Administration of antenatal corticosteroids (AC) is the standard of care during pregnancy for women who are at risk of early delivery. Evidence indicates that AC improve survival and reduce morbidity for preterm infants. However, research suggests that infants whose mothers receive AC have an altered hypothalamic-pituitary-axis (HPA) response to stressors in early life. Results are mixed regarding the nature of these effects, with studies showing both suppressed and augmented HPA activity. In addition, research is very limited beyond the 4th month of life. The purpose of this study was to determine if AC exposure was associated with infant cortisol levels in a resting state or in response to a stressor at 1, 6 and 12 months postnatal. We also evaluated the moderating role of preterm birth in this association. 181 women and their infants participated in the study. Women were recruited during the 3rd trimester of pregnancy; at this time, they completed the Perceived Stress Scale and provided 8 salivary samples over a 2-day period for cortisol assay. They provided these data again at 6 and 12 months postnatal. At 1, 6, and 12 months postnatal, salivary samples were collected from infants to examine their cortisol levels before and after participation in a ‘stressor protocol’. Data were extracted from the medical record on AC exposure, gestational age, maternal obstetric risk, and neonatal morbidity. Mixed effects multilevel regression modeling was used to examine the aims. Infants whose mothers received AC had significantly lower resting state  and post-stressor  cortisol levels across the first year of life than infants whose mothers did not receive AC. There was no moderating effect of preterm birth on the relationship between AC exposure and cortisol. Results indicate a state of dampened HPA activation and cortisol hypo-arousal that persists across the first year of life among infants who were exposed to corticosteroids in utero. Further research is needed to examine mechanisms responsible for any alterations that occur during development of the fetal HPA axis, including epigenetic and biochemical factors that control hormonal secretion, negative feedback, and glucocorticoid receptor function throughout the HPA axis. Findings warrant careful consideration by obstetric clinicians of the benefits and risks of prescribing AC. Guidelines have expanded recently to include women at later gestation with select risk factors . These s1.2.Human research examining the effects of AC on the HPA axis has focused primarily on infancy, although evidence is beginning to accumulate about longer-term effects in school-age children. Evidence in the early postnatal period has been somewhat mixed, with the majority of studies demonstrating that AC exposure is associated with a suppressed HPA axis response in infants shortly after birth . HoweverStudies of older infants and school-age children demonstrate that suppression of basal cortisol levels does not appear to be present beyond the postnatal period. Instead, those exposed to AC show either elevated  or similEvidence is more limited for associations between AC exposure and stress reactivity. Infants exposed to AC exhibit alterations to stress reactivity in response to immunization, demonstrated by either suppression  or upregTaken together, most studies show an altered cortisol response in children exposed to AC, evident in the first days of life as HPA suppression and then re-emerging in school age and adolescence as increased stress reactivity. However, differential effects of AC on basal levels of cortisol versus effects on cortisol reactivity to stressors remain unclear. In addition, progression of potential cortisol dysregulation over the first year of life is understudied. Further research is needed to ascertain how long suppression may persist and explore the trajectory of HPA axis alteration from birth to one year of life. Virtually no studies have included samples of both term and preterm infants large enough to examine differential effects of AC on cortisol levels or reactivity of these two groups. Examining the potential moderating role of prematurity in any effects of AC is critical to better understand how prematurity and AC may interact in eliciting alterations in the HPA axis and to whom any effects of AC can be generalized.Our study had two aims. Our primary aim was to determine if AC exposure is associated with infant resting state cortisol, post-stressor cortisol, or cortisol reactivity to a stressor at 1 month, 6 months, or 12 months postnatal. Our hypothesis was that infants exposed to corticosteroids would have significantly dampened cortisol at rest and in response to a stressor across all time periods, when compared with infants who were not exposed to AC during gestation. Our secondary aim was to determine if preterm birth status moderates the relationships between AC exposure and infant cortisol in a resting state or in reactivity to a stressor. We hypothesized that preterm infants would experience greater dampening of their cortisol levels and cortisol reactivity than term infants from exposure to AC.2.2.1.This research was part of a larger longitudinal study that examined the effect of various risk factors during pregnancy on birth outcomes and the stress regulation of infants over the first year of life. We performed a power analysis to determine needed sample size. Based on findings from our previous research and that of others, we estimated that the difference between AC exposed and not exposed infants would be a medium effect at baseline (d =0.5) and between a medium and large effect over time (d =0.64). The interaction between AC group and preterm status was estimated as a medium effect at baseline (d =0.5), and between a weak and medium effect over time (d =0.35). Power estimations were obtained with Monte Carlo simulations and carried out with 10,000 random draws. We determined that effects specified in our aims could be detected by enrolling a sample size of 136, with power at or above .80 and two-sided alphas at .05. Our actual sample size for this analysis was n = 181 infants.Mothers who were 18 years of age or older were recruited from two Obstetrics and Gynecology clinics affiliated with a large university medical center. Inclusion criteria were that women spoke English or Spanish, were in their third trimester of pregnancy (between 24 and 34 weeks’ gestation) and were identified by their obstetric clinician as being at increased risk for preterm labor. Risk for preterm labor was based on factors such as history of preterm birth and current obstetric health status. However, women who delivered at term were retained in the study along with those who delivered prematurely. Women who had ongoing steroid use or a history of endocrine conditions, those who smoked, and women with serious medical problems  or cognitive impairment were excluded. Additionally, infants with chromosomal and genetic anomalies, chronic lung disease, congenital heart disease, or other major neonatal illness were excluded. Data of children being treated with synthetic glucocorticoids after birth (e.g. for asthma) or who developed endocrine problems  were not included in the analysis. A member of the research team contacted eligible women to provide details about the study and obtain informed consent if they were interested. Proxy consent for their infants to participate was obtained from the pregnant women. The study was approved by the University Institutional Review Board for Human Research Protection.During the week of recruitment, women completed a baseline sociodemographic questionnaire and the Perceived Stress Scale (PSS). They also received written, verbal, and behavioral guidance on how to provide saliva samples into a small vial. They provided saliva samples 4 times each day over a 2-day period during pregnancy, and again for 2 days at 6 and 12 months postpartum. After the infant’s birth, we collected data from women and their infants during home visits at 3 timepoints: 1, 6, and 12 months of infant age. At each of these visits, a ‘stressor’ protocol was implemented (described below) to measure infant cortisol levels before and after the stressor. The researcher prepared mothers for the stressor ahead of the visit and discussed exactly what the protocol would involve. Mothers identified a suitable, quiet time and area in the home where the protocol could take place without disturbance by others living at the residence.Data were extracted from the electronic medical records of mothers and infants to identify information about covariates for potential inclusion in testing of the study aims. Information was acquired regarding receipt of antenatal corticosteroids as part of obstetric care, overall maternal obstetric risk, infant gestational age, preterm birth status, extent of neonatal morbidity, and infant sex.2.2.Our standardized ‘stressor’ protocol differed in its format at 1 month versus 6 and 12 months of infant age to be developmentally appropriate. Research Assistants (RA) were trained in each protocol, including oral and written instructions, practice of the protocol until proficient in a simulated setting with feedback, and then accompanying an expert Clinical Research Coordinator on at least 2 actual visits where the protocol was administered. Each protocol had distinct baseline, stressor, and post-stressor periods and was administered between 10 am and 12 pm to control for effects of circadian rhythm on cortisol levels. Mothers were present during the protocol but were only directly involved during the 6- and 12-month stressors.2.2.1.SalivaBio infant swab was placed in the infant’s mouth between the cheek and bottom gum on the side facing downwards to collect pooled saliva. The swab was left in place for 5 min, being repositioned if needed to assure that the swab was saturated. After the swab was removed and placed in a storage tube, infants were positioned on their back. The RA then took the infant’s temperature, removed the diaper, performed peri-care, and applied a new diaper. Near the end of this caregiving period, the infant was again positioned on the back with head to one side. A second saliva sample was then acquired for DNA analyses unrelated to the aims described in this paper using the same procedure described above. The stressor protocol lasted 15 min. After collection of this sample, the infant was covered with a blanket and a ‘post-stressor’ period began during which the infant was left undisturbed for 5 min. Then the final saliva sample was acquired, using the same method as described previously. Total time from the onset of the caregiving stressor until we began collection of the final salivary sample was 20 min.This protocol involved a previously tested caregiving procedure shown to elicit a broad distribution of stress responses from infants in their first month of life . Three E2.2.2.The ‘Repeated Still Face Paradigm’  was used2.2.3.Because our research involved infants, we developed a procedure  for managing any undue distress that might occur during the stressor protocols. If an infant became excessively distressed (e.g. strong