancy, including low birth weight, reduced birth size, and intrauterine growth retardation 1234. Moreover, reduction in fetal growth has been associated with poor neurological development as well as with an increased risk for chronic diseases later in life 56.

A cohort study is the design of choice for evaluating the impact of air pollution on fetal growth as pregnancy is a process in which the relationship between a given type of exposure and an associated effect may be observed in a limited period of time 7. Some of the studies carried out on this topic have included large populations using birth data from health care registries 8910 whereas other cohort studies had smaller samples, but more detailed, primary data 111213. Authors of recent methodological reviews 7141516 agree that new prospective studies should allow for adequate assessment of air pollution exposure, consider different time windows of exposure, and collect sufficient information on confounding variables.

Nitrogen dioxide (NO₂) is the air pollutant most frequently used as a surrogate for traffic-related pollution in prospective studies, both in adults and in children 1718. This is due to the fact that outdoor NO₂ levels correlate well with pollutants generated by traffic, they can be easily measured using passive samplers, and they are routinely measured by air quality networks, which allows for correction for seasonality.

The INMA study (Spanish Children's Health and Environment) is a prospective multi-centre pregnancy and birth cohort study that seeks to evaluate the role of the environment on fetal development and children's health in the general population in Spain 19. The objective of this report is to assess the association between residential exposure to outdoor NO₂ during pregnancy and anthropometric measures at birth.

Characteristics of the newborns, the mothers and their pregnancy, and the fathers' height in relation to size measures and SGA are described in Additional file 1. In brief, older mothers, mothers who had higher pre-pregnancy weight and/or BMI, who were taller, of higher social class, non-smokers, and of Latin American origin had infants with a higher birth weight and a lower proportion of SGA (in weight) babies. Primiparous mothers, those with low weight gain, those with only primary school education, and those who still smoked at week 12 had infants with a lower birth weight and a higher proportion of SGA (in weight) babies. Boys weighed more than girls. Similar patterns were found for birth length and head circumference adjusted for gestational age, and for SGA (in length) except that there were no differences by country of origin, and in the case of length, no differences by either social class or education were observed. Finally, taller fathers had bigger babies and a lower proportion of SGA babies.

The spatial distribution of NO₂ levels throughout the study area showed a gradient from the urban zone to the rural one with the two motorways crossing the area playing an important role (Figure 1). The mean residential outdoor NO₂ level corresponding to the 785 pregnancy periods was 36.9 μg/m³ (Table 1). For 43.2% of the women, the outdoor NO₂ levels at their residences during the pregnancy period were above 40 μg/m3, the World Health Organization guideline for annual NO₂ concentration 24. Individual NO₂ levels for each trimester correlated well with NO₂ levels for the whole pregnancy, and moderately between themselves (Table 1).

Results from this mother and child cohort living in a large, heterogeneous area in Valencia, Spain, suggest an association between maternal exposure to outdoor air pollution and birth outcomes. The odds of being SGA-weight increased by 37% when ambient NO₂ levels increased 10 μg/m³ during the second trimester of pregnancy. For anthropometric measures in continuous form, an association with air pollution appeared for women living in zones with ambient NO₂ levels above 40 μg/m³. The first and second trimesters seem to be the relevant window of exposure.

