Obesity is an escalating health problem worldwide, and hence the causes underlying its development are of primary importance to public health. There is growing evidence that suboptimal intrauterine environment can perturb the metabolic programing of the growing fetus, thereby increasing the risk of developing obesity in later life. However, the link between early exposures in the womb, genetic susceptibility, and perturbed epigenome on metabolic health is not well understood. In this study, we shed more light on this aspect by performing a comprehensive analysis on the effects of variation in prenatal environment, neonatal methylome, and genotype on birth weight and adiposity in early childhood.In a prospective mother-offspring cohort (N = 987), we interrogated the effects of 30 variables that influence the prenatal environment, umbilical cord DNA methylation, and genotype on offspring weight and adiposity, over the period from birth to 48 months. This is an interim analysis on an ongoing cohort study.ANK3, CDKN2B, CACNA1G, IGDCC4, P4HA3, ZNF423 and MIRLET7BHG) were significantly associated with birth weight, with a subset of these in genes previously implicated in metabolic pathways in humans and in animal models. Methylation levels at three of seven birth weight-linked loci showed significant association with prenatal environment, but none were affected by polygenic risk score. Six of these birth weight-linked loci continued to show a longitudinal association with offspring size and/or adiposity in early childhood.Eleven of 30 prenatal environments, including maternal adiposity, smoking, blood glucose and plasma unsaturated fatty acid levels, were associated with birth weight. Polygenic risk scores derived from genetic association studies on adult adiposity were also associated with birth weight and child adiposity, indicating an overlap between the genetic pathways influencing metabolic health in early and later life. Neonatal methylation markers from seven gene loci  contains supplementary material, which is available to authorized users. The epidemic of obesity is a major public health issue. The risk of obesity appears to begin in utero, as a suboptimal intrauterine environment can have a lasting impact on metabolic control –5. A majBirth weight is often used as a surrogate outcome to evaluate the overall quality of the in utero environment. Firstly, both low and high birth weights have been implicated in childhood and adult onset of chronic diseases such as obesity, impaired glucose tolerance, type 2 diabetes mellitus (T2DM) and coronary artery disease . SecondlTo date, only four epigenome-wide studies have been reported that examined the association between methylation marks in neonate tissues and birth weight –22, and RXRA promoter methylation in umbilical cord DNA correlates with childhood obesity in replicate cohorts, and that the level of methylation is associated with maternal nutrition in the first trimester [HIF3A) gene (a gene previously associated with adult adiposity [We previously reported that rimester . Using adiposity ) associadiposity . It folldiposity , prompteIn the current study we take a comprehensive approach to understand the genesis of adiposity in early life by interrogating the effects of prenatal environment, genotype and DNA methylation, and we report four important findings. First, we identify prenatal environments that influence birth weight. Second, we report associations between child weight/adiposity (at birth and during early childhood) and polygenic risk score derived from adult adiposity genetic association studies. Third, we find variations in the neonate DNA methylome that associate with birth weight and size/adiposity measures in early childhood. Last, we determine SNPs and specific prenatal environments contributing to this variability in the neonate epigenome. This study is the first large sample size EWAS (N = 987) that assesses the impact of prenatal environment and genetic and epigenetic factors on birth weight and size/adiposity in early childhood. It is also the first neonate EWAS conducted in an Asian population.This work is part of the Growing Up in Singapore Towards healthy Outcomes (GUSTO) study, a prospective mother-offspring birth cohort designed to investigate developmental origins of health and disease (DOHaD). The GUSTO cohort has been described previously . Pregnan2 (m2) at all time points. Subscapular and triceps skinfolds were measured at birth, 18, 24, 36 and 48 months, and taken in triplicate using the Holtain skinfold callipers  on the right side of the body, and recorded to the nearest 0.2 mm. Subscapular to triceps skinfold ratio was derived by dividing subscapular skinfold (mm) by triceps skinfold (mm). BMI is used as a proxy for adiposity in the analyses and to be concise, the terms BMI and adiposity have been used interchangeably in this study. Some caution should be exercised in interpreting the findings on BMI, because while BMI is widely accepted as an indirect measure of adiposity, it has its limitations. For example, elevated BMI levels may arise as a result of extra muscle mass or stunted linear growth [Child weight and recumbent length/standing height were measured at birth and at nine subsequent time points . Child weight was measured using calibrated scales  and recorded to the nearest gram. Recumbent length  was measured using a SECA infant mat  and recorded to the nearest 0.1 cm. Standing height (36 and 48 months) was measured using a stadiometer  from the top of the child’s head to his or her heels, and recorded to the nearest 0.1 cm. Weight and length/height measurements were taken in duplicates for reliability. BMI was derived as weight (kg) divided by lengthr growth . We haveAn interviewer-administered questionnaire was conducted at 26–28 weeks of gestation to obtain information on occupational activity during pregnancy, alcohol usage before and during pregnancy, and smoking patterns before and during pregnancy. Maternal height and weight were measured during the same time period. Pre-pregnancy weight was self-reported during study recruitment in the first trimester of pregnancy. Gestational weight gain (GWG) was calculated as the difference between the pre-pregnancy and 26–28 week weights. Maternal ppBMI was derived as pre-pregnancy weight divided by height squared. Maternal glucose levels (2-h post-glucose and fasting) were ascertained at 26–28 weeks using an oral glucose tolerance test of 75 g after an overnight fast (8–14 hours). Maternal plasma fatty acids, including n-6 polyunsaturated fatty acids (PUFA), n-3 PUFA, monounsaturated fatty acids (MUFA), and saturated fatty acids, were measured using gas chromatography–mass spectrometry, and expressed as percentage contribution to total plasma phosphatidylcholine (PC) fatty acid. Specifically, plasma lipids were extracted using chloroform–methanol  and PC was isolated by solid phase extraction. Fatty acid methyl esters were generated from PC after reaction with methanol containing 2% (v/v) sulfuric acid, extracted into hexane and separated by gas chromatography. Fatty acid methyl esters were identified by comparison with retention times of previous standard runs and quantified using ChemStation software (Agilent Technologies). Maternal micronutrient levels  were measured from serum drawn at 26–28 weeks of gestation. Maternal calorie intake at 26–28 weeks gestation was calculated from both 24-h dietary recall and 3-day food diary. Maternal depressive symptoms were assessed using the Edinburgh Postnatal Depression Scale, which was designed and normed expressly for depressive symptoms over the peripartum period , 30. SymP > 0.01 were set to missing. Probes from sex chromosomes were removed. Colour adjustment and normalisation of Type 1 and 2 probes was performed. Methylation beta values were first converted to M-values before applying COMBAT to remove batch (plate) effects [Methylation profiling of umbilical cord samples was performed using the Infinium HumanMethylation450 array, following standard protocol, and processed using in-house quality control procedure . Raw met effects , and the effects , 36, as  effects , 38.Genotyping was performed using the Illumina Omniexpress + exome array. Non-autosomal SNPs, SNPs with call rates < 95%, or minor allele frequency < 5%, or those that failed Hardy–Weinberg Equilibrium were excluded from the analysis. Principal components analysis was used to confirm self-reported ethnicity/ancestry. Samples with call rate < 99%, cryptic relatedness and sex/ethnic discrepancies were excluded. Alleles on the positive strand were reported as per the hg19 build of the human genome assembly. After quality control filtering 577,204 SNPs were available for downstream analysis.The overall analysis framework is summarised in Additional file Linear regression models were used to examine the association of 30 prenatal environment variables with infant birth weight. Eleven of these 30 prenatal environment variables that associated with birth weight were used for subsequent analysis. We first separately studied the association of each prenatal environment variable with birth weight, adjusted for infant sex, ethnicity and GA. This was followed by the association of prenatal environment variables with birth weight, adjusted for each other along with infant sex, ethnicity and GA. We examined the distribution of infant birth weight, and subsequently decided to use a log-transformation on infant birth weight to improve normality and reduce the impact of outliers. Following Gelman , binary P values for adult BMI reported by the Genetic Investigation of ANthropometric Traits (GIANT) consortium [P value-informed clumping procedure implemented in PLINK. To reduce the inclusion of SNPs in linkage disequilibrium (LD), two rounds of clumping were performed. We first used a cut-off of R2 = 0.5 within a 250-kb window to identify potential index SNPs; in each 250-kb window, the SNP with the smallest P value from GIANT was kept while SNPs in LD (R2 > 0.5) were removed. Second, to further exclude SNPs in long-range LD with potential index SNPs, the clumping procedure was repeated with a cut-off of R2 = 0.2 within a 5-Mbp window. For each individual, the cumulative score was computed by summing the number of score alleles, weighted by the regression coefficients reported by the GIANT consortium. We computed PRS for each ethnic group separately, and at different P value thresholds pT for the index SNPs (pT from 10–10 to 1). For each ethnic group, PRS was standardised to mean 0 and variance 1 (Z-score) separately. We then regressed PRS against log-transformed child anthropometric measures, adjusted for child sex and GA, for each ethnic group. We examined ethnicity-stratified associations of PRS with child anthropometric outcomes for different P value thresholds. For each ethnic group, we selected the P value threshold that gave the best-fit score (defined as PRS showing consistent associations with child weight and BMI at multiple time points). This best-fit PRS was then used for subsequent analysis. We did not use other child outcomes  for evaluating the best-fit score as these outcomes were measured at fewer time points and generally had larger measurement error. We