ongoing crying with mottling or red face) during the stressor, we followed a series of steps which were discussed with the mother before the protocol began. The mother would briefly place her hand on the infant’s shoulder until the distress was minimized. During the stressor at 1 month postnatal only, mothers could alternately use their voice to soothe the infant. If this did not adequately address the distress, the mother would hold the infant briefly and then the protocol would be continued. If holding was necessary beyond 60 s and did not regulate the distress, the stressor protocol was discontinued and rescheduled for another day. During the stressor at 1 month postnatal, 16.7 % of the infants required help with their distress. At 6 months, 4.5 % required intervention, and 13.4 % at 12 months. We employed Mann-Whitney U tests to determine whether there were any differences in cortisol values for infants who deviated slightly from the standard protocol and those who did not. There were no significant differences between the two groups in any cortisol values. For baseline cortisol across the 3 timepoints, significance levels ranged from p = .37 to p = .77. For post-stressor cortisol, they ranged from p = .50 to p = .63, and, for cortisol reactivity, significance levels for differences between groups ranged from p = .23 to p = .86.2.3.2.3.1.Information about women’s age, education, race and ethnicity, partnership and employment status, and financial security was acquired through a sociodemographic questionnaire. These data were used for description of the sample.2.3.2.Mothers’ receipt of betamethasone during pregnancy  was identified through review of the medical record. The infant’s gestational age was also acquired from the medical record, with preterm birth status defined as less than 37 weeks’ gestation at birth.2.3.3.Cortisol is a downstream measure of the body’s HPA axis response to stress. The first salivary sample (baseline resting state) and the final salivary sample (post-stressor) from each stressor protocol were used to assay average cortisol level of the infant prior to and after the stressor. To avoid contamination of cortisol samples, infants were not fed within 30 min prior to the RA’s arrival nor during the procedures. Biospecimens were stored at 20° Centigrade until they were sent to Salimetrics biochemical laboratory for analysis. At the lab, they were thawed to room temperature, vortexed, and centrifuged at 3000 RPM for 15 min prior to assay. Assays were performed with the Salimetric Salivary Cortisol Assay Kit, and high sensitivity salivary cortisol enzyme immunoassay (ELISA) used to analyze samples in duplicate. Sensitivity of the assay had a lower limit of .007 μg/dl, a standard curve ranging from 0.012 to 3.0 μg/dl, an average intra-assay coefficient of variation of 4.6, and an average inter-assay coefficient of variation of 6 %.Infant cortisol level at resting state was the ug/dL value identified during the baseline period prior to the stressor while post-stressor cortisol was the ug/dL from the final sample after completion of the protocol. Infant cortisol reactivity was the difference between the ug/dL value at baseline and the value identified for the final post-stressor salivary sample. This change in cortisol levels indicates the magnitude of an infant’s response or amount of reactivity to the stressor.2.3.4.Data on four covariates were acquired to examine their potential confounding effects and adjust for these in final analysis if needed. Covariates included obstetric medical risk, mothers’ perceived stress during pregnancy, and mothers’ endogenous cortisol levels during pregnancy .G) for the 2-day samples. AUCG measured the amount of total cortisol output across the day, considering the difference between individual cortisol samples and the time between each sampling period. We calculated AUCG with the trapezoidal formula from The Obstetric Medical Risk Index  was used2.3.5.Descriptive statistics were used to characterize the sample. We used mixed effects multilevel regression to determine if fetal AC exposure during gestation was associated with infant resting state cortisol level, cortisol reactivity to a stressor, or post-stressor cortisol level at 1 month, 6 months, or 12 months postnatal. We employed Full Information Maximum Likelihood (FIML) methods to achieve unbiased estimates of the effects over time for data missing at random. A bias-corrected nonparametric bootstrap was employed with 1000 replications to accommodate non-normality. We employed nonparametric bootstrapping since standard approaches such as winsorizing of outliers and log transformation of cortisol variables were insufficient to correct for lack of normality in cortisol distributions. Excluding extreme values solely due to their extremeness can distort results by removing important information about variability inherent in the sample. Regardless, their removal did not effectively address skew for some variables. Bootstrapping techniques enable retention of all values when log transformations do not effectively address a skewed distribution. Thus, bootstrapping allowed us to capture the full variability of our dataset without violating assumptions of normality and to interpret results on the original scale. A Bonferroni adjustment to the confidence intervals (98.33 %) was also applied to account for multiple comparisons. Initial Spearman correlations were examined between each infant cortisol metric and the four continuous covariates: obstetric medical risk, maternal pregnancy stress, and maternal endogenous cortisol during pregnancy . Covariates showing a significant relationship at p = .05 or less were included in the final regression models. In addition, we assessed the potential for infant sex differences in preliminary models, computing its main effect on cortisol levels and cross-level interactions between AC exposure and sex. To test Aim 2, we examined a cross-level interaction in the models between AC exposure and preterm birth status. We carried out statistical analyses with Stata 16 . We evaluated all tests of significance with a two-sided alpha of 0.05.3.3.1.Data from 181 mothers and infants were included in this analysis. Descriptive statistics for the sample are presented in 3.2.2 = 3.47 (df=2), p = .17), post-stressor cortisol (χ2 = 6.86 (df=2), p = .03), and cortisol reactivity (χ2 = 0.946 (df=2), p = .62) were not significant across the 3 timepoints. Pairwise comparisons between timepoints did indicate trends toward a significant difference between 1 and 6 months of age for both level of baseline cortisol  and post-stressor cortisol  but not for cortisol reactivity. We examined the stability of infant cortisol levels from 1 month to 12 months of infant age. First, we calculated Intraclass Correlation Coefficients (ICC) for infant baseline and post-stressor cortisol as a measure of intra-individual stability over 1, 6 and 12 months of infant age. The ICC for baseline cortisol was .33 . For post-stressor cortisol, the ICC was .26 . Cortisol reactivity had an ICC of .06 . All coefficients indicate a high level of cortisol variability for infants across time, likely due to changes in environmental exposures and effects of development. We also used Friedman’s non-parametric test for repeated measures to assess stability of the entire sample’s cortisol data over time. After Bonferroni corrections for multiple testing, differences in baseline cortisol . For the stressor protocol at 6 months postnatal, we did not find a significant increase from baseline to post-stressor for the entire sample of infants . On average, infants increased by .062 (0.07) from baseline cortisol (0.587 μg/dl) to post-stressor cortisol (0.649 μg/dl). In contrast, the 12-month stressor protocol did show a significant mean increase of .259 (0.15) μg/dl  from baseline (0.437 μg/dl) to post-stressor (0.697 μg/dl). We ran all bootstraps with 5000 repetitions.For the sample as a whole, the stressor protocol at 1 month postnatal elicited a significant mean increase of .197 (0.08) from baseline (0.578 μg/dl) to post-stressor (0.775 μg/dl) based on 3.4.G) for their cortisol during pregnancy was significantly associated with any infant cortisol variable. Infants whose mothers had a higher AUCG during pregnancy had a higher baseline resting state cortisol level as well as a higher post-stressor cortisol level at 12 months of age. Based on this relationship, we did include AUCG in our multilevel regression modeling to examine the aims. However, AUCG had no relationship to any of the infant cortisol metrics when considered with other variables. For example, the direct effect of AUCG on infant resting state cortisol was B = 0.00 . The moderating effect of AUCG on the relationship between AC exposure and infant resting state cortisol was B = 0.00 . Similarly, the direct effect of AUCG on infant post-stressor cortisol was B = 0.03 . The moderating effect of AUCG on the relationship between AC exposure and infant post-stressor cortisol was B = 0.01 . Consequently, AUCG was not included in the final models.Preliminary correlations between maternal prenatal covariates and infant cortisol variables are shown in 3.5.Results of the regressions for infant resting state (baseline) cortisol and cortisol after the stressor (post-stressor) are presented in Findings for post-stressor infant cortisol support the same significant association with AC exposure as for infant resting state . Infants whose mothers received AC during pregnancy had significantly lower post-stressor cortisol levels across the first year of life (x‾ = 0.43) than infants whose mothers did not receive AC (x‾ = 3.09). There was no effect of preterm birth status on infants’ post-stressor cortisol (neither a direct nor moderating effect).For cortisol reactivity, there was no association of either AC exposure or preterm birth to infant cortisol reactivity in response to a stressor . The reaIn examining the potential role of infant sex, we found no significant differences between male and female infants in any of the cortisol variables across the 3 timepoints, including average baseline cortisol , post-stressor cortisol , or cortisol reactivity . In addition, infant sex did not moderate the relationship between AC exposure and either baseline , post-stressor , or cortisol reactivity levels .4.4.1.Our results indicate that infants who were exposed to AC had lower resting cortisol levels and dampened cortisol levels after exposure to a stressor. These responses occurred at 1, 6, and 12 months of age, indicating a persistent hypo-arousal of their HPA axis throughout the first year of life compared with infants who were not exposed to AC. Our findings support previous research linking AC exposure to suppression of the HPA axis in the early postnatal period and provide new evidence that this effect may persist longer than previously known. Changes observed in exposed infants may result from fetal adaptations that influenced development of their HPA axis. In typical HPA axis development, the placenta protects the fetus by modulating exposure to maternal cortisol via the 11βHSD2 