Smoking during pregnancy is related to fetal constraint and accelerated postnatal growth. However, the pathways between these factors have not been clarified. Pathway analyses that link these factors can help us better understand the mechanisms involved in this association. Therefore, this study aimed to examine pathways between maternal smoking during pregnancy and growth in infancy.Participants were singletons born between 1993 and 2006 in rural Japan. The outcome was the change in weight z-score between birth and 3 years of age. Pathways from maternal smoking and other maternal factors  to growth in infancy via birth factors  and breastfeeding were examined using structural equation modeling.P = 0.002). Taking all pathways into account, the standardized total effect of maternal smoking on growth in infancy was 0.11.Complete data were available for 1524 children (775 boys and 749 girls). The model fit appeared adequate. Lower birth weight and non-exclusive breastfeeding mediated the association between maternal smoking during pregnancy and rapid growth in infancy. Maternal smoking was also directly linked to rapid growth in infancy (standardized direct effects 0.06, Maternal smoking during pregnancy may both indirectly, through birth weight and breastfeeding status, and directly influence growth during infancy; however, there may be other pathways that have not yet been identified. Therefore, numerous studies have explored the factors that may influence growth during this period.Appropriate growth during infancy, particularly the rate of growth, is critical to health in later life. Systematic reviews have consistently identified an association between rapid weight gain in infancy and later obesity.6 in addition to birth and early-life factors such as birth weight,7 gestational age, and feeding methods.8 However, these factors may also be interrelated. In addition, birth factors might mediate the effect of parental factors. These maternal and early-life factors work together to influence growth in infancy. To better understand the effect of each factor and the potential interactions among factors, we attempted to identify the pathways between maternal factors, birth and early-life factors, and growth in infancy using pathway analysis.Relationships have been reported between growth in infancy and maternal factors such as maternal smoking during pregnancy,9 However, information on mechanisms through which maternal smoking during pregnancy affects fetal and postnatal growth is lacking; further, other factors that may influence these relationships have not been thoroughly studied. Therefore, in this study, we examined the pathways between maternal smoking during pregnancy and offspring growth in infancy after controlling for other factors that could also influence growth during infancy.Among these factors, maternal smoking during pregnancy has been reported as a major cause of fetal undernutrition and has been shown to be related to later obesity.10,11The study participants comprised children born between April 1, 1993, and March 31, 2006, in the Enzan area of Koshu City, Yamanashi Prefecture, Japan. The participants were from Project Koshu, which is a dynamic Japanese community-based prospective cohort study. Project Koshu is an ongoing study that started in 1988, in which all expectant mothers who responded to a survey during the obligatory visit at the city office for the registration of pregnancy were recruited into the cohort. Their children were followed from birth onwards. Further details of the project have been reported elsewhere.This study was approved by the ethics review board of the University of Yamanashi’s School of Medicine and was conducted in accordance with the Guidelines Concerning Epidemiological Research . The Koshu City administrative office collaborated with the authors of this study.Maternal characteristics and early-life factors were collected from participants’ mothers using a self-reported questionnaire at their pregnancy registration, and interviews were conducted at follow-up by public health nurses in Koshu City.12 Growth in infancy was assessed using the change in weight z-score from birth to 3 years of age. We defined rapid growth in infancy according to the standard proposed by Ong et al,7 who indicated that a change in weight z-score exceeding +0.67 between birth and 2 years of age by gender defines rapid growth in infancy. As indicated by Ong et al, z-scores of 0.67 represent the width of each percentile band on standard growth charts ; thus, a weight gain exceeding +0.67 SD indicates an increase across at least one of these centile bands.7 In our analysis, we used the same cut-off value but a longer age period (birth to 3 years).Participants’ anthropometric data were collected from their birth registration and medical check-up at 3 years of age. Weight z-score /standard deviation [SD]) at birth and at 3 years of age were calculated by gender. Means and SDs were calculated based on all available data from the study sample. Distribution of weights in this dataset was similar to the data of National Growth Survey on Preschool Children in 2000.2, 18.5–24.9 kg/m2, or ≥25 kg/m2), maternal work status (working or not working), maternal smoking habit and alcohol consumption during pregnancy (current smoker/drinker or does not smoke/drink), maternal education level , and paternal education level . Participants’ early-life factors included birth weight (<2500 g or ≥2500 g), gestational age (<37 weeks or ≥37 weeks), primiparity (1st birth or other), and breastfeeding during the first 3 and 6 months (exclusive breastfeeding or non-exclusive breastfeeding). The mother provided information on breastfeeding every month from delivery. Exclusive breastfeeding throughout the first 3/6 months was defined as exclusive breastfeeding during the first 3/6 months.We used SAS version 9.3  for statistical analysis. We used analysis of variance to compare anthropometric indicators at birth and 3 years of age by groups classified according to change in weight z-score. We then examined the crude associations between parental/early-life factors and rapid growth in infancy using logistic regression analysis. The parental factors included maternal age , maternal height , maternal body mass index . Included independent variables were maternal smoking during pregnancy, maternal work status, maternal height and maternal BMI; the mediators were birth weight , gestational age , primiparity, and breastfeeding during the first 3 months. Breastfeeding during the first 6 months was not used for analysis because of the large proportion of missing data. The association between the independent variables and the pathways that linked the independent variable to the outcome were determined using an explorative model.A structural equation model was constructed to examine the pathways between maternal smoking during pregnancy and growth in infancy using AMOS 7.0 . The complete-case dataset was used for this analysis. The outcome was growth in infancy (continuous); the independent variables were determined according to the results from the univariate logistic regression analysis above  had complete information on maternal and early-life factors. As shown in Table 2 vs. 20.8 kg/m2, P = 0.4; maternal height 157.5 cm vs. 157.9 cm, P = 0.1; maternal working 45.0% vs. 48.7%, P = 0.1; and maternal smoking during pregnancy 7.9% vs. 5.8%, P = 0.06) were found between these 774 cases and the 1524 cases included in the pathway analysis.Overall, 774 cases from the original dataset were excluded from the pathway analysis due to lack of information on maternal factors (262 cases), birth characteristics (9 cases), or information during infancy (183 cases). No significant differences in maternal factors , primiparity, non-exclusive breastfeeding (for the first 3 and 6 months), high maternal height, maternal working, and maternal smoking during pregnancy were significantly associated with rapid growth in infancy Table .P = 0.002). Taking all of the pathways into account, the standardized total effect of maternal smoking on infancy growth was 0.11.The model fit in the pathway analyses was adequate . The pathways determined using the exploratory methods, in addition to the standardized regression coefficients and significance, are depicted in 10 we also carried out a sub-analysis stratified by gender. However, we did not find a significant gender difference in the association between maternal smoking and infant growth (data not shown).In this study, we examined the association between growth in infancy and maternal, birth, and early-life factors. We also explored pathways between maternal smoking during pregnancy and rapid growth in infancy. The results suggested that maternal smoking during pregnancy might be related to accelerated growth in infancy both directly and indirectly through fetal development and early breastfeeding practices. Since previous studies indicated a gender difference in the association between maternal smoking and childhood BMI trajectories,7 Another study by Regnault et al demonstrated that fathers’ heights and BMI as well as their children’s gestational age and feeding methods were related to weight-growth velocity at three months of age.8 Further, a study by Nafstad indicated that weight gain during the first year was slower in breastfed children than in those who were not breastfed and faster in children of smoking mothers than those of non-smoking mothers.6 Finally, a study by Baird also indicated that infants who were breastfed gained weight more slowly.13 These results were similar to those of the present study. However, since these factors were correlated to each other in previous studies, it was difficult to determine the separate contribution of each factor.Previous studies have discussed factors related to rapid growth in infancy. Ong et al reported that children with rapid growth during infancy had lower birth weight and length and were more likely to be the first child than children without rapid growth during infancy. Further, their mothers were more likely to smoke during pregnancy.14 and low birth weight followed by rapid growth in infancy.15 It has been reported that maternal smoking during pregnancy affects fetal development by influencing uterine, umbilical, and fetal cerebral artery blood flow.16 Further, catch-up growth occurs in most low-birth-weight infants and is likely to result in childhood obesity.7 Birth order may also influence the association between maternal smoking and growth in infancy. Previous studies have confirmed the association between birth order and growth in childhood.17,18 On the other hand, reviews have demonstrated that women who smoke are less likely to breastfeed and breastfeed for a shorter duration than nonsmokers.