also did not consider child length for evaluating the best-fit score as it did not capture adiposity. However, we report associations between PRS for all child outcomes. For concision, the result sections report only the conclusive findings (child weight and BMI), while the rest (e.g. skinfolds) are provided under Additional file To determine whether genetic variation at loci previously associated with adult adiposity was associated with newborn size/adiposity, polygenic risk score (PRS), or cumulative genetic risk profile, was computed for each infant in the GUSTO cohort using regression coefficients and nsortium . PRS was pT from 0–10 to 1. We further applied genomic control to the P values if the genomic inflation factor computed across 174,211 CpGs was greater than 1. A genomic control correction could help correct for residual confounding due to cellular heterogeneity; however, it could also be too conservative, as a global inflation in epigenome-wide P values in response to increased adiposity in adults has been previously reported [To interrogate the association between perinatal methylome and birth weight, we performed linear regression of log-transformed birth weight against methylation at each CpG site, adjusted for child sex, GA, ethnicity, cellular proportions and interactions between ethnicity and cellular proportions. Cellular proportions for fibroblasts, B-cells and T-cells were estimated  using a reported  and coulreported . This suP < 0.05/(8 × 11) ~ 5 × 10–4. Genomic inflation factor was computed for each prenatal environment across all 174,211 variable CpGs, and genomic control was applied if the inflation factor for the variable was above 1. This simple Bonferroni correction for multiple testing across the 11 prenatal environments was likely to be conservative as the 11 prenatal environments were associated with one another . FDR was not used for multiple testing adjustments here and for the top CpGs and offspring size/adiposity in early childhood because of the relatively small number of tests  and dependency between the tests [P value) with the CpG.We then characterised the influences of the prenatal environment and SNPs on the variability in methylation at CpGs showing association with birth weight. First, to investigate the influence of the prenatal environment on methylation levels, we regressed methylation at each CpG site against (standardised) prenatal environment variables, adjusting for child sex, GA, ethnicity, cellular proportions and interactions between ethnicity and cellular proportions. To adjust for multiple testing across eight CpGs and 11 prenatal environments, a CpG was defined to be influenced by the prenatal environment if the most significant association with the prenatal environment variables had genomic control-adjusted he tests , 49. Forcis-SNPs (defined here as SNPs on the same chromosome as CpG), using an additive genetic model, adjusted for child sex, GA, ethnicity, cellular proportions, and interactions between ethnicity and cellular proportions. For SNPs where the minor homozygote genotype group had ≤ 50 individuals, the minor homozygote and heterozygote genotype groups were combined (dominant genetic model). A total of approximately 5 × 105 CpG-SNP tests were conducted, corresponding to testing eight CpGs across 8,392 to 47,298 cis-SNPs for each CpG . A CpG was defined to be influenced by the genotype (SNPs) if the most significant association between the CpG and cis-SNPs attained P < 1 × 10–7, the Bonferroni threshold to maintain a family-wise Type 1 error rate of 0.05 across approximately 5 × 105 tests.Second, to interrogate the influence of SNPs on methylation levels, we regressed each CpG against P < 0.05/(8 × 45) = 1 × 10–4. The genomic inflation factor was computed for each offspring anthropometry measure across all 174,211 CpGs, and genomic control was applied if the inflation factor for the anthropometry outcome was above 1. This simple Bonferroni correction for multiple testing across different size/adiposity measures was likely to be extremely conservative as the 45 size/adiposity measures were strongly associated with each other. For concision, the result sections describe only the conclusive findings, while the rest are reported in Additional file Finally, we examined whether these methylation marks at birth were associated with offspring weight in early childhood (3–48 months) and offspring length and adiposity  from birth to 48 months. We also examined BMI change in early childhood , where BMI Z-score at birth and 48 months were calculated using WHO child growth charts. Child anthropometric measures (except BMI change) were log-transformed to improve normality and reduce the impact of outliers. Each offspring anthropometric measure at each assessment time point was analysed separately. This was done by linear regression of (log-transformed) anthropometric measures at each time point against the methylation at each CpG site, adjusted for child sex, GA, ethnicity, cellular proportions and interactions between ethnicity and cellular proportions. To account for multiple testing across the eight CpGs and 45 child size/adiposity measures, a CpG would be associated with offspring size/adiposity if the genomic control-adjusted We used different multiple testing methods (FDR vs. Bonferroni) at different analysis steps in the sections above. The reason for the use of different methods was due to the vastly different number of tests to be adjusted for multiple testing in each analysis step. To adjust for multiple testing across 174,211 CpGs in birth weight and neonatal DNA methylome, we used FDR. For the environmental influences on top CpGs and for the top CpGs and offspring size/adiposity in early childhood sections, FDR could not be used for multiple testing adjustments because of the relatively small number of tests  and dependency between the tests , 49. InsThe infant methylation data analyzed in the current study is available as Additional files This analysis used 987 of 1177 singleton deliveries in the GUSTO cohort study. The subject selection criterion included live singleton term births with Apgar score ≥ 9, and availability of anthropometric measures, covariates/confounder information, as well as genotyping and methylation data for all subjects . There was also a suggestive association with parity . Greater maternal adiposity (ppBMI and GWG), height, glucose levels (fasting and 2-h post-glucose), n-6 PUFA levels, age and parity were associated with higher birth weight, while higher MUFA levels and maternal smoking (before and during pregnancy) were associated with lower birth weight. Birth weight changed by 2.2–5.5% for every 2 SD change in maternal adiposity (ppBMI and GWG), height, glucose levels (fasting and 2-h post-glucose) or FA levels (n-6 PUFA and MUFA). The effect sizes for parity (non-first born vs. first born), maternal age (≥35 years vs. < 35 years) and smoking (yes vs. no) were similar and ranged from 1.4% to 6.5% ; (2) 3’-UTR of Cyclin-Dependent Kinase Inhibitor 2B ; (3) intron of Immunoglobulin Superfamily, DCC Subclass, Member 4 ; (4) intron of Prolyl 4-hydroxylase, Alpha Polypeptide III ; (5) intron of Calcium Channel, Voltage-Dependent, T Type, Alpha 1G Subunit ; and (6) intron of Zinc Finger Protein 423 , while the remaining two CpGs mapped to the non-coding gene MIRLET7BHG (P = 9.9 × 10–7 and P = 2.2 × 10–6).DNA from umbilical cord tissue of the 987 neonates was interrogated on Infinium HumanMethylation450 BeadChip arrays. A total of 174,211 CpGs were identified to vary in methylation by more than 10% across the subjects. These CpGs were more likely to be located in open seas and intronic/intergenic regions  was modest with an interquartile range (IQR) of 4.6% to 9.6%. DNA methylation levels at five of seven loci (five CpGs) were positively associated with birth weight, while DNA methylation at the remaining two loci (three CpGs) were negatively associated with birth weight. The effect sizes were modest, with a 3.7–9.2% change in birth weight associated with a 10% increase in methylation (corresponding to approximately 0.4–2 IQR). Together, these eight CpGs accounted for an additional 9.5% of the total variation in birth weight, in addition to the 32% accounted by infant sex, ethnicity, GA, 11 prenatal environments and PRS. Sensitivity analysis using a reference-free method to adjust for cellular heterogeneity gave similar results  were significantly associated with the prenatal environment after adjusting for multiple testing . Methylation levels at cg25685359 (MIRLET7BHG) showed a significant inverse association with maternal n-6 PUFA levels , and a significant positive association with maternal smoking before pregnancy . Methylation levels at cg25487405, which also mapped to MIRLET7BHG, showed modest associations (P < 0.05) with these two prenatal environment variables, though the associations did not survive multiple testing adjustments. The directionality of the associations between methylation and prenatal environments is consistent  showed a negative association with birth weight . Methylation levels at three of seven loci (IGDCC4) showed a significant positive association with maternal fasting glucose levels , and it also showed positive association with maternal ppBMI (P = 8.1 × 10–4). Likewise, methylation at cg22383874 (CACNA1G) was significantly and positively associated with maternal fasting glucose levels , and was also positively associated with maternal ppBMI (P = 2.9 × 10–2) and maternal 2-h post-glucose levels (P = 4.2 × 10–3). The directionality of associations between cg23671997 (IGDCC4) and cg22383874 (CACNA1G) and the prenatal environments is consistent  and cg22383874 (CACNA1G) with maternal fasting maternal glucose levels were similar but slightly reduced . Three loci were significantly associated with cis-SNPs after adjusting for multiple testing . The CpG-SNP distances ranged from 12 to 168 kb  was similar with and without adjustment for genotype at the SNP, and the genotype at the SNP was not associated with birth weight , we regressed each CpG against all P < 0.05). The effect sizes (associations between methylation and child weight) were either (1) strongest at birth and decreased from 3 to 48 months, or (2) strong at birth, decreased initially, and then increased from 18 to 48 months, or (3) strongest at birth, but remained the same (approximately) from 3 to 48 months  with BMI at birth ; the change in effect sizes of BMI with child age showed a similar pattern as that of child weight . The associations between neonate methylation and child size/adiposity in early childhood (3–48 months) were not independent of birth weight (data not shown). Methylation levels at cg25685359 (MIRLET7BHG) showed a suggestive association with BMI change in early childhood .Methylation levels at all seven loci (eight CpGs) showed association with child weight in at least one time point in early childhood (3–48 months), even though these associations did not survive multiple testing adjustments  also showed suggestive association with child BMI at 48 months. Even though the associations in early childhood did not survive multiple testing corrections, these CpGs still hold potential as biomarkers of adverse