enzyme, which oxidizes cortisol into its inactive metabolite, cortisone. However, synthetic GC such as betamethasone and dexamethasone are not readily metabolized by 11βHSD2 . They crResearch using animal models has shown that AC administration can have significant effects on the developing HPA axis . The fetPrevious evidence demonstrates that exposure to prenatal stress may also impact fetal HPA axis development, through exposure to elevated levels of the mother’s endogenous cortisol . Our fin4.2.Despite our findings that AC exposure significantly affected resting and post-stressor cortisol levels, this effect was not true for cortisol reactivity at any assessment time point. This could suggest that perturbations to fetal HPA axis development induced by AC likely targeted basal or tonic levels of activation rather than mechanisms underlying responsiveness to a specific stressor. Different types of exposure can be linked to distinctly different alterations in cortisol regulation . Based oAlternatively, contrasting patterns of cortisol reactivity among infants may have masked any effects of AC on our sample’s overall response to the stressor. In a review and meta-analysis of infant cortisol reactivity, 4.3.To our knowledge, this was the first study to examine the moderating effects of prematurity on AC exposure in a large sample that included term infants. Preterm birth status had no direct or interaction effects with AC exposure on infant resting state, cortisol reactivity, or post-stressor cortisol levels at any assessment time point. The lack of any moderating effect is congruent with findings from previous studies of preterm infants that gestational age does not moderate AC effects on cortisol , and ext4.4.Our results support previous research that AC exposure may have adverse consequences for fetal and infant health, calling attention to the need for careful consideration when prescribing AC and for administration guidelines that maximize benefits while minimizing risks for the fetus. More robust research is needed to examine the interaction between exogenous and endogenous glucocorticoid exposure and their effects on stress regulation of children, avoiding attribution of fault to mothers by better accounting for external influences on fetal development of adrenocortical function. While our results support previous evidence of glucocorticoid programming effects on fetal HPA axis development , little Our findings provide new evidence that the impact of AC exposure on the HPA axis persists throughout the first year of life, but future research is needed to explore these associations in toddler and early childhood age groups where evidence remains sparse. While cortisol levels provide physiologic evidence of the impact of AC, studies need to relate alterations of the HPA axis to potential effects on infant emotional regulation, which has been closely linked to psychopathology in later childhood . A bette4.5.Findings should be interpreted in the context of the study’s limitations and strengths. Our sample consisted of mothers receiving prenatal care, with relatively high levels of education and income; generaliz-ability of our findings is limited to similar populations. The majority of preterm infants in our sample was late preterm, which may have limited our ability to see interaction effects for gestational age that might be more pronounced in younger and more vulnerable age groups. Finally, we performed the Still Face Paradigm on only one occasion at each of the 6- and 12-month time periods; multiple assessments would have increased the reliability of results.However, the size as well as racial and ethnic diversity of our sample are strengths; many of the previous studies examining AC and infant cortisol involve smaller, more homogenous European populations. Experiences of stress and the circumstances of preterm birth are known to vary among diverse populations in the U.S. ; therefo5.AC may significantly reduce morbidity and mortality for infants delivered preterm; but the potential risks associated with their use must be considered along with their benefits during clinical decision making. Our results indicate a state of dampened HPA activation and cortisol hypo-arousal that persists across the first year of life among infants who were exposed to corticosteroids in utero. More research is needed to understand critical developmental periods in HPA axis and stress regulation during which interventions may be most beneficial.


Globally, preterm birth affects more than one in every 10 live births. Although the short‐term cardiopulmonary complications of prematurity are well known, long‐term health effects are only now becoming apparent. Indeed, preterm birth has been associated with elevated cardiovascular morbidity and mortality in adulthood. Experimental animal models and observational human studies point toward changes in heart morphology and function from birth to adulthood in people born preterm that may contribute to known long‐term risks. Moreover, recent data support the notion of a heterogeneous cardiac phenotype of prematurity, which is likely driven by various maternal, early, and late life factors. This review aims to describe the early fetal‐to‐neonatal transition in preterm birth, the different structural and functional changes of the preterm human heart across developmental stages, as well as potential factors contributing to the cardiac phenotype of prematurity. Over 15 million neonates are born prematurely each year, which corresponds to more than 10% of live births worldwide  is a fetal condition that occurs in more than 20% of preterm births Gardosi, . It is tAn additional complication and the most common morbidity of prematurity is bronchopulmonary dysplasia (BPD), although incidences vary between regions and institutions ; writing – original draft (lead); writing – review and editing . Adam Lewandowski: Conceptualization ; supervision (lead); writing – original draft (supporting); writing – review and editing .


More than 6% of babies are born with a structural or functional defect, and many of these need special care and treatment to survive and thrive. Such defects can be inherited, arise through exposure to altered conditions or compounds in the womb, or result from a combination of genetic and environmental factors. Since the 1940s, animal experiments and epidemiological studies have identified many environmental factors that can cause particular birth defects. More recently, advances in genomics have allowed a simple genetic diagnosis in ∼ 30% of birth defects. However, the cause of the remainder is a mystery. I believe that a key limiter to successful identification of new environmental factors is that clinicians, epidemiologists and developmental biologists all approach the topic from different angles. I propose that better communication between such experts will further increase our understanding of the environmental causes of birth defects, and potentially reduce their global burden. About 6% of babies are born with a birth defect. Historically, many environmental risk factors causing these defects have been identified. However, this research has stalled in recent years. I believe that the time is now right to re‐invigorate this field through closer collaboration between clinicians, epidemiologists and developmental biologists. Many of these result in infant mortality, and many of those who survive face lifelong disability and treatment. The burden of mortality is particular high in middle‐ and low‐income countries, where perinatal medical interventions and on‐going treatments may not be routinely available. Since ancient times, people have speculated about the causes of birth defects, discussing the various merits of inherited, environmental or supernatural causes. The latter two explanations were in vogue until the late 19th century, when the rediscovery of Gregor Mendel's work shifted researcher attention to genetics. This focus has largely remained to the present day, especially over the past 30 years with the revolution in genetic and genomic technologies. As a result, simple genetic causes have been found for many types of birth defect, and sequence variants in hundreds of genes have been associated with individual cases in particular families. Such genetic diagnosis is incredibly useful for individual families to provide information on issues such as prognosis, treatment and recurrence rate. However, understanding the genetic causes of birth defects has had relatively little impact on the population prevalence of birth defects. This is because in most cases the causative sequence variants are unique to each family. Thus, beyond identifying candidate genes for sequencing in new patients, this knowledge has limited applicability to other families or the general population. Furthermore, even with the great success of genetic diagnosis, the causes of two‐thirds of birth defects remain unknown. This is because birth defects can also be caused by environmental factors that cause altered conditions in the womb during embryonic development. This can be due to alteration of maternal physiology, or exposure of the developing embryo to teratogenic compounds. In contrast to genetic studies, identification of such environmental risk factors has scope for reducing the population prevalence of these birth defects. Despite this great potential, studies of the environmental causes of birth defects have stalled in recent years. This is despite the great advancements of our understanding of human embryogenesis, and the increasing use of electronic health records in primary health care. Even though an interdisciplinary approach may be self‐evident, I believe that the time is now right to re‐invigorate this field of research through closer collaboration between clinicians, epidemiologists and developmental biologists.Birth defects are a major global problem, affecting ∼ 6% of live births, and more than 10% of miscarriages.1 ManyIn general, the association of particular environmental factors with specific birth defects can be identified in two ways, either via experimental teratology in animal models, or by epidemiology of human populations.ICH S5 (R3) guideline on reproductive toxicology: Detection of toxicity to reproduction for human pharmaceuticals. (2020). Retrieved from https://www.ema.europa.eu/en/ich‐s5‐r3‐guideline‐reproductive‐toxicology‐detection‐toxicity‐reproduction‐human‐pharmaceuticals#current‐version‐section). This is because reliable in vitro models for embryogenesis have not been developed. However, these studies have become increasingly refined. In the last 30 years, developmental biology has made great strides in understanding how and when many tissues and organs are formed during embryogenesis. In addition, many of the underlying molecular mechanisms have been identified. This knowledge is now being used to plan new studies, as well as re‐examine the findings of old teratological studies. For example, in the 1950s exposure of pregnant mice to lowered oxygen levels was shown to cause malformation of the embryo's heart and vertebrae. 