Results for the different air pollutants varied in the different studies. Besides particulate matter (PM) [either of diameter <10 μm -PM₁₀- or <2.5 μm -PM_2.5-] and carbon monoxide (CO), NO₂ appears as one of the pollutants more frequently associated with birth outcomes. In a previous review 2 we identified six articles reporting associations between NO_x or NO₂ with either birth weight, low birth weight (LBW, measured as birth weight <2500 g), or SGA. The three articles that included nitrogen oxides (NO_x) were ecological in design and used data from central monitors. None of them found an association between NO_x and birth weight. For NO₂, results from the literature reviewed suggested some association with birth weight, but were still not conclusive 82526. In recent years a considerable number of articles have been published in this field. We have identified 12 articles studying the association of NO₂ exposure with birth weight that were published after our previous review (Additional file 2) 101227282930313233343536. Of the four studies analyzing birth weight, an association was found in three of them: Bell et al. in Massachusetts and Connecticut (USA) 10, Mannes et al. in Sydney (Australia) 32, and Gouveia and cols in Brazil 29, but no relationship was observed in the Children's Health Study 31. Interestingly, all but one of the articles 36 studying SGA found an association with NO₂; in the study in question, however, NO₂ was the only pollutant studied to be associated with head circumference. As an example, in their study in Vancouver, Brauer et al. 35 estimated residential exposures to air pollution and the risk of SGA. Of the seven air pollutants studied, the association with NO₂ was the most robust. On the other hand, only three studies found an association between LBW and NO₂ 102835. This discrepancy may be due to the fact that the number of cases of SGA is greater than that of LBW term babies, which gives the study more statistical power. Moreover, the use of SGA, calculated for each week of gestation, enables the effect of gestational length to be more effectively controlled than LBW, which is estimated simply by selecting births that take place after a certain period of gestation (i.e. between weeks 37- 44).

Few studies have examined the relation between air pollution exposure during pregnancy and other anthropometric indicators such as birth length or head circumference (HC). Studies of two cohorts of pregnant women in Poland and in New York described a relationship between prenatal exposure to airborne polycyclic aromatic hydrocarbons (PAH) and fetal growth 37. Regarding prenatal NO₂ exposure and birth length or HC, a birth register-based study assessed birth length and HC among 26,617 term births in Brisbane, Australia 36. An IQR range increase in NO₂ (11.1 μg/m³), but not in other pollutants, during the third trimester was associated with a reduction in crown-heel length: -0.15 cm (95%CI: -0.25 to -0.05). Moreover, in the French Eden cohort 38 a reduction of -0.31 cm in HC at birth was found when comparing NO₂ exposure in the highest tertile (>31.4 μg/m³) to that in the lowest tertile. Our results are consistent with the findings of these two studies.

Up to now a clear window of susceptibility for growth retardation has not been identified. In our study we found that exposure during the first trimester is most closely related to a decrease in birth weight and length. In the case of SGA (both, in weight and in length) however, the strongest relationship was found with exposure in the second trimester. Regarding reduced HC, when exposure was evaluated above vs. below 40 μg/m³, exposure throughout the pregnancy was the most clearly related. This may indicate that exposure during the entire pregnancy plays the most important role for reduction in the growth of the infant head.

Very few studies have completely assessed the shape of the relationship between air pollution exposure and reproductive outcomes. Instead, most have analyzed the relation using air pollution variables in the continuous form or comparing only two levels. Some have attempted to examine the shape using tertiles or quartiles and observed an increased risk of LBW at higher quartiles 1228. Regarding NO₂ exposure and birth weight, only Ha et al. 25 examined this relationship using GAM models, as did we in the present study. In the former study, although the authors considered the relationship to be relatively linear, a change in the slope may be observed in the figures, with a higher negative gradient after NO₂ values of around 32 ppb (60 μg/m³). In our study, we found some indication of a reduction in birth length starting at a threshold of approximately 40 μg/m³. For HC and the risk of SGA we found a monotonic relationship with air pollution exposure.

The biological mechanisms by which air pollutants may affect fetal growth are still unclear. There is some evidence that NO₂ alters fetal growth and thus may play a causal role. NO₂ is a potent oxidant and increased lipid peroxidation in the maternal and/or fetal compartment has been found in preterm births 39. Tabacova et al. investigated the relationship between exposure to nitrogen-oxidizing species and pregnancy complications in an area in Bulgaria highly polluted by oxidized nitrogen compounds 40. Methemoglobin, a biomarker of individual exposure, was determined, and glutathione balance and lipid peroxide levels were used as measures of oxidant/antioxidant status. A high percentage of women suffered from pregnancy complications, the most common being anaemia (67%), threatened abortion/premature labour (33%), and signs of preeclampsia (23%). Methemoglobin was significantly elevated in all three conditions, in comparison with normal pregnancies. Reduced:total glutathione, an indicator of maternal antioxidant reserves, decreased, whereas cell-damaging lipid peroxide levels increased. More recently, Mohoroviz found similar results for methemoglobin in a polluted area of Croatia 41. These results suggest that maternal exposure to environmental oxidants can increase the risk of pregnancy complications through stimulation of methemoglobin formation, which may lead to hypoxia and hypoxemia in pregnant women and has an important influence on maternal health as well as on placental and fetal development.