metabolic trajectory as the prevalence of obesity increases with age and might become more apparent later in the life-course. Lastly, methylation levels at three of seven loci associated with birth weight  also showed significant associations with the prenatal environment; however, similar analyses with childhood weight and adiposity measures showed suggestive associations. Together, these findings provide evidence that birth weight is influenced by both genetic and prenatal environment factors, possibly acting through different mechanisms, either by altering the epigenome  or independently of the epigenome (e.g. the PRS).We have demonstrated that genetic, epigenetic and prenatal environmental factors are linked to offspring size and adiposity at birth and in early childhood. Firstly, we identified individual prenatal environmental influences on birth weight; we have previously reported that some of these prenatal environment variables  continued to associate with offspring size and adiposity in early childhood , 51. SecANK3, CDKN2B, CACNA1G) and the miRNA let-7b host gene (MIRLET7BHG) that have been previously implicated in metabolic disorders in human adults and animal model systems. ANK3 encodes a protein from ankyrin family, and ankyrins have been associated with age dependent adiposity and insulin resistance in a rat model system [CDKN2B is known to be involved in metabolic processes since it is highly expressed in subcutaneous adipose tissue, and its expression alters with energy balance  [CDKN2A/B 9p21.3 locus were previously found to be associated with risk for CVD and T2DM in adults [CACNA1G antagonists to obese rodents results in reduced body weight and fat mass, and increased lean muscle mass [MIRLET7BHG host gene, belongs to the let-7 family of miRNA that is known to play an important role in adipocyte differentiation (3T3-L1 mouse cells) by targeting HMGA2, a transcription factor that regulates growth and proliferation [Igf1r), insulin receptor (Insr) and insulin receptor substrate-2 (Irs-2) in skeletal muscle and liver tissues [Notably, four of seven methylation loci were located in coding genes (l system . CDKN2B ht-loss) . Furthern adults . T-type cle mass . MicroRNferation , 58. Fur tissues . Let-7 i tissues .MIRLET7BHG, IGDCC4, CACNA1G) or suggestive association with child BMI at age 48 months . Collectively, our findings fit within the paradigm of epigenetic mediation in the DOHaD hypothesis. According to the DOHaD hypothesis, the predisposition to adulthood diseases is primed in utero by specific antenatal environments [As mentioned earlier, some of these loci also showed association with either the prenatal environment  gene, was statistically significant after adjustment for multiple testing in our cohort . However, the absence of any single loci achieving the conventional genome-wide significance at 5 × 10–8 was more likely to be due to a lack of statistical power than caused by a lack of genetic influences on birth weight. Therefore, we used a genetic risk profiling approach and genetic variants reported by the GIANT consortium to form a single composite measure/score of genetic risk, and used this risk score to investigate genetic influences on birth weight. Third, the GUSTO cohort study was primarily designed to obtain extensive prenatal environment measures at mid-pregnancy. Consequently, we were unable to examine trimester-specific effects on the growing fetus. Since late pregnancy weight gain has been linked to suboptimal metabolic outcomes in offspring, we analysed maternal weight measures in late pregnancy derived from medical records . As gestational weight gain from pre-pregnancy to mid-pregnancy already showed a significant association with birth weight, we restricted the late pregnancy analysis to the weight gain between mid-pregnancy and 36–41 weeks. Unlike the gestational weight gain up to mid-pregnancy, the additional weight gain during late pregnancy did not associate with infant birth weight (P = 0.12). It is unclear whether the absence of significance is an indication of trimester-specific effects or a result of low statistical power due to the reduced sample size. Future studies require detailed pre-pregnancy and trimester-specific information to reflect better on the temporal influences of prenatal environment on the growing fetus. Lastly, while we have longitudinal measures of anthropometry, we do not have longitudinal measures of methylation in early childhood and during fetal development, which would be important for determining causality and directionality of the effects. For example, to investigate if DNA methylation mediates effects of the prenatal environment on offspring adiposity one would need to first establish the temporality/directionality of the effects, i.e. whether (1) increased child adiposity leads to the alterations in DNA methylation, or (2) DNA methylation changes lead to increased child adiposity. DNA methylation is a possible mediator in the latter scenario but not the former. Moreover, examining further how DNA methylation levels at these loci change with age, body size, adiposity during childhood and environmental exposures during childhood will allow for better evaluation of these candidate loci for stratification and obesity prevention strategies. A comparison of methylation measurements collected in utero, at birth and in early childhood, across different tissue types, is an important area of investigation for future studies.There are limitations of this study. First, residual confounding is a concern in any epidemiological investigation. In the context of EWAS, one of the major sources stems from cellular heterogeneity of the tissue being surveyed, as different cell types can have distinct methylation profiles. Cord tissue, like other infant tissues examined in a neonate EWAS, is heterogeneous in its cellular content and consists of stromal, epithelial and endothelial cells (and possibly cord blood contamination) , 63. To Childhood obesity has both immediate and long-term effects on the health and well-being of an individual. Children who are obese are more likely to become obese adults –66. In tDevelopmental pathways to adiposity begin before birth and are influenced by genetic, epigenetic and prenatal environment factors. These pathways may have lasting effects on offspring size, adiposity and metabolic trajectory, and have utility in identifying individuals who are susceptible to obesity and metabolic disease later in life.


In observational epidemiology, both low and high birth weights are associated with later type 2 diabetes. The mechanisms underlying the associations are poorly understood. We review evidence for the roles of genetic and non-genetic factors linking both sides of the birth weight distribution to risk of type 2 diabetes, focusing on contributions made by the most recent genome-wide association studies (GWAS) of birth weight.There are now nine genetic loci robustly implicated in both fetal growth and type 2 diabetes. At many of these, the same alleles are associated both with a higher risk of type 2 diabetes and a lower birth weight. This supports the Fetal Insulin Hypothesis and reflects a general pattern for type 2 diabetes susceptibility alleles: genome-wide, there is an inverse genetic correlation with birth weight, and initial estimates suggest genetic factors explain a large part of the covariance between the two traits. However, the associations at individual loci show heterogeneity; some fetal risk alleles are associated with higher birth weight. For most of these, the association reflects their correlation with the maternal risk allele which raises maternal glucose, thus increasing fetal insulin-mediated growth.GWAS have improved our understanding of the mechanisms underlying associations between type 2 diabetes and birth weight but questions remain about the relative importance of genetic versus non-genetic factors and of maternal versus fetal genotypes. To answer these questions, future work will require well-powered analyses of parents and offspring. The rising global prevalence of type 2 diabetes presents a major challenge for human health. Both environmental and genetic factors contribute to this complex disease. The roles of obesogenic diet and low physical activity as major adult risk factors are well established. Moreover, in any given population, there are individuals whose profile of genetic variation confers greater susceptibility, for example by predisposing to pancreatic beta cell dysfunction. Most of the genetic variance in type 2 diabetes susceptibility is attributable to variants of common frequency in populations •.An additional associated factor, which has attracted much attention during the past 25 years, is birth weight. Observational studies have consistently reported associations between type 2 diabetes risk in adulthood and either low or high birth weight . Taken tThe observation that individuals born with lower birth weights were more likely to have impaired glucose tolerance in middle-age was first demonstrated 25 years ago , and hasGCK), which reduce glucose sensing by the pancreas, cause both a reduction in insulin-mediated fetal growth and hyperglycemia after birth. Based on this and other rare, monogenic examples of low birth weight with either impaired insulin secretion or greater insulin resistance, the Fetal Insulin Hypothesis was proposed [The principle that genetic factors could be important was established in studies of monogenic diabetes. In 1998, Hattersley et al.  demonstrproposed ; allelesEvidence to support a role for polygenic effects in the link between type 2 diabetes risk and birth weight has come from epidemiological studies, e.g., , which sCDKAL1, ADCY5, and HHEX-IDE loci were also associated with lower birth weight. It is likely that these associations were mediated through their effects on insulin secretion. The most recent GWAS of birth weight [P < 5 × 10−8), nine of which had also been associated with type 2 diabetes , coupled with increasingly well-powered genetic studies of birth weight, enabled the first evidence to emerge that multiple common genetic variants could contribute both to variation in birth weight and type 2 diabetes risk. Evidence from candidate gene studies –17, and h weight ••, in 15tes Fig. . Most ofGenetic variation at the 60 birth weight loci and approximately 100 type 2 diabetes susceptibility loci explain approximately 2 and 5–10% of the variation in these traits, respectively. Assays of common fetal genetic variation across the whole genome have been estimated collectively to explain 15% of the variance in birth weight and at least 49% of liability-scale variance in type 2 diabetes risk , 22. HowTo estimate the overall genetic correlation between type 2 diabetes and birth weight, genome-wide summary statistics may be combined using a technique known as LD score regression •. This mWhile genome-wide correlation and covariance estimates lend support for a substantial role of common genetic effects contributing to the low birth weight–type 2 diabetes association, it is important to consider their limitations. The methods used assume a linear relationship between the two phenotypes, which is not strictly applicable to the overall