60 years later, the application of modern molecular techniques including candidate gene analysis and untargeted transcriptomics showed that the lack of oxygen perturbs the Fibroblast growth factor (FGF) signaling pathway in the developing embryo to cause the defects.Mammalian embryonic development is a complex process that cannot be easily modelled in vitro. For this reason, the best way to test whether a particular environmental factor can cause birth defects is by animal experimentation. Early studies took a sledgehammer approach, exposing pregnant animals to different compounds or environmental conditions, then examining the effects on the resultant offspring. Animal studies are still used today when new therapeutic compounds are in the process of being approved for use in humans . This was confirmed in the 1980s by several large‐scale epidemiological studies, and finally in 1990 a large scale clinical trial proved conclusively that maternal folate supplementation significantly reduced the risk of having a child with a neural tube defect.Epidemiology takes large populations of people, and compares the exposures to potential risk factors of cases with a birth defect and matched controls who lack the defect. Environmental factors for analysis by epidemiology are generally selected in one of two ways. Firstly, anecdotal observation of increased rates of birth defects in a small population can be used to generate a testable hypothesis. One of the first examples of using this approach to identify an environmental factor causing birth defects is maternal infection with the  in 1941.6 Howe Therefore, public health education can only be effective if it leads to all women of child‐bearing age actively avoiding environmental risk factors. Thirdly, therapeutic drugs with well‐known teratogenic side effects ideally should not be used to treat pregnant women. However, in some cases the health benefits to the mother may be judged to outweigh the risks of offspring birth defects. A prominent example of this is valproic acid (VPA), which is used as an anti‐seizure medication for epilepsy and increasingly for bi‐polar syndrome. Withdrawal of VPA during pregnancy can have dramatic and life‐threatening effects on the mother. Lastly, avoidance of some risk factors requires active public health intervention. For example, it is well‐documented that maternal infection with the Rubella virus in early pregnancy carries a high risk of offspring birth defects. Population vaccination is an effective way of limiting the chances of Rubella exposure during pregnancy. However, for Rubella ∼ 90% of the entire population needs to be vaccinated to provide sufficient herd immunity to prevent disease outbreaks. Sadly, growth of the anti‐vaccination movement in some developed nations in recent years has led to a resurgence of Rubella. Some environmental risk factors, such as maternal disease or altered physiology, cannot be avoided. Instead, the relative risk can be reduced by maternal behavior change. A good example is maternal pre‐existing type 1 or type 2 diabetes mellitus (PGDM). It is well‐established that PGDM substantially increases the risk of having a child with one or more specific types of birth defect. The teratogen is likely to be hyperglycemia itself, with offspring defect rate increasing with increasing maternal glycated hemoglobin (Hb1ac) levels, a routine measure of glycemic control. Fittingly, pre‐pregnancy care by specialist health professionals to improve maternal glycemic control has been shown to significantly reduce the risk of offspring birth defects. Finally, the effects of some environmental risk factors can be overcome by dietary alteration or medical intervention. The best known example is folic acid supplementation. It is now well known amongst the general public that folic acid deficiency increases the risk of having a child with a neural tube defect. This can be readily corrected by peri‐conceptional maternal folic acid supplementation, as shown in a landmark clinical trial published in 1990. Subsequently, changes in public health policy to recommend folate supplements for pregnant women, and/or mandated addition of folate to basic foodstuffs, has reduced the population prevalence of neural tube defects (NTDs) by up to 70%.Once an environmental risk factor has been identified, this knowledge can be translated by either avoidance or by mitigation. The simplest approach to minimize the risk of having a child with a birth defect is to simply avoid exposure to environmental teratogens. However, in practice this may be more complicated than it first appears. Firstly, to avoid a risk factor, it must be identified in advance of the pregnancy. Although dozens of environmental risk factors are known or suspected, it is likely that yet others remain to be discovered, and these cannot be avoided. Secondly, half of all pregnancies in developed countries are unintended.9 TherIn conclusion, understanding of how each particular environmental risk factor causes birth defects can be used to design strategies to reduce the global incidence of birth defects. However, to achieve this aim most efficiently, cross‐disciplinary collaboration is needed. Unfortunately, communication between clinicians, epidemiologists and developmental biologists is not as good as it could be. Below, I summarize some of the most common misunderstandings between the different fields. each potentially with a different embryological origin. However, many researchers investigating animal models of congenital heart disease (CHD) have an inadequate knowledge of normal embryonic heart morphology and do not accurately or comprehensively describe abnormal anatomy. For example, when using mouse models of birth defects, embryos are often described as having a “ventricular septal defect” (VSD). However, subtypes of VSDs include peri‐membranous, muscular, inlet, outlet, or as part of a common atrioventricular junction. If the precise subtype of VSD is identified, this may give important clues to the embryological origins of these defects.It is essential to accurately describe the precise anatomy of an individual with a structural birth defect. This may seem pedantic, but simplification of description can lead to erroneous assumptions about the embryological origins of the defect. For example, congenital heart disease is not a single entity. The nomenclature suggested by the International Pediatric and Congenital Cardiac Code contains no less than 318 distinct entries,13 eacSolution: To maximize the translational potential of basic research, developmental biologists should ask a clinical anatomist to assist them to accurately determine and describe the embryonic phenotypes in their animal models.Likewise, some epidemiological studies test for an association between a particular environmental factor and the broad term “CHD”. However, both embryological and molecular origins of different types of heart defects can vary considerably. In addition, if smaller sub‐groups of CHD are used, these may not be grouped on the basis of embryological or molecular origin, but on final phenotype. Any particular environmental teratogen may well only perturb one particular process of embryonic heart development, and thus might only increase the risk of a few subtypes of CHD.Solution: Epidemiologists should consult developmental biologists and clinical anatomists to identify sub‐groups of defects likely to have a similar embryological or molecular origin. Although, separating case groups into smaller sub‐types of defects will reduce case numbers, paradoxically this may well increase statistical power to detect risk factors.Most organs and structures in the human embryo form very early in gestation, over a short defined period. For example, in human embryos, the heart forms between weeks 3–8 of embryogenesis . By the end of this period, the architecture of the embryonic heart is almost identical to that of an adult. Thus, there is only a short window in which the developing heart is susceptible to external factors to cause structural heart defects.effects of altered development, rather than the underlying causes. Furthermore, a lack of understanding of embryonic development and metabolism can lead to poor hypothesis building. One particularly prominent example is the case of diabetic embryopathy. As mentioned above, pre‐existing maternal diabetes carries a significant risk of having a child with one or more severe birth defects. This phenomenon has been studied for many years in animal models. One particularly popular hypothesis is that increased glucose uptake causes “increased reactive oxygen species (ROS)” in the developing embryo, and that this leads to specific effects on organ formation. It is certainly true in adult tissues that hyperglycemia causes elevated ROS. Indeed, this has been proposed as being the underlying common pathogenic mechanism for all adult diabetic complications. This is because excess glucose levels lead to an overproduction of superoxide in the mitochondrial electron transport chain during oxidative phosphorylation. However, in most tissues of the mouse embryo oxidative phosphorylation does not begin until after the chorioallantoic branching (CB) stage (∼E9.5), when maternal‐embryo gas and nutrient exchange begins. Thus, elevated glucose levels during early embryogenesis are unlikely to cause elevated ROS at the same time as the key events of organ formation that are disrupted by hyperglycemia.One of the most important issues in designing animal studies is when during embryogenesis the analysis is performed. This requires a detailed knowledge of basic developmental biology. Exposure to an environmental teratogen will have more than one effect on a developing embryo. The initial exposure is likely to have an immediate effect. Here, changes in the expression of specific genes, proteins, or signaling pathways can lead to altered cell metabolism and physiology, proliferation rate, migration or polarity. However, these initial effects generally do not cause immediately visible changes in the embryo. In many cases, any morphological changes will only become apparent at a later time, culminating in a structural or functional defect. Thus, the aim of any molecular investigation should be to analyses the embryos at, or immediately after, the teratogenic insult. However, many studies examine changes in gene, protein or signaling pathway expression when an altered phenotype has become apparent in the developing embryo. This likely to be long after the original molecular events causing altered embryogenesis have occurred. Therefore, these analyses will only identify the Solution: Clinicians planning using animal models to investigate the causes of birth defects should consult developmental biologists to identify the best strategy for determining the molecular origins of these defects.A poor understanding of human development also devalues some epidemiological studies. In general, epidemiological studies of birth defect risk factors include exposures during any stage of pregnancy. However, as discussed above, developing embryos are only likely to be vulnerable to environmental teratogens in the first