Our study has several limitations. The number of women participating in the study is small compared with that in other studies. Subsequently, the power of the study is fairly low and the estimates have wide confidence intervals. In addition, we had no information available on other important pollutants such as PM₁₀, PM_2.5, sulphur dioxide (SO₂), and CO, for which some associations with fetal growth have been described in other studies. Consequently, we cannot affirm that NO₂ is the air pollutant definitively associated with birth measurements. Due to the colinearity between pollutants, NO₂ may simply be a proxy for other toxins. Still, NO₂ has been shown to be a marker of air pollution from road traffic 42 and could be a reasonable marker of ultrafine particulates or PAH from this source. Unfortunately, we did not have information on indoor levels of air pollutants. However we did have information on environmental tobacco smoke exposure, an important source of indoor air pollution, and we controlled for this.

Notwithstanding the aforementioned weaknesses, our study has several important strengths. In this prospective study we followed a pregnant cohort from early pregnancy and assessed exposure, health outcomes, and covariates in great detail. In addition, the statistical approach using GAM models allows us to examine the shape of the relationship while the use of robust methods permits the minimization of the influence of extreme values. Moreover, we developed a protocol combining measurements from NO₂ passive samplers, kriging, and LUR in order to obtain estimates of individual exposure to ambient NO₂ for each woman. We also performed four different campaigns to assess the stability over time of the spatial NO₂ distribution in the study area, as recommended by Ritz and Wilhelm 15. Our method allowed us to address local heterogeneity in order to assign an individual estimate of the exposure, a problem that has been reported to affect other studies 1530. Lastly, our study had access to detailed information about each woman's residence throughout pregnancy, including changes of location and address.

Findings from this mother and birth cohort study in Valencia, Spain, suggest that prenatal exposure to outdoor air pollution, measured as NO₂, affects the anthropometric development of the fetus, reducing its length and head circumference and increasing the risk of having a small for gestational age (in weight) baby.

We found an association between exposure to levels of NO₂ above 40 μg/m³ during the first trimester of pregnancy and a reduction in birth weight. This association was only marginal for birth length.

For head circumference (HC) reduction and the risk of SGA, a monotonic relationship with air pollution exposure was observed. The relevant period of exposure for the risk of SGA was the second trimester. Exposure throughout the pregnancy played the most important role in decreased HC.

Compared with other recent studies, NO₂ levels in the study area occupy an intermediate position; therefore, the results are not due to extreme exposure conditions. Taking into account the relationship between fetal growth reduction and child development and health, strategies should be developed to reduce air pollution in order to prevent these risks.

BMI: Body mass index; BSP: Black smoke particles; CI: confidence interval; CO: carbon monoxide; GAM: generalized additive models; GIS: geographical information system; HC: head circumference; INMA: Spanish Children's Health and Environment study; IQR: Interquartile range; LBW: low birth weight (measured as birth weight <2500 g); LR: likelihood ratio; LUR: land use regression; NO₂: nitrogen dioxide; NO_x: nitrogen oxides; OR: odds ratio; PAH: polycyclic aromatic hydrocarbons; PM: particulate matter; PM₁₀: particulate matter of diameter <10 μm; PM_2.5: particulate matter of diameter <2.5 μm; ppb: parts per billion; PR: prevalence ratio; SES: socio-economic status; SGA: small for gestational age; SO₂: sulphur dioxide.

The authors declare that they have no competing interests.

Authors contributed to the article as follows: FB conceived the study, supervised the data collection and data analysis, and prepared the manuscript. ME contributed to data collection, conducted the data analysis of the association of interest, and helped with manuscript preparation. CI prepared the outcome variables, developed the land use regression analysis, assisted with data analysis, and helped with data interpretation and manuscript preparation. SL, AE, RR, ML, and MR contributed to data collection, provided critical revision of the manuscript, and helped with data interpretation and manuscript preparation. All authors have read and given final approval of the version to be published.