non-linear relationship that has been observed between type 2 diabetes risk and birth weight.The fact that mother and child share 50% of their genes also means that care must be taken in interpreting estimates of genetic covariance between birth weight and type 2 diabetes. The correlation (r ≈ 0.5) between maternal and fetal genotype means that shared genetic effects are not necessarily directly attributable to the fetus. Fetal genetic associations may in fact indicate effects of the intrauterine environment, influenced by maternal genotype , than if inherited from their father [Studies of monogenic diabetes and type 1 diabetes have provided additional evidence in support of such intrauterine effects; an 8-year earlier age of diagnosis was observed for individuals inheriting a diabetes-causing mutation in r father ; and in r father .MTNR1B locus, both the fetal and maternal risk allele at SNP rs10830963 have been identified in GWAS as associated with higher birth weight [MTNR1B has one of the largest effects of common variants known to influence fasting glucose levels [As illustrated in Fig. h weight ••, 27. Te levels , thus thGCK and TCF7L2 loci [GCK is likely to represent an association signal distinct from the nearby low frequency variant that has been associated with birth weight when present in the fetus [Similar patterns of association have been observed between maternal type 2 diabetes risk alleles and higher offspring birth weight at the 7L2 loci –37, both7L2 loci . Howeverhe fetus   also have relatively large effects on type 2 diabetes risk. These three loci are all classified as associated with reduced beta cell function [TCF7L2, is not associated with lower birth weight [The variety of associations observed between known type 2 diabetes variants and birth weight enable us to make a number of inferences about their mechanisms of action. Of the nine loci in Fig. function , and theh weight , stronglMTNR1B, GCK) are known to have relatively large effects on fasting glucose and more moderate effects on type 2 diabetes risk. The balance of maternal birth weight-raising vs. fetal birth weight-lowering effects might be viewed as a bioassay on the life-course timing of physiological effects on beta-cell function. For example, a larger maternal than fetal effect suggests the allelic effect is less prominent in early life than in later life. To disentangle the contribution of the mother’s genotype from that of the offspring, and to estimate accurately the maternal and fetal effects at individual loci will require extremely large samples of mother-child pairs.Alleles showing the largest effects on raising birth weight through the maternal genotype  [Type 2 diabetes loci have been identified that express effects on disease risk in a parent-of-origin specific manner . To datend DLK1) ••. FuturThe U-shaped association between type 2 diabetes and birth weight is underpinned by multiple genetic and non-genetic factors. Recent GWAS data have greatly improved our understanding of the genetic factors contributing to the two traits, providing examples of individual loci that explain both positive and inverse covariation between them. Moreover, genome-wide genotype data in individuals with life-course phenotype data has enabled initial estimates of the overall role of genetic factors, suggesting they may play a major role in the association between type 2 diabetes and lower birth weight. Many questions remain unanswered, however, such as the relative importance of maternal and fetal genetic effects both genome-wide and at individual loci, and it is likely that great future gains will be made from well-powered analyses of parents and offspring.


Adults born very preterm have increased cardiac mass and reduced function. We investigated whether a hypertrophic phenomenon occurs in later preterm infants and when this occurs during early development.Cardiac ultrasound was performed on 392 infants (33% preterm at mean gestation 34±2 weeks). Scans were performed during fetal development in 137, at birth and 3 months of postnatal age in 200, and during both fetal and postnatal development in 55. Cardiac morphology and function was quantified and computational models created to identify geometric changes.P<0.001; right 39.3±38.1 vs. 16.6±40.8, P=0.002). Greater changes were associated with lower gestational age at birth .At birth, preterm offspring had reduced cardiac mass and volume relative to body size with a more globular heart. By 3 months, ventricular shape had normalized but both left and right ventricular mass relative to body size were significantly higher than expected for postmenstrual age (left 57.8±41.9 vs. 27.3±29.4%, Preterm offspring, including those born in late gestation, have a disproportionate increase in ventricular mass from birth up to 3 months of postnatal age. These differences were not present before birth. Early postnatal development may provide a window for interventions relevant to long-term cardiovascular health. Worldwide, ~1 in 10 infants are born preterm, and owing to improved maternal–fetal and neonatal intensive care the majority now survive to adulthood , 3. In eex utero environment in the preterm infant, we performed serial echocardiograms at birth and 3 months of age to assess differences in maturational patterns of cardiac structure and function between preterm and term infants. We then compared the changes in cardiac mass, volumes, and shape with those expected during term pregnancies from early gestation, taking account of variation in body growth and other perinatal factors.To test the hypothesis that there is evidence of postnatal changes to cardiac performance in response to the Between 2011 and 2015, mothers being cared for by the Oxford University Hospitals NHS Foundation Trust were identified by their clinical care team and invited to take part in one or more of a portfolio of studies coordinated by the Oxford Cardiovascular Clinical Research Facility. These studies were designed to investigate the impact of pregnancy complications on cardiovascular development during gestation and after birth.A stratified recruitment approach to ensure balanced representation of preterm and term birth, as well as hypertensive and normotensive pregnancies, was used through the EPOCH  study  . The phen=192 fetuses scanned, 110 fetuses were scanned once; 43 were scanned twice; 21 were scanned three times; 12 were scanned four times; and six were scanned five times.The final 55 mothers from our neonatal cohort were recruited antenatally and fetal echocardiography performed starting at 15 weeks of gestation and at multiple time points until delivery . Fetal eAll mothers gave written informed consent and assent for involvement of their children, including permission to access maternal and offspring clinical records. Singletons and multiples were included. Mothers below the age of 16 years were excluded from the study as were those with chronic cardiovascular conditions prenatally, including hypertension, although those who suffered from hypertensive disorders of pregnancy were included. Infants were excluded if they had evidence of any severe malformations, congenital cardiovascular disease, chromosomal abnormalities, or genetic disorders, but those with persistent features of a fetal circulation at birth, i.e., patent ductus arteriosus (PDA) and/or atrial septal defect (ASD), were included.Z-scores for weight were calculated using the international standard size at birth reference charts from the INTERGROWTH-21st Project .Gestational age at the time of measurements was calculated relative to gestational age defined at first trimester ultrasound. At both the birth and 3-month assessments, weight was measured using digital scales (Charder Model MS4200) to the nearest 0.01 kg with the infant fully naked. During gestation, head circumference was taken from a cross-sectional view of the fetal head at the level of the thalami as close as possible to the horizontal as previously described  and the  Project , 12 usinData collection methods and clinical characterization have previously been reported  and are Fetuses were scanned on a Philips HD9 with a C6-3 curved-array transducer. At birth, a Phillips CX50 was used with a 3-month follow-up on a Philips iE33 with an S12-4 transducer. At each time point, a 2D transthoracic echocardiography protocol that included acquisition of a four-chamber view optimized for the LV was performed according to standard guidelines . PostnatInvestigators performing the assessments were not blinded to participant group, but those involved in image analysis were.Statistical analysis was performed using SPSS Version 20  and GraphPad Prism Version 6.0 . In addition, STATA, Version 11.2, software  was used to analyze developmental trajectories and cardiac shape analysis was performed with Matlab . Values are presented as mean±SD unless stated otherwise.t-test and Mann–Whitney U test were used as appropriate. Tests of associations were performed using the χ2 test. Linear regression models were performed with a forced entry method. Pearson correlations (r) were used for bivariate associations, and unstandardized regression coefficients (B) were used for bivariate and multivariate regression models. Variables were selected from bivariate regression models to be included in multivariate regression models. The sample size n=134 for term offspring and n=121 preterm offspring provided us with 80% power at a significance level of α=0.05 to detect a difference of at least 0.38 SDs between groups.To compare mean differences in cardiac size postnatally, Student’s z-scores across gestational ages.To further explore the timing of mass and volume changes in preterm infants, we studied trajectories of cardiac development using the combined data set of echocardiographic measures of LV and RV mass at different time points during gestation and postnatal development. As volumes pre- and postnatally would not be comparable because of circulatory changes at birth, we limited combined gestation and postnatal analyses to mass. Smoothed centiles of right and left ventricular mass, left and right EDV, and ratio of mass as a function of HC according to gestational age were constructed using fractional polynomials. To account for body size differences between preterm and term infants at different time points, we adjusted the left and right ventricular mass for head circumference  on the basis that this measure would be the most consistently and accurately quantifiable body measure throughout gestation and postnatal development rather than weight or body surface area. This is because weight cannot be directly or accurately measured in gestation and therefore pre- and postnatal values cannot be directly compared. In addition, equations for body surface area have not been validated for fetuses. Where appropriate, we applied a multilevel, linear regression analysis to account for repeated measures , but thet-test. Shape differences were first tested in each Principal Component Analysis mode under three conditions: (1) at birth, (2) at follow-up, or (3) by their growth. Then, the Principal Component Analysis modes, which accounted for the most variation within the experimental sample, were combined through a