trimester. Once the critical developmental period is past, exposure to a teratogen will have no effect. Thus, studies which include exposures at any time during pregnancy may dilute any signal, reducing the statistical power of the study. Ideally, further restriction of the exposure window for each particular type of birth defect would maximize the chances of detecting a teratogenic effect, although this may be technically challenging.Solution: Prior to designing epidemiological studies, epidemiologists should consult developmental biologists to identify the critical period during pregnancy when the embryo is likely to be most vulnerable to exposure to an environmental teratogen. and in many cases these events have occurred before the mother is aware she is pregnant. However, diagnosis of a birth defect does not usually occur until prenatal scans at 20 weeks or perhaps not even until birth. This means that an accurate determination of maternal physiology or exposures to potential teratogens several months before may be difficult.Epidemiological research is conducted in real‐life situations, not under controlled conditions. Therefore, study design is complicated, and is critical to the success of the study. There many different ways of performing epidemiological studies, and each has distinct advantages and disadvantages. For example, the best quality data comes from studies where the data is collected prospectively, specifically for that study. Here, participants are recruited into the study, and thus data can be collected in a uniform and standardized way. However, this is costly and studies may take years. It also requires participants time and engagement over a long period of time, and thus runs the risk of individuals dropping out of the study. Retrospective data collection is easier and quicker, and can take advantage of existing data sources such as electronic health care records, employment records, and social care data; as well as participant surveys and interviews. However, some of this latter data can be subject to recall bias. This is because the key events of embryogenesis occur in the first 8 weeks of pregnancy. ∼ 50% of pregnancies are unintended,9 and Solution: Clinicians or developmental biologists should not attempt an epidemiological study without an expert collaborator. To maximize the chances of generating reliable and useful results, they need to be very carefully designed to fit the research question under investigation. This requires taking into account a wide variety of factors, including whether a cohort or case‐control study would be more appropriate, data source quality, minimizing selection and information bias, and choosing an appropriate statistical analysis.Exposure to teratogens conditions in utero remains an important cause of human birth defects globally. Understanding how these environmental factors cause birth defects is important. This is because there is great potential to significantly reduce the prevalence of birth defects globally, through changes to influence public health policy, as demonstrated by the remarkable success of folic acid supplementation in reducing NTDs. To do this more efficiently, I believe that closer communication between clinician, epidemiologists and developmental biologists is now required to efficiently harness the advances in molecular and “big data” techniques to increase our understanding of the environmental causes of birth defects, and potentially reduce their global burden.No conflicts of interest, financial or otherwise, are declared by the author.


Ambient air pollution has been recognized as one of the most important environmental health threats. Exposure in early life may affect pregnancy outcomes and the health of the offspring. The main objective of our study was to assess the association between prenatal exposure to traffic related air pollutants during pregnancy on birth weight and length. Second, to evaluate the association between prenatal exposure to traffic related air pollutants and the risk of low birth weight (LBW).2.5, soot, NOx, and NO2 was assessed by land use regression (LUR) models at individual level. Multiple linear regression models were built to evaluate the association between air pollutants with birth weight (BW) and birth length (BL). Logistic regression was used to assess the risk of low birth weight (LBW) associated with all air pollutants.Three hundred forty mother-infant pairs were included in this prospective cohort study performed in Jakarta, March 2016–September 2020. Exposure to outdoor PM2.5 concentration was almost eight times higher than the current WHO guideline and the NO2 level was three times higher. Soot and NOx were significantly associated with reduced birth length. Birth length was reduced by − 3.83 mm  for every IQR (0.74 × 10− 5 per m) increase of soot, and reduced by − 2.82 mm  for every IQR (4.68 μg/m3) increase of NOx. Outdoor air pollutants were not significantly associated with reduced birth weight nor the risk of LBW.The average PMx during pregnancy was associated with reduced birth length. Associations between exposure to all air pollutants with birth weight and the risk of LBW were less convincing.Exposure to soot and NOThe online version contains supplementary material available at 10.1186/s12940-023-00973-0. An e2.5 concentrations that are low to moderate on a global scale [2.5 concentration were highest in the WHO South-East Asia Region, followed by the WHO Eastern Mediterranean Region [2 concentration is quite different from those of PM2.5 with the highest in Eastern Asia, the Middle East, North America and much of Europe [2.5 concentration of 18.47 (95% CI: 15.58–22.90) μg/m3 for urban and rural areas combined and 19.11 μg/m3 for urban areas only in 2018 [In low- and middle-income countries (LMIC), attention has focused mostly on indoor air pollution from unvented cooking and heating stoves and to a more limited extent, environmental tobacco smoke . Most ofal scale . In 2019 in 2018 . This reThe neonatal mortality rate in Indonesia has not substantially improved in the past two decades . Other tThis prospective birth cohort study was performed in Jakarta from March 2016 until September 2020. The study recruited 413 mothers in early pregnancy from nine primary health care centers  in Jakarta, Indonesia. There were 69, 132, 122, and 90 pregnant women recruited in year 2016, 2017, 2018, and 2019, respectively. This study was ethically approved by the Institutional Review Board of the Faculty of Medicine University of Indonesia/Cipto Mangunkusumo General Hospital, Jakarta, Indonesia (reference number: 895/UN2.F1/ETIK/2015). Written informed consent was obtained from all the participants prior to study enrolment.Pregnant women were recruited in their first trimester or early in the second trimester of pregnancy  during their antenatal care visit. They were included in the study if they were residing in the catchment area of the primary care centers and could be contacted by phone. Recruitment was performed by midwives in each health care center. After enrolment, pregnant mothers were followed-up in line with routine antenatal care (ANC) guidelines and underwent several examinations, including anthropometrics, blood pressure, and ultrasound. We excluded (1) subjects who refused to continue their participation in this study and (2) subjects who moved out of town during follow up period. Participating women were followed up until delivery and their infants were followed up until the age of 6 months.Maternal characteristics including maternal age, parity, history of abortion, working status, family income, level of education, any diseases prior to pregnancy, alcohol or illicit drug use prior to pregnancy, smoking history prior to pregnancy were obtained using structured questionnaires at enrolment. Paternal characteristics, including age, working status, level of education, and smoking habits prior to their spouse’s pregnancy were also obtained at recruitment.A structured questionnaire was performed in every trimester to obtain any pregnancy complications and medications, current smoking habit, and exposure to indoor pollutant such as cooking fuel, insecticides, garbage burning, as well as dietary intake. Pregnant women who did not smoke but were exposed to household members who smoked on a daily basis at home in the presence of the pregnant women were considered as exposed to passive smoking, while pregnant women who were smoking throughout pregnancy were considered as active smoker. Data regarding the average number of cigarettes smoked per day throughout the duration of pregnancy for each smoker were recorded. Most of the pregnant woman had their delivery in primary care. Referral to the secondary health care center for complicated pregnancy or delivery was performed in accordance with the national health care policy.Body weight, fundal height and blood pressure of the pregnant mothers were measured at every ANC visit. Weight gain during pregnancy (expressed as Δ BMI) was calculated using the formula of BMI at delivery-BMI prior to pregnancy. At the time of delivery, maternal blood pressure and body temperature were measured; gestational age and complications during pregnancy and delivery  were also recorded.Infant weight, length, and Apgar score were measured at birth. Birth weight and length were measured using a standardized protocol twice and then averaged. The averaged value was used for the final analysis. Infants who developed complications were examined by the attending physician and received standard care including referral to secondary health care center. Neonatal morbidities, including respiratory distress, asphyxia, bacteremia/sepsis, hyperbilirubinemia, and congenital anomalies and all causes of neonatal mortality were recorded. Neonatal mortality was defined as any mortality occurring within the first 28 days after birth. Other adverse birth outcomes were defined as preterm birth if gestational age < 37 weeks and low birth weight if birth weight < 2500 g .2.5), soot (a measure of black carbon), nitrogen dioxide (NO2) and the sum of nitrogen dioxide (NO2) and nitrogen oxide (NO), denoted as NOx. Measurements were made at 88 sites across the study area. We selected 37 urban background sites, 7 urban green sites and 44 traffic sites, based upon the assumption that motorized traffic is an important source of spatial variability in the study area. Annual average concentrations were calculated after correction for temporal variation with continuous measurements from a reference site [Exposure assessment for the cohort was based upon land use regression (LUR) models  developence site .Fig. 1Stx, NO2, PM2.5 and soot respectively. The models thus explain a moderate amount of the measured variability. Exposure to air pollution was assessed at individual level, calculated for each member of the cohort by applying the LUR model to the residential address [LUR models were developed using predictor variables obtained from direct systematic field observations of traffic counts and street configuration and global GIS databases of road data from Open Street map and impervious surface  