LDA in an attempt to identify the morphological signature of a premature birth. Cross-validation (leave-1 out) was used to test the generality of differences found in the LDA, and to select the optimal set of Principal Component Analysis modes to be combined to differentiate groups.Analysis of shape variation between preterm and term infants was performed by a non-paired P-values<0.05 were considered statistically significant.Maternal and offspring demographic and anthropometric characteristics in the preterm and term groups are presented in P<0.001). These volumetric mass changes were supported by the linear measures based on posterior wall thickness (PWd) and associated formulaic mass estimates (P<0.001). Preterm neonates already had an increased right ventricle (RV) mass/EDV ratio at birth (P=0.002). Changes in LVMI and RV mass index (RVMI) were proportional to gestational age at birth  and for the preterm group alone (P=0.003). Both parameters were associated with mass change, although neither were significant in multivariable models (P=0.001), whereas exposure to antenatal steroids was not significant (P=0.67). Similarly, for RVMI, gestational age remained significant (P=0.03), whereas exposure to antenatal steroids was not significant (P=0.93). Furthermore, we have performed a subgroup analysis in our preterm group for LVMI and RVMI at birth and 3 months of postnatal age to compare those who were (n=93) and were not (n=28) exposed to antenatal steroids  mass and end-diastolic volume (EDV), indexed to body surface area, at birth compared with those born at term . As the stimates  Table 2P<0.001. s change . Similare models . Antenatsteroids . These rz-scores within the group modeled from the expected cardiac fetal mass being −0.16±0.53 for the LV and −0.13±0.50 for the RV. By follow-up, however, several of the preterm infants were exceeding the 95% centile for cardiac mass with an absolute difference in the 50th centile at 49 weeks of postmenstrual age  between preterm and term LV mass being 1.61 g. To allow for the change in body size during this period, we additionally created trajectories based on mass indexed to head circumference as a consistent measure between gestation and postnatal development .There was a reduced LV stroke volume and ejection fraction at birth in the preterm group related to both the reduction in EDV index and an increase in end-systolic volume (ESV) index. LV stroke volume, but not ejection fraction, remained reduced at 3 months of age . In the ral E/E′ . InteresP<0.001), but this difference between groups had disappeared by 3 months of age (P=0.24).Ventricular shape analysis suggested a unique preterm cardiac developmental pattern. The major mode that differentiated groups was, as expected, a general size mode —mode 1. This study shows for the first time that preterm infants have a greater increase in both LV and RV mass over the first three postnatal months that is disproportionate to increases in body and total cardiac size. Indeed, average cardiac mass changes are double that of their term-born counterparts during this period. Furthermore, there is a reduction in LV diastolic function with a persistent reduction in RV systolic function in the preterm infants. Finally, the hypertrophic patterns and global dysfunction are postnatal processes that are not evident during gestation or at birth, but rather emerges during the early postnatal period.The increase in ventricular mass at 3 months of age is strikingly similar to our previous reports in adults born preterm , 5 and iOur observations are consistent with findings from a preterm-born sheep model, which had a five- to sevenfold increase in cardiomyocyte hypertrophy during postnatal development, as well as altered interstitial myocardial fibrosis and cardiomyocyte maturation . Similarin utero and is thicker walled than the LV in fetuses up to 2.7 kg in weight (Our measures of mass at birth were similar to levels previously reported in babies of similar size, born at different gestations , 26, 27,n weight , 29. Ourn weight , this con weight . Howevern weight .z-score on the LV, consistent with a previous finding of mass increase proportional to birth size in very preterm infants (in utero hypoxia, which has been shown to cause cardiac remodeling in animal models (Other perinatal factors linked with preterm birth might have influenced our findings. The use of antenatal steroids was not included in multivariable analyses because of the significant co-linearity with preterm birth. However, although we cannot be sure of an independent effect on mass, our results suggest that degree of prematurity, rather than antenatal steroids, has a more substantial contribution to the observed changes in cardiac mass. Multivariable analyses highlighted a small independent influence of birthweight  infants . Maternal models . Blood pl models  and sligl models , 34, we l models . The myol models . Therefol models .per se (Multiples were not excluded in this study. It is known that twins are at an increased risk of congenital heart disease, in particular in monozygotic pairs . Of the per se , and theper se .Longitudinal follow-up of this cohort will establish whether changes in mass and associations with function persist into later life. Furthermore, their relation to reduced exercise capacity  and highIn the neonatal cohort, because of our stratified recruitment, the incidence of maternal hypertension was similar between groups , which mAnother potential limitation is our use of automated software to derive measures from single 4-chamber views. Neonatal and fetal echocardiography is technically challenging, although four-chamber views can be acquired in most fetuses by 13 weeks of gestation . Therefoin utero, which is associated with cardiac dysfunction. These changes reflect those observed in experimental models of preterm delivery and in adults who were born preterm. The postnatal period appears to be a critical period of cardiac development and may offer a window for interventions to prevent long-term cardiovascular consequences of preterm birth.Preterm infants undergo a disproportionate increase in ventricular mass in the postnatal period, over and above what would be expected


Supplemental Digital Content is available in the text. Daily changes in aeroallergens during pregnancy could trigger early labor, but few investigations have evaluated this issue. This study aimed to investigate the association between exposure to aeroallergens during the week preceding birth and the risk of early delivery among preterm and term pregnancies.We identified data on 225,234 singleton births that occurred in six large cities in the province of Ontario, Canada, from 2004 to 2011 (April to October) from a birth registry. We obtained daily counts of pollen grains and fungal spores from fixed-site monitoring stations in each city and assigned them to pregnancy period of each birth. Associations between exposure to aeroallergens in the preceding week and risk of delivery among preterm , early-term (37–38 weeks), and full-term (≥39 weeks) pregnancies were evaluated with Cox regression models, adjusting for maternal characteristics, meteorologic parameters, and air pollution concentrations, and pooled across the six cities.w = 22.1 grains/m3) increase in weed pollen the day before birth among early-term  and full-term pregnancies . Exposure to fungal spores cumulated over 0 to 2 lagged days was associated with increased risk of delivery among full-term pregnancies only . We observed no associations among preterm deliveries.The risk of delivery increased by 3% per interquartile range width (IQRIncreasing concentrations of ambient weed pollen and fungal spores may be associated with earlier delivery among term births. However, no study has investigated this issue specifically.Short-term increases in aeroallergens have been associated with allergic and asthma exacerbations in the general population,15 Other studies found associations between warm season of births and reduced gestational length.17 However, these studies did not include measures of aeroallergens as possible factors affecting the duration of pregnancy.Fluctuation of aeroallergens has been closely related to climate, with increasing levels as warm seasons are expected to last longer. Although a number of studies have evaluated exposure to ambient temperature during pregnancy and the risk of earlier delivery, less is known about the potential impact of increasing levels of aeroallergens on pregnancy outcomes. Most studies showed an effect of exposure to high ambient temperature during the few days preceding delivery as possible factors affecting gestational length.20 Thus, this study aimed to evaluate the association between aeroallergen exposures during late pregnancy and the risk of early delivery among preterm and term births.Little is known regarding exposure to aeroallergens during pregnancy and global climate change will likely increase aeroallergen concentrations, such as pollen and fungal spores.http://www.bornontario.ca) to extract all singleton births occurring in six cities where an aeroallergen monitor was available  in the province of Ontario, Canada, from April 1, 2004 to October 31, 2011. This real time, internet-based system captures hospital births and home births in the province of Ontario and contains information on maternal and prenatal characteristics, health services, intrapartum interventions, and maternal and infant outcomes. Between 2004 and 2011, data capture for all births across the province of Ontario improved from an estimated 82% to 100% of births. In fact, a quality assurance project conducted in 2008 showed that 96% of all births in Ontario were captured in the birth registry.21 Missing births were due to a small number of hospitals and midwifery practices, spread across the province, that were not participating in the birth registry in the early years.We used the provincial birth record system BORN Ontario  were not considered events, but were included as fetuses at risk if they were at risk of preterm or term delivery during the risk period. This approach has been previously used in an article on maternal exposure to extreme heat for which the risk period, similar to ours, was limited to the summer months only.11 In addition, we had no information on birth outcomes for some women who were pregnant at the same time as participants of our study, but gave birth before the study started or after the study ended. To account for the noninclusion of these women, which has been described previously as the “fixed cohort bias,”25 we included only births with estimated conception dates ranging from 19 weeks  before the study started to 44 weeks  before it ended. The final sample consisted of 225,234 births plus an additional 145,762 fetuses born after October, but at risk during April to October. We evaluated the risk of early delivery among preterm and term births based on prior literature that used a similar analysis approach.25 Specifically, we investigated whether exposure to aeroallergens during the week before birth was a trigger of early delivery resulting in preterm birth or resulting in early-term birth for those at term. Preterm delivery was defined as gestational age < 37 weeks, early term as 37–38 weeks, and full term as ≥39 weeks of gestation. Gestational age was determined from the mother’s last menstrual period, so there is potential for misclassification of gestational age, but there is no reason to suspect that misclassification differed by aeroallergen levels.A time-to-event study design was used by including all fetuses at risk during the time period under study. This approach has been previously suggested to account for potential biases due to short-term variation in seasonal patterns of conception that cannot be accounted for in time series or case-crossover studies.Ethics approval for this study was granted by the Health Canada and the Public Health Agency of Canada’s (PHAC) Research Ethics Board, the Children’s Hospital of Eastern Ontario and the Ottawa Health Science Network Research Ethics Board.http://links.lww.com/EDE/B120). The number of airborne allergen particles per cubic meter (m3) of air sample was estimated by analyzing the particles adhering to the silicon grease-coated sample rods. Aeroallergens are expressed as the total counts of pollen grains per m3 and the total number of spores per m3. Technicians doing the measurements for aeroallergens are trained palynologists and quality control between technicians is done throughout the year. Exposure to aeroallergens during pregnancy was defined as the total counts of pollen grains or spores on the day of birth or any of the six preceding days . We restricted the exposure window to 1 week because there is little evidence that earlier exposures could directly trigger labor via an inflammatory process. To improve accuracy and precision of exposure measurement, we included only pregnant women whose residence was within 20 km of the closest monitoring station, which is consistent with prior literature assessing the health impacts of exposure to aeroallergens across cities.4 We also conducted a sensitivity analysis that included only pregnant women whose residence was within 5 km of the closest monitoring station. There were no changes in the location of the monitoring stations during the study period.We obtained daily airborne allergens data from April to October of each year from Aerobiology Research Laboratories, which operates one aeroallergen monitoring station in each city under investigation. The original purpose of starting aeroallergen monitoring was due to the demand by allergists and the public for this kind of information across Canada. For each city, samplers are located in neighborhoods that have been identified by Aerobiology Research Laboratories to be representative of the biota in a 50-mile radius and are at about 6–7 feet from the ground, which is assumed to be representative of the air that people are breathing. The vegetation surrounding the local collecting station will have an impact on the pollen and fungal spore levels. However, the mixing of the air in the atmosphere does minimize this to a degree. Therefore, all samplers are located away from structures and as far away from the local trees as possible. This is to ensure the air can freely mix the aeroallergens that are present and make the collections more representative. At each station, rotational impaction methods were used to obtain 24-hour averages of aeroallergens . A description of the dominant species/taxa for each type of aeroallergens is provided in eTable 1 . Because we did not have any individual-level socioeconomic status (SES) variables, we evaluated confounding SES by extracting three SES variables from the 2006 Canada Census at the dissemination-area (DA) level: median family income; proportion of population in the DA who are visible minority; and percentage of the adult female population who were 25–64 years old with a university degree. A dissemination area is a small geographical unit composed of one or more neighboring dissemination blocks, with a population of 400 to 700 persons. All of Canada is divided into dissemination areas. These area-based variables have been shown to be reasonable measures of neighborhood-level SES.2.5, NO2, and O3, which are also potential confounders, were obtained for each city through automated fixed-site monitoring stations from the National Air Pollution Surveillance (NAPS) network provided by Environment Canada. There were two air pollution monitoring stations in Ottawa, 12 in Toronto, two in Hamilton, two in London, two in Windsor, and one in Thunder Bay. Both meteorologic and air pollution factors were measured continuously and were captured for the week preceding birth based on previous research.25 We also included a time-dependent categorical variable for month/year of follow-up  to account for seasonality in the analysis. This approach is similar to a previous article investigating maternal exposure to aeroallergens, which adjusted for year and season of birth.9 In addition, a previous study evaluating effects of ambient heat on early delivery using a time-to-event approach used a similar adjustment to ours for seasonality and long-term trends.11 In addition, we included a natural cubic spline with four degrees of freedom for day of the year to remove residual confounding due to within-month variation.Daily mean temperature, relative humidity, and barometric pressure, considered potential confounders in the current analysis, were provided by Environment Canada using the average of measurements of all weather stations within each city. Daily average measurements of ambient air pollutants PM27 We obtained city-specific hazard ratios (HRs) with their 95% confidence intervals (CIs) to evaluate the effect of short-term exposure to aeroallergens during pregnancy on the risk of early delivery. For each gestational period, we calculated the HR associated with changes in aeroallergens on the probability of giving birth (compared with remaining pregnant). Similar to a previous study evaluating the relationship between high ambient temperature and risk of early delivery, we applied different censoring approaches depending on the gestational period.11 Specifically, when investigating the risk of early delivery during the preterm period, we censored pregnancies >37 weeks of gestation. For the risk of early delivery during the early-term period, we censored pregnancies ≥39 weeks of gestation and excluded births <37 weeks. We also evaluated whether aeroallergens could trigger delivery among full-term births and for this analysis we excluded births <39 weeks of gestation. In particular, a HR above one among preterm pregnancies represents an increased risk of preterm birth associated with changes in aeroallergens, while a HR above one during the early-term period represents an increased risk of early-term birth due to changes in aeroallergens. Finally, a hazard ratio above one among full-term pregnancies corresponds to an increased risk of early delivery due to the effect of aeroallergens. For each gestational period, we also censored pregnancies that delivered during November to March that were part of the pool of fetuses at risk from April to October.Our analysis consisted of two stages. In the first stage, we applied Cox proportional hazards models separately in each city to investigate the risk of early delivery in three gestational periods  using gestational age in days as the time scale.28 We accounted for the potential lagged effects, as well as for the potential nonlinear effect of daily mean ambient temperature, the linear effect of relative humidity and the linear effect of ambient air pollution on the hazard of birth using the distributed lag nonlinear methodology,27 A 6-day lag period was specified to evaluate the lagged effect of all environmental measures on the hazard of birth. All environmental factors were available as time-dependent variables for each day of gestation starting at week 19 until birth. The relationship between aeroallergens and the risk of early delivery is presented for an interquartile range width (IQRw) increase for each type of aeroallergen. We also modeled aeroallergens in categories based on quintiles, as did previous work investigating aeroallergens as an exposure.2 The lagged effect of aeroallergens was modeled with a natural cubic spline with an intercept and two internal knots placed at equally spaced values in the log scale. This represents the temporal change in risk after a specific exposure, and it estimates the distribution of immediate and delayed effects that cumulate across the lag period. We fitted the potential nonlinear effect of daily mean temperature over a week period on the hazard of birth using a natural cubic B-spline with three internal knots placed at the 10th, 50th, and 90th percentiles of city-specific temperature distributions.11 Relative humidity and air pollution variables were also evaluated on a continuous basis over a week period. The Akaike information criterion and Bayesian information criterion adapted to survival analysis were used for optimal model selection.27 Finally, in a second stage, we pooled the estimated city-specific overall aeroallergens-hazard of birth associations using a multivariate meta-analytical model.30For each location, we specified the aeroallergens-associated risk of early delivery relationship with a distributed lag linear model.2.5, NO2, and O3), weather variables, neighborhood-level factors, and maternal characteristics in the multivariable models using a backward deletion approach.31 This was accomplished by adjusting for all potential confounders and then removing the least significant confounding variables one by one in a stepwise manner as long as the total proportional change in the estimate compared with the fully adjusted model was less than 10%. We tested the proportional hazards assumption using aeroallergens-by-gestational age interaction terms. Analyses were performed with the R software, version 3.1.3, using packages dlnm, version 2.1.4, and mvmeta, version 0.4.5.We evaluated potential confounders such as markers of air pollution (PMhttp://links.lww.com/EDE/B120). Among them, 9% reported smoking during pregnancy, 8% had medical comorbidities, and the majority (55%) previously given birth. A total of 24,776 women were pregnant more than once over the period under investigation (data not shown). During the study period, total pollen concentrations ranged from 0 to 5,002.0 grains/m3 and total fungal spores from 0 to 108,241.1 counts/m3 (Table http://links.lww.com/EDE/B120). Weak correlations were observed between aeroallergens variables, except between total pollen and tree pollen . Less than 5% of days throughout the study period had missing information on aeroallergens.Among a total of 225,234 births, there were 14,064 preterm births, 56,897 early-term births, and 154,273 full-term births (Table http://links.lww.com/EDE/B120). Similar results were observed among full-term births, with increased HRs at lags 1  and 2 . In addition, increasing levels of fungal spores were associated with an increased risk of early delivery among full-term births at lags 1  and 2  per IQRw . No associations were observed among preterm deliveries.Lagged associations between each subtype of aeroallergens and the risk of early delivery among preterm, early-term, and full-term births are presented