i.e. household income and level of education, maternal age, parity , 18, 19,2.5, soot, NOx, and NO2 with birth weight and birth length. Logistic regression was used to explore the association between outdoor air pollutants concentration with low birth weight, expressed as OR with 95% confidence interval. We performed sensitivity analysis to test the robustness of our results when restricting the cohort to women with infant born at gestational age ≥ 37 weeks. We additionally calculated the difference between PM2.5 and soot to further differentiate effects of PM2.5 and soot. This difference was highly correlated (R = 0.99) with PM2.5. We also isolated the effect of soot from PM2.5 by generating new variable as the residual of soot [3 for PM2.5, 0.75 × 10− 5 per m for soot, 4.68 μg/m3 for NOx, and 3.74 μg/m3 for NO2. Statistical significance was assumed if 95% confidence intervals did not include the estimation of null values, corresponding to two-sided p values < 0.05. Statistical analyses were conducted using IBM SPSS version 24 for Mac, while the LUR models were developed using R 4.1.2.Multiple linear regression models adjusted for gestational age and subsequently all potential covariates were specified to evaluate the association between outdoor concentration of PM of soot  and furtInitially, 413 pregnant women were enrolled in this study, yet 73 mother-infant pairs were not included in the analysis i.e. 52 dropped for various reasons (refused to continue or moved out of town), 13 experienced miscarriages at an early stage of the pregnancy, one subject had ectopic pregnancy, and 7 had incomplete birth anthropometric measurement. Further, one baby with extremely low birth weight  was excluded. In the end, 340 mother-infant pairs were included in this study.The pregnant women in our study were 27.9 (SD 5.37) years of age on average, most were high school graduates and on their first pregnancy Table . Around 2.5 concentration was almost eight times (36.54 μg/m3) as high as the recommended WHO guideline level  [2 were at least three times higher (32.62 μg/m3) than the recommended WHO guideline level of 10 μg/m3 annually [2.5 which was moderate (0.51), and the correlation between NOx with NO2 which was high (0.87)  . The meax were significantly associated with reduced length at birth in fully adjusted regression model. The birth length was significantly reduced by − 3.83 mm  for every 0.74 × 10− 5 per m (IQR) increase of soot, and significantly reduced by − 2.82 mm  for every 4.68 μg/m3 (IQR) increase of NOx concentration. We also observed an inverse association between PM2.5 and NO2 with birth length, but those association were not significant statistically. The beta coefficient of the final model (model 2) of the association between the residual of soot [2.5.Table  of soot  with bir2.5, soot, NOx, NO2) were inversely but not statistically significantly associated with birth weight in fully adjusted regression models. Birth weight was reduced by 54.4 g  for every 7.14 μg/m3 increase in PM2.5, 48.76 g  for every 0.75 × 10− 5 per m soot, and 17.20 g  for every 3.74 μg/m3 increase in NO2 concentration. There was a positive association between all outdoor air pollutants concentration  with the risk of LBW as shown in Table All outdoor air pollutants , the association between outdoor air pollutants with birth anthropometric was robust Table . The birx was significantly and consistently associated with reduced length at birth. All air pollutants  were also associated with reduced birth weight though not statistically significant. Confidence intervals for associations with the risk of low birth weight were wide. Few studies have explored the effect of soot on birth anthropometric and to our knowledge, ours is the first study in low-middle income countries to include the role soot as one of the important determinants of air pollution.Our study provides evidence regarding the effect of exposure to traffic related air pollution during pregnancy on birth anthropometry in low-middle income countries. Exposure to soot and NO2 > 40 μg/m3 during the first trimester was associated with a 2,7 mm  reduction in birth length. A study in Brisbane Australia, reported that IQR increase in NO2 (11.1 μg/m3) during the third trimester was associated with a reduction in crown-heel length of − 1.5 mm  [2 and − 2.3 mm  associated with PM2.5, while Jedrychowski et al. [2.5 above 36.3 μg/m3. Our result is consistent with these studies, to an extent that the effect on the birth length reduction is more pronounced in our study. Every IQR increase in NOx (4.68 μg/m3) concentration result in birth length reduction of − 2.82 mm. Even though we did not observe the same result for NO2, but our findings expand observations on NO2 to NO and other nitrous compound that build NOx on the birth anthropometric. NOx is more related to primary combustion emissions, where NO2 also has a substantial secondary component (formation in the atmosphere once emitted).Birth length has been associated with intrauterine well-being of the fetus. Newborns are considered as small for their gestational age (SGA) if their birth length is less than 10th percentile according to their gestational age. Babies born SGA have negative long term outcomes including the risk of early cardiovascular diseases, diabetes, obesity , early p.5;-0.5) . However.5;-0.5)  in the N.5;-0.5)  found bii et al.  also fou− 5 per m increase of soot result in − 3.83 mm reduction in birth length and this association is independent of PM2.5 concentration. To our knowledge none of the previous studies focus on the effect of soot on the birth outcomes and birth anthropometric. Soot is elemental carbon emitted by fuel combustion of gas and diesel engines/ vehicles such as motorcycles, cars, and trucks. It has been postulated that (nano)particles, including soot can pass the placental barrier. However, most of the studies originated from in vitro cell cultures or animal studies. The recent evidence on soot translocation to human placenta following inhalation under real-life comes from the ENVIRONAGE birth cohort study [3) and high (1.70–2.42 μg/m3) levels of residential soot during pregnancy [We also found a consistently inverse association of prenatal exposure to soot on birth length, every 0.74 × 10rt study . The ENVregnancy . Soot paregnancy .2.5 and NO2 during pregnancy resulted in 14.7 g and 8.9 g reduction in birth weight. Malmqvist et al. [3 increase of NOx, while Steib et al. [3) NO2 in birth weight of term infant.Although not statistically significant, the reduction in birth weight in our study were greater than those previously reported by other studies in high income countries or other Asia-Pacific countries. Similar results were observed by Augilera et al.  in the It et al.  reportedn = 21). Liu et al. [2. The ESCAPE study reported a OR of LBW for every 5 μg/m3 increase in PM2.5 of 1.18  and the OR was 1.09  for every 10 μg/m3 increase in NO2 [We did not find significant association between all outdoor traffic related pollutants with the risk of LBW although the ORs were slightly higher than previously reported. Likely, the wide confidence interval is due to small number of infants with LBW  [2.5 in this study was 8 times as high as the recommended level by WHO and the mean NO2 level was 3 times higher than the WHO recommendation [2.5 concentration that we measured between 2016 and 2018 (36.5 μg/m3) was very similar to the average concentration measured at a central site by the US embassy (35.2 μg/m3). Correlations between the four pollutant exposures were lower than observed in previous European studies [The concentration of all outdoor and indoor air pollutants in this study was significantly higher than those measured in the birth cohort studies in high income countries i.e. the ESCAPE study (PM2 μg/m3) . The mea3 increase in PM2.5 was associated with an odds ratio of 1.47 of increased c-reactive protein (an inflammatory marker) during early pregnancy. Veras et al. [ONAGE birth cohort study showed that soot particles were detected on the maternal and fetal sides of the placenta, suggesting that soot may be transported to the developing fetus [2.5 and PM10 during pregnancy.The biological mechanisms underlying the association between air pollutant and birth anthropometrics are not entirely understood. The pregnant women and her unborn child are considered to be more vulnerable towards exposure to air pollution due to pregnancy-related physiological adaptions and the intense prenatal growth and development . Air pols et al.  observedng fetus . Anotherng fetus  determin2 during pregnancy induces lipid peroxidation in the placenta and disturbs postnatal development. NO2 may also have direct toxic effects on the fetus [2 in Bulgaria [Studies in animal models showed that exposure to NOhe fetus . A studyBulgaria  suggesteBulgaria .2.5, soot, NOx, NO2) [We consider our prospective cohort study design with detailed air pollution measurements to be a major strength of this study. To date most of the epidemiological studies on the effect of air pollution to birth anthropometrics come from secondary data and retrospective trace of the exposure in the same area. To best of our knowledge this was the first study assessing the effect of early life exposure to air pollutants on birth anthropometrics in Indonesia and other LMIC in South-East Asia using LUR modelling to assess the individual exposure to all important air pollutants  , 43, 44.Ox, NO2) , 30. TheOx, NO2) , 30.Our study limitations relate to the relatively small sample size, potentially lacking the robustness to explore possibly subtle effects of early life exposure of air pollutants on birth anthropometrics. Technical errors in anthropometric measurements were minimized by performing the measurements twice, but might not fully be excluded, if any technical errors occurred we assume this was independent of the exposure. We did not measure the postnatal air pollution exposure, but Esplugues et al.  indicatex consistently results in birth length reduction. Our study may have implications not only for the health and development of children but also for adult health. Epidemiologic studies showed that prenatal hazards that restrict fetal growth may be associated with delays in motor and social development through childhood [In summary, the result of our study provides convincing epidemiological evidence that antenatal exposure to traffic related air pollution in pregnant woman result in adverse birth outcomes. Our study showed that soot and NOhildhood , reducedhildhood , early phildhood  and alsohildhood . This fiAdditional file 1. Methods of outdoor air pollutant measurement.Additional file 2: Supplemental Table 1. LUR models for air pollutant concentration assessment.Additional file 3: Supplemental Table 2. Correlation between air pollutants concentration at the measurement sites.Additional file 4: Supplemental Table 3. Correlation between air pollutants concentration in the cohort.Additional file 5: Supplemental Table 4. The association between exposure to indoor air pollutants during pregnancy with birth anthropometrics in a-indoor subset group.