in Table w (22.1) increase among early-term  and full-term  pregnancies. Total pollen  and fungal spores  also were associated with increased risk of early delivery among full-term pregnancies when the effect was cumulated over 0 to 2 days. When restricting to births within 5 km of the monitors , all associations imprecise, but were all stronger in magnitude. Additional analyses based on quintile categories of aeroallergens showed that the highest categories (Q5) of exposure to total pollen and weed pollen were associated with early delivery among early-term births (Table http://links.lww.com/EDE/B120). For all overall effects calculated, there was no heterogeneity across cities. For instance, eFigure 1 (http://links.lww.com/EDE/B120) shows that estimated effects of weed pollen cumulated over lag 0 to 2 days among early-term pregnancies were similar in magnitude across each city . Aeroallergen-by-gestational-age interaction terms suggested that hazards were proportional over the preterm, early-term, and full-term periods.The cumulated estimated effect over lags 0 to 2 days showed that weed pollen was associated with increased risk of early delivery by 2% for every IQR34In this study, we investigated the risk of early delivery in association with increasing airborne pollen and fungal spores among a retrospectively created profile of exposure among pregnant women living in six large cities in the province of Ontario, Canada. We found that increasing levels of weed pollen were associated with increased risk of delivery among early- and full-term pregnancies and that total pollen and fungal spores were positively associated with the risk of delivery among full-term pregnancies. Findings of this study suggest that exposure to aeroallergens may shorten gestational length of pregnancies that reach term during the pollen season, but not enough to discernibly affect preterm birth rates. This study may have implications in reducing risks of early-term births as evidence is emerging regarding increased risk of childhood morbidity and mortality among those born early term compared with those born later term.16 Authors found that in studies conducted in the northern hemisphere, there was a relationship between season of birth and rate of preterm birth with a peak in January and another in June.16 However, the evaluation of the seasonality of birth is limited by the difficulty of disentangling the potential factors driving the risk of delivery. In addition, analyses based on seasonality of birth neglect that it is fetuses that are at risk and not only newborns. For instance, the annual pattern of birth may be affected by seasonal preferences in pregnancy planning or increases in coital frequency during holidays, which can affect the seasonal variation in the numbers of fetuses at risk of delivery.17 Therefore, it is difficult to directly compare this with previous studies investigating seasonality of birth.To our knowledge, this is the first study to investigate the association between specific types of aeroallergens and the risk of delivery. Previous studies have investigated mostly the association between season of birth, including pollen season, and birth outcomes. The relationship between season of birth and gestational length has been examined in a recent review.35 The latter is characterized by a balance between innate and adaptive immune cells between the fetus and the mother creating an anti-inflammatory environment which sustains pregnancy.10 Labor occurs when an inflammatory response is initiated through the secretion of cytokines/chemokines infiltrating reproductive tissues to create a proinflammatory microenvironment, which leads to delivery.37 It is likely that pregnant women sensitive to elevated levels of airborne pollen and/or fungal spores may suffer from allergy symptoms. Airborne pollen and fungal spores could act as a triggered stimulus of the immune system that could induce an earlier activation of cytokines/chemokines infiltrating reproductive tissues, eliciting a shift from an anti-inflammatory to a proinflammatory microenvironment and consequently earlier labor among term pregnancies. In fact, season of birth has been associated with a range of differences in cord blood cytokine response profiles, which may support our hypothesis.38The underlying mechanism by which airborne pollen and fungal spores may affect the risk of earlier delivery is still uncertain. One possible mechanism could be related to an immune response triggering labor following exposure to short-term fluctuations in pollen. During pregnancy, the fetus develops within the mother without succumbing to immunological rejection because of the establishment of a fetomaternal tolerance.39 Thus, our findings may reflect an adverse immune response among pregnant women who have seasonal allergies to weed pollen that triggers labor late in gestation. Consequently, women who have allergic responses to weed pollen may be more vulnerable to an adverse reaction to aeroallergens and consequently earlier delivery during pregnancy. However, in this study, we did not have information on allergy history and we could not stratify analyses by allergy status.Weed pollen was the only type of pollen associated with an increased risk of delivery among early- and full-term pregnancies. In fact, weed pollen is the most important cause of allergic rhinitis in the north-east part of North America and is likely responsible for 50% to 90% of seasonal allergies in this area.32 Another study showed increased risk of hospital admissions during childhood among infants born early term compared with those born later term.33 Thus, the triggering of labor by weed pollen resulting in early-term birth could carry lifelong consequences.In this study, weed pollen was associated with the risk of delivery among early-term pregnancies. The health impacts of children born early term are still not clear, but some evidence is emerging regarding long-term health impacts. For instance, a national cohort study in Sweden showed increased risk of mortality in infancy, early childhood, and young adulthood among early-term births compared with later-term births.This study has some limitations. First, we relied on fixed-site regional monitors to estimate personal exposures to ambient aeroallergens, weather variables, and markers of air pollution which may have resulted in exposure misclassification due to spatial heterogeneity. However, this misclassification was likely nondifferential and thus likely led to an underestimation of the effects. For instance, effect estimates, although imprecisely measured, were stronger when restricting analysis to births  within 5 km from an aeroallergens monitoring station. Second, while we included several important confounding factors, we cannot rule out potential residual confounding by unmeasured factors, such as individual ethnicity or socioeconomic status information. Third, we did not have information on pollen allergy status among pregnant women as those women might be more vulnerable to the effects of pollen on risk of earlier delivery. This is an important avenue for future research to investigate the potential modifying effect of this condition in the associations under investigation. Notable strengths of this study include the large sample size, the use of a distributed lag linear model in a time-to-event analysis to characterize fully the exposure–response relationship, the range of maternal risk factors to account for potential confounding, and the availability of ambient aeroallergens data across several regions.This study showed that increasing levels of ambient weed pollen were associated with risk of delivery among early- and full-term pregnancies and that increasing levels of fungal spores were associated with risk of delivery only among full-term pregnancies. If shown to be causal, these results suggest that certain types of ambient aeroallergens could trigger labor late in gestation. Future studies should be conducted to verify these findings.The authors thank Dr. Ann Sprague and Daniel Bedard for facilitating access to the BORN database.


Skin-to-skin contact (SSC) is an important factor to consider in the care of late preterm infants (born between 34 0/7 and 36 6/7 completed weeks of gestation). The literature suggests that SSC between preterm infants and their mothers facilitates breastfeeding. However, more studies are needed to explore potential dose-response effects between SSC and breastfeeding as well as studies that explicitly investigate SSC by fathers among late preterm infants. The aim was to investigate the duration of healthy late preterm infants’ SSC with the mother and father, respectively, during the first 48 h after birth and the associations with breastfeeding , clinical and demographic variables.This was an observational cohort study in which parents to healthy late preterm infants, born between 34 5/7 and 36 6/7 completed weeks of gestation, recorded duration of SSC provided by mother and father, respectively. Demographic and clinical variables were retrieved from the medical records and were used as predictors. Multiple linear regression analysis was used to assess the association between the predictors and the outcome, SSC (hours), separately for mothers and fathers.n = 64) and fathers (n = 64), was 14.7 (5.6) and 4.4 (3.3) hours during the first day (24 h) after birth and 9.2 (7.1) and 3.1 (3.3) hours during the second day (24 h), respectively. Regarding SSC with mothers, no variable was significantly associated with SSC during the first day, while the mean  time of SSC during the second day was 6.9 (1.4–12.4) hours shorter for each additional kg of birthweight (p = 0.014). Concerning SSC with fathers, the mean (95% CI) time of SSC during the first day was 2.1 (0.4–3.7) hours longer for infants born at night (p = 0.015), 1.7 (0.1–3.2) hours longer for boys (p = 0.033), 3.2 (1.2–5.2) hours longer for infants born by caesarean section (p = 0.003), and 1.6 (0.1–3.1) hours longer for infants exclusively breastfed at discharge (p = 0.040). During the second day, the mean (95% CI) time of SSC with fathers was 3.0 (0.6–5.4) hours shorter for each additional kg of birthweight (p = 0.014), 2.0 (0.5–3.6) hours longer for infants born during night-time (p = 0.011), 2.9 (1.4–4.4) hours longer if the mother was primipara (p < 0.001), and 1.9 (0.3–3.5) hours shorter if supplementary artificial milk feeds were given. None of the other predictors, i.e., mother’s age, gestational age, or induction of labor were significantly associated with infants’ SSC with mothers or fathers during any of the first two days after birth.The mean (standard deviation [SD]) time per day spent with SSC with mothers (Future studies are warranted that investigate duration of SSC between late preterm infants and their parents separately and the associations with breastfeeding and other variables of clinical importance. Late preterm infants, defined as infants born between 34 0/7 and 36 6/7 completed weeks of gestation , are to Skin-to-skin contact (SSC), the infant placed naked (with a diaper and with or without a cap) in skin-to-skin contact in a vertical position on the caregiver’s bare chest , is an iIt is important to distinguish between SSC for healthy infants and kangaroo mother care (KMC). Continuous or intermittent SSC is one of three components of KMC. The other two include exclusive or nearly exclusive breastfeeding and early discharge from hospital . The WorSo far, research has mainly focused on psychological and physiological effects related to early mother-infant SSC . HoweverTo summarise, there is a need for further studies that explicitly investigate SSC by fathers . AdditioThis was an observational cohort study in which parents to late preterm infants recorded duration of SSC provided by mother and father, respectively.Late preterm infants born between 35–36 completed weeks of gestation were screened for inclusion. Eligibility criteria included: Swedish-speaking parents of a healthy late preterm infant cared for in family rooms at the postnatal ward of Uppsala University Hospital in Uppsala, Sweden. For the purpose of this study, “healthy” means that the infant spent no time in the neonatal intensive care unit, i.e., had no respiratory morbidity, neonatal infection or feeding difficulties that required nasogastric feeds. Neither did the infant require active management for hypothermia (incubator) or support with intravenous fluids due to hypoglycaemia. Exclusion criteria included: multiple births, infants whose mother chose not to breastfeed and infants who needed neonatal intensive care.The proportion of infants born prematurely has been relatively constant during the past decades in Sweden, that is, nearly 5 percent of all single births. Approximately 4 percent are born between 33–36 completed weeks, whereas one percent is born between 22–32 completed weeks of gestation . ApproxiAt the time of the study, healthy late preterm infants born at ≥35 completed weeks of gestation were routinely cared for at the postnatal wards, i.e., together with term infants. Late preterm infants born <35 completed weeks of gestation were routinely cared for at the neonatal intensive care unit, and thus were excluded from the present study. Neonatologists directed the infants’ medical care plan at the postnatal wards, whereas a midwife working with a nurse assistant  provided the midwifery care. The midwife was responsible for providing care for the infant and the mother, including the detection of complications, the accessing of medical care, breastfeeding advice and support, as well as postnatal health counselling and education. The care was family centered, i.e., the majority of fathers/partners stayed together with the mother and the new-born baby in family rooms during the postnatal care period. Siblings had free visiting hours, but were not allowed to stay overnight. Mothers intending to breastfeed were offered individualised support from midwives to facilitate the establishment of lactation and exclusive breastfeeding. According to standard care, the infants were placed SSC on their mother immediately after birth to enable the occurrence of the first breastfeed as soon as possible. If the mother was restricted due to medical reasons from holding the baby after birth, the infant was placed SSC with the father/partner. Health professionals recommended SSC between the infant and the mother/father, i.e., to hold the baby naked (with a diaper and with or without a cap) in the kangaroo position  on the cEthical approval to conduct the study was obtained from the Regional Ethical Review Board in Uppsala, Sweden .See During the inclusion period, between the 15th of November 2010 and the 30th of November 2011, parents of 86 late preterm infants agreed to participate in the study. However, five infants were excluded as they were referred to the neonatal intensive care unit and parents of 17 infants did not complete the data collection. Thus, parents of 64 (61%) late preterm infants completed the data collection. Forty-three infants were born between 36 0/7 and 36 6/7 completed weeks of gestation, 20 infants between 35 0/7 and 35 6/7 and one infant was born at 34 5/7 completed weeks. Due to the real life design of the study, no power calculation was performed.Potential participants i.e., late preterm infants cared for in family rooms at the postnatal wards, were identified through the log book at the hospital’s maternity ward. The book includes name of the ward where the mother/infant were cared for after birth, the mother’s name, Swedish identification number, address, date of birth and completed weeks of gestation. Written and oral information about the study was given to parents by the authors EM, ELF, and HV within 24 h after birth at the postnatal ward. Parents who agreed to participate and completed a written informed consent form began completing the diary, one chart per day, commencing from the time they gave their consent, during the infant’s stay at hospital. Data from the time of birth to the initiation of study participation were entered retrospectively in the diary by the parents. When the family was discharged from the hospital, the diary was left in a special study mailbox at the postnatal ward. One of the authors (EM) retrieved the study variables from the medical records.n = 2), vacuum extraction (n = 2), manual extraction of placenta (n = 1), large perineal tears (n = 1), and postpartum bleeding >1,000 ml (n = 1) were assessed but were not used in the analyses due to too few observations to perform any relevant statistical analyses. Demographic and clinical variables were chosen on the basis of the available literature at the time of the study , respectively, after birth and the associations with breastfeeding, clinical and demographic variables.n (%), for categorical variables and by means and standard deviations (SD), supplemented by median and range, for continuous variables. Linear regression analyses were used for assessing the associations between the predictors and the outcome, separately for mothers and fathers. Three kinds of regression models were used: (i.) the unadjusted model, using each predictor in separate regression models; (ii.) the full model, including all predictors simultaneously in the same regression model; and (iii.) the full model, using a backward elimination approach starting with the full model and step-by-step deletion of the variable with the highest p-value, re-estimating the model and deleting the variable which now had the highest p-value, and so on, with the process continuing until only variables with p-values <0.20 remained in the model  age was 30.5 (19–40) years. Thirteen mothers (20.3%) gave birth by caesarean section, of which two (3.1%) were elective. The median (range) of infants’ gestational age was 36 2/7 (34 5/7–36 6/7) weeks. No infant had an Apgar score below 7 points at 5 min after birth.n = 64) was 14.8 h (range 0.0–24.0) and 4.3 h (range 0.0–14.0) with fathers. The second day the corresponding figures were 9.0 h (range 0.0–24.0) SSC with mothers and 2.5 h (range 0.0–13.0) SSC with fathers.During the first day after birth, the infants’ median time spent SSC with mothers , i.e., infants with lower birthweights had more SSC than infants with higher birthweights  of 6.9 (1.4–12.4) hours shorter SSC for each additional kg of birthweight for the trimmed model (hweights .p = 0.015), 1.7 (0.1–3.2) hours longer SSC if the infant was a boy (p = 0.033), 3.2 (1.2–5.2) hours longer SSC if the infant was born by caesarean section (p = 0.003), and 1.6 (0.1–3.1) hours longer SSC if the infant was exclusively breastfed at discharge (p = 0.040). During the second day, the infant’s birthweight, time point of birth, parity, and if supplemental feeding were given, were all significantly associated with SSC with the father in the trimmed model, with a mean (95% CI) of 3.0 (0.6–5.4) hours shorter SSC for each additional kg of birthweight (p = 0.014), 2.0 (0.5–3.6) hours longer SSC for infants born at night (p = 0.011), 2.9 (1.4–4.4) hours longer SSC if the mother was primipara (p < 0.001), and 1.9 (0.3–3.5) hours shorter SSC for infants given supplementary artificial milk feeds (p = 0.024)  of 2.1 (0.4–3.7) hours longer SSC for infants born at night (= 0.024) .None of the following variables were associated with infants’ SSC with mothers  or fatheThis is one of the first studies that explicitly investigate duration of SSC between healthy late preterm infants and their mothers and fathers separately and its associations with breastfeeding, clinical and demographic variables.Breastfeeding at discharge was not associated with the extent of infants’ SSC with mothers during any of the first two days after birth. However, all mothers in our study were breastfeeding to some extent at hospital discharge, 47% exclusively. Research regarding dose-response effect between duration of SSC and breastfeeding is ambiguous . In cont, with longer SSC if the infant was born by caesarean section. Following caesarean birth, early SSC between mother and her newborn infant may be limited due to practical and medical safety reasons for both infant and woman  did not complete the data collection about SSC. It could be speculated that these parents found the documentation in the diary complex and time-consuming. Another aspect to consider is that the data regarding SSC were based only on self-reports and that data from birth to study initiation was reported by the parents retrospectively. Our choice of data collection in the present study, i.e., the diary, was based on a previous study concluding that parents are able to document variables related to SSC of their infant in a complete and reliable manner . FinallyThe present study raises questions regarding dose-response effects between duration of SSC and breastfeeding. Exclusive breastfeeding at discharge was significantly associated with infants’ duration of SSC with fathers during the first day after birth, whereas no associations were found with mothers. Future studies are warranted that investigate duration of SSC between late preterm infants and their parents separately and its associations with breastfeeding. Taken together, a gender perspective needs to be implemented in paediatric research as the inclusion of mothers alone or parents as a couple may hinder firm conclusions regarding variables of importance for the infant’s care.10.7717/peerj.3949/supp-1Data S1Dataset for late preterm infants.Click here for additional data file.


There are considerable inconsistencies in the results of Haraux et al. . For oneAnother unusual feature is that the univariate odds ratio of 1.8, (or 0.74), for hair cosmetics changes to an OR of 6.11 on multivariate analysis. For occupational exposure to EDCs, the univariate OR of 3.1 changes to 9.64 in the multivariate analysis. Such a large change is unusual and shows severe negative confounding; it would be interesting to know the responsible confounders. In a similar study of hypospadias and occupational exposure to hair sprays, the univariate and adjusted ORs were similar, 2.30 and 2.39, with both studies adjusting for birth weight and folate intake [The participation rates in this study are undocumented, and the results depend on questionnaires completed by the parents, presumably in the hospital environment, shortly after birth, when anxiety about the child would be high and the potential for differential responses considerable.