When is the sensitive window of ambient air pollution exposure in association with birth weight?In this cohort study of 628 predominately low-income Hispanic women who were pregnant, exposures to particulate matter and nitrogen dioxide in early pregnancy to midpregnancy were significantly associated with lower birth weight, particularly for mothers experiencing higher perceived stress during the prenatal period and living in a neighborhood with a high level of stressors from environmental pollution.The findings of this study suggest that protecting women who are pregnant from air pollution may improve birth weight, particularly among mothers with high levels of psychological stress or environmental pollution. Fetal growth is precisely programmed and could be interrupted by environmental exposures during specific times during pregnancy. Insights on potential sensitive windows of air pollution exposure in association with birth weight are needed.To examine the association of sensitive windows of ambient air pollution exposure with birth weight and heterogeneity by individual- and neighborhood-level stressors.Data on a cohort of low-income Hispanic women with singleton term pregnancy were collected from 2015 to 2021 in the ongoing Maternal and Developmental Risks from Environmental and Social Stressors cohort in Los Angeles, California.10) and aerodynamic diameter less than 2.5 μm (PM2.5), nitrogen dioxide (NO2), and 8-hour maximum ozone were assigned to residential locations. Weekly averages from 12 weeks before conception to 36 gestational weeks were calculated. Individual-level psychological stressor was measured by the Perceived Stress Scale. Neighborhood-level stressor was measured by the CalEnviroScreen 4.0.Daily ambient particulate matter with aerodynamic diameter less than 10 μm (PMz score (BWZ). The associations between air pollutant and BWZ were estimated using distributed lag models to identify sensitive windows of exposure, adjusting for maternal and meteorologic factors. We stratified the analyses by Perceived Stress Scale and CalEnviroScreen 4.0. We converted the effect size estimation in BWZ to grams to facilitate interpretation.Sex-specific birth weight for gestational age 2.5 exposure during 4 to 22 gestational weeks was associated with a −9.5 g  change in birth weight. In stratified models, PM2.5 from 4 to 24 gestational weeks was associated with a −34.0 g  change in birth weight and PM10 from 9 to 14 gestational weeks was associated with a −39.4 g  change in birth weight in the subgroup with high Perceived Stress Scale and high CalEnviroScreen 4.0 scores. In this same group, NO2 from 9 to 14 gestational weeks was associated with a −40.4 g  change in birth weight and, from 33 to 36 gestational weeks, a −117.6 g  change in birth weight. Generally, there were no significant preconception windows for any air pollutants or ozone exposure with birth weight.The study included 628 pregnant women  and their newborns . On average, an interquartile range (IQR) increase in PM2.5, PM10, and NO2 were associated with lower birth weight, particularly for mothers experiencing higher perceived stress and living in a neighborhood with a high level of stressors from environmental pollution.In this cohort study, early pregnancy to midpregnancy exposures to PM This cohort study examines the association between ambient air pollution and infant birth weight by maternal individual- and neighborhood-level stressors. Such sensitive windows, if well identified, could be a compelling period of targeted intervention and provide a better understanding of environmental influences on fetal development.17 However, previous findings regarding sensitive windows of air pollution exposure with birth weight have been inconsistent, with each of the 3 trimesters having been identified.21 Although the whole prenatal period could be a sensitive window to air pollution exposure, erroneous estimation and inconsistencies could also be due to a temporal trend in exposure levels in adjacent time windows.22 Statistical approaches, such as distributed lag models (DLMs), can better handle temporal trends, thus identifying true sensitive windows.24 Applying DLMs is therefore needed to better assess the critical periods of air pollution exposure during pregnancy in association with birth weight.Newborns with low birth weight face increased risks of neonatal mortality and long-term disease risk.26 Stressors at the individual level, such as psychological perceived stress in women who are pregnant, has been assessed in few longitudinal cohort studies.28 Generally, high levels of prenatal psychosocial stress exposure have been shown to increase children’s susceptibility to asthma and neurocognitive underdevelopment in association with air pollution exposures, possibly through altered maternal allostatic load or placental function.29 Moreover, neighborhood-level stressors could arise from the total environmental pollution  , as well as the population’s vulnerability to poor health.30 However, whether individual-level psychological stress and neighborhood-level cumulative burden could be associated with the susceptibility to air pollution exposure with birth weight remains unknown.Robust literature reports an enhanced susceptibility to the adverse effects of prenatal air pollution exposure on birth outcomes in populations with socioeconomic disadvantage and associated stressors.31 we used DLMs to explore sensitive windows of ambient air pollution exposure in association with birth weight. Furthermore, we evaluated whether the association between air pollution and birth weight differed by individual-level psychological stress, neighborhood-level cumulative burden, or both.In a cohort of predominately low-income Hispanic women who were pregnant,31 Briefly, MADRES participants were recruited from clinical sites that serve low-income populations in Los Angeles, California. Women were eligible if they were aged 18 years or older, within 30 weeks of gestation, singleton pregnancy, and spoke English or Spanish fluently. Exclusion criteria included HIV infection; physical, mental, or cognitive disabilities that would prevent participation; or current incarceration. Maternal written consent was obtained at the time of recruitment. Participants were compensated for their time. The institutional review board at the University of Southern California approved all aspects of this study. This analysis is reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The Maternal and Developmental Risks from Environmental and Social Stressors (MADRES) study is an ongoing prospective pregnancy cohort study started in 2015.32 we found our final sample size was sufficient to detect a greater than 4-g difference in birth weight (Cohen 2f>0.01) with a power of 80% and the type I error of 0.05.As of September 1, 2021, 710 participants had reached delivery and had high-quality birth outcome information abstracted from electronic medical records. We excluded 12 participants who reported smoking during pregnancy and 70 participants who delivered prematurely . A flowchart describing the final sample size of 628 women is provided in eFigure 1 in the 2), particulate matter with aerodynamic diameter less than 2.5 μm (PM2.5) and less than 10 μm (PM10), and 8-hour maximal ground-level ozone (O3) were assigned to each participant’s residential location from 12 weeks before conception to 36 weeks 7 days during pregnancy, using inverse-distance-squared weighted spatial interpolation from ambient air quality monitoring data , with an average of 4 monitoring stations within 8 to 14 km to each residential address .33Birth weight and newborn sex were abstracted from electronic medical records. Gestational age at birth  was estimated based on ultrasonography measurement (87.6%), medical consensus (11.5%), or last menstrual period (0.9%). We calculated sex-specific birth weight for gestational age 34 to represent individual-level psychological stressor, and the cumulative impact score from the California statewide environmental justice screening tool, CalEnviroScreen 4.0 (CES), to represent the neighborhood-level cumulative burden from both total environmental pollution and population vulnerability.30 The PSS is a widely used, validated 10-item psychological instrument to assess stressors that may possibly influence mental health of the respondent (how the stressor is perceived),34 which may be more relevant to a list of stressors that will affect one’s mental health. All available measurements of PSS scores  in the first , second (12.2 [6.0]), or third (11.2 [5.8]) trimesters were averaged as the final PSS score across pregnancy. We chose the upper quartile (75th percentile) to dichotomize a high-PSS group (≥16 PSS score) and a low-PSS group (<16). The CalEnviroScreen 4.0 was developed by the California Office of Environmental Health Hazard Assessment to identify census tracts in California that are disproportionately burdened by both multiple sources of pollution  and high population vulnerability .30 The CES cumulative impact score ranges from 0 to 100, with a higher score indicating a higher cumulative burden, and the score was assigned to each participant based on residential census tract.30 We chose the California state median to dichotomize a high CES group (≥50) and a low CES group (<50).We a priori selected the following modifiers: perceived stress during pregnancy measured by the Perceived Stress Scale (PSS)35 Participants self-reported prepregnancy weight, race, Hispanic ethnicity, birth country, and marital status. The enrollment time point was determined based on the gestational week of consent (<20 vs 20-30 weeks). Maternal standing height was measured twice by a stadiometer. Maternal prepregnancy body mass index was calculated using self-reported prepregnancy weight and measured height at a prenatal study visit. We created a combined variable indicating ethnicity by birthplace, which had 3 categories: non-Hispanic, Hispanic born in the US, and Hispanic born outside the US. Marital status was categorized to indicate cohabitation status, including cohabitate, not cohabitate, and decline to respond. Birth order was combined as first, second, and third or more. To maximize statistical power, we coded missing observations of categorical covariates as a separate category. Weekly outdoor temperature was calculated using daily average temperatures at the residential location extracted from a high-resolution (4 × 4 km) gridded surface meteorologic data set.36Potential confounders were selected a priori based on literature review and analyses  and tested for differences by covariates using analysis of variance. We used DLMs to examine the association of weekly exposure to each of the air pollutants  with BWZ.23 The DLMs simultaneously included exposure to each air pollutant’s weekly levels from 12 weeks before conception to 36 gestational weeks with a cross-basis, which combined a linear dose-response function and a nonlinear lag-response function with a 3-knot natural cubic spline to constrain correlated weekly exposures.23 The number and location of knots for each air pollutant were chosen based on the Akaike information criterion and visual inspections.23 Distributed lag models were adjusted for the aforementioned potential confounders and weekly temperature. Effect size estimates are presented as changes in BWZ per each IQR increase in air pollutant . To facilitate interpretation, we converted the difference in BWZ to the difference in birth weight (grams), with 1 z score = 450 g . We used the bayesian distributed lag interaction models and identified that a heterogeneity of both a sensitive window and within-window estimate fitted our data best.37 Therefore, we stratified the analyses by PSS (high vs low), CES (high vs low), and their joint distribution. We conducted sensitivity analyses, including multipollutant DLMs with all air pollutants included as separate cross-bases, complete-case analysis by removing participants with missing values of covariates, DLMs with different number and location of knots in the cross-basis, and DLMs adjusting for PSS, CES, or both as covariates. A P value <.05 was deemed statistically significant. Analyses were conducted with in R, 4.2.0 with package dlnm, version 2.4.7 and regime ; all others were performed with SAS, version 9.4 (SAS Institute LLC). Further details are presented iMen the eMethods and eTable 1 in the We examined frequencies and proportions to describe the distributions of categorical variables. Birth weight for gestational age The study included 628 pregnant women and their newborns . The 2.5 exposure during the window from 14 to 22 gestational weeks was associated with lower birth weight. On average, birth weight changed by −9.5 g  per IQR increase in PM2.5 exposure in each week across this sensitive window, with the largest change  in gestational week 18. An IQR increase in NO2 exposure in each week from 9 to 14 gestational weeks was associated with birth weight . There was no association of PM10 and O3 exposure with birth weight. The results were robust in sensitivity analyses of multipollutants , but no associations in the low-PSS group (n = 411). In stratification analysis by CES (2.5 and PM10 with birth weight in the high-CES group (n = 396), but no associations in the low-CES group (n = 230). We further stratified the analysis by the combined groups of PSS and CES scores  and in PM10 exposure in each week from 9 to 14 gestational weeks . Also in this group, an IQR increase in NO2 exposure was associated with birth weight in each week from 9 to 14 weeks , and from 33 to 36 weeks  in birth weight. An IQR increase in NO2 exposure in each week from 23 to 26 weeks also was associated with birth weight . Some sparse significant differences in BWZ by PM10 in the low-PSS, high-CES group and by NO2 in the high-PSS, low-CES group were also seen, both within the window from 5 to 10 gestational weeks. There were no associations between O3 and birth weight.In stratification analysis by PSS , we obses by CES , we obseS scores . In the 2, PM2.5, and PM10 exposures in the window from early pregnancy to midpregnancy. The inverse associations were generally stronger, and the sensitive windows were generally wider within subgroups with either high individual-level perceived stress, high neighborhood-level cumulative burden, or both. We found no evidence for associations of air pollution exposure during the preconception period with birth weight nor for prenatal O3 exposure with birth weight.In the predominately low-income Hispanic population that comprises the MADRES cohort, we observed robust associations of lower birth weight with NO2.5 and PM10 exposure with birth weight, and our results are consistent with these overall findings. In a meta-analysis of 17 studies, increases in PM2.5 exposure during the entire pregnancy were associated with lower birth weight with an effect size similar to ours.38 We found exposure to PM10 from 5 to 17 gestational weeks was associated with lower birth weight in women who had high perceived stress, lived in a neighborhood with a high cumulative burden, or both, but not in the overall cohort, suggesting outcomes of PM10 may be sensitive to population characteristics that have also been noted in previous studies.20 Other studies, including a meta-analysis, reported inconsistent associations of NO2 with birth weight.39 We found an association of NO2 exposure during the sensitive window from 9 to 14 gestational weeks with lower birth weight in the overall cohort that was strengthened among the subgroup with high perceived stress but attenuated toward the null in the low perceived stress subgroup. These findings together suggest a critical role for stressors at individual and neighborhood levels in determining the associations between sensitive windows of various air pollutants and birth weight.A robust literature exists reporting the inverse association of ambient PM2.5 exposure and birth weight have identified inconsistent sensitive windows ranging from early to late pregnancy.11 In the present study, our identification of early pregnancy to midpregnancy as a sensitive window is consistent with other studies.15 Registry-based studies based in China13 and Texas21 have also used DLMs to identify sensitive windows in finer periods , and generally have found the exposure window of 20 to 35 gestational weeks to be important, which partly overlaps with our findings. Early pregnancy to midpregnancy is the critical period of organogenesis and functional initiation that lays the foundations for fetal growth, such as the beginning of hematopoiesis, brown fat formation, and thyroid hormone secretion.16 Therefore, exposure to air pollution during early pregnancy could affect fetal and placental development and eventually lead to lower birth weight, particularly for mothers with high psychosocial stress.Previous studies aiming to identify associations between sensitive windows of PM2.5, PM10, and NO2 exposure with birth weight. Our findings are consistent with studies that found neighborhood-level socioeconomic deprivation is associated with prenatal exposure to air pollution and offspring health outcomes, including birth weight.41 Among a handful of studies that have measured individual-level psychological stress, susceptibility to asthma, poor lung function, or accelerated biological aging associated with air pollution exposures was greater in a higher stress group than in the lower stress group.28 To our knowledge, our study is the first to report that individual-level maternal perceived stress may increase susceptibility to changes in birth weight associated with exposure to PM2.5, PM10, and NO2 exposure and widens the sensitive windows of exposure.Both individual-level psychosocial stress and neighborhood-level cumulative burden could amplify the inverse association of PM45 Nevertheless, further studies are needed to elucidate the mechanisms of the joint effects of individual and neighborhood stressors on the association between air pollution and reduced fetal growth.The CES cumulative impact scores capture the neighborhood-level cumulative burden from both environmental pollution and population vulnerability, thus providing further evidence that susceptibility to changes in birth weight associated with air pollution was greater among populations with a high cumulative neighborhood burden. The population with both high individual-level psychosocial stress and high neighborhood-level cumulative burden faced the greatest susceptibility and the widest sensitive window of air pollution exposure in association with birth weight, suggesting that individual-level and neighborhood-level stressors may compound one another. Both individual- and neighborhood-level stressors might act on some known biological pathways that are also involved in responses to air pollution exposures, including heightened oxidative stress and systemic inflammation, hormonal interruption, and reduced placental function.A strength of our study is the use of DLMs to identify sensitive windows of exposure and the use of individual- and neighborhood-level stressors to investigate outcome modification.46 Sample size was reduced in subgroup analyses, especially in the case of PSS missingness (13.5%). We excluded preterm births owing to the missingness of exposure before 37 weeks, which could potentially introduce collider bias. We attempted to evaluate such bias by comparing exposure and covariates between preterm and full-term infants (eTable 5 in the The study has limitations, including lack of information on the source and constituents of particulate matter. Neighborhood-burden CES cumulative impact score is only available in California, but similar measures, such as the Environmental Justice Screening, are available for all US states.2.5, PM10, and NO2 during sensitive windows primarily in early pregnancy to midpregnancy were associated with lower birth weight. Wider sensitive windows and greater changes were seen in children born to mothers with high perceived stress or who lived in a health-disparity neighborhood with a high burden from environmental pollution and population vulnerability.In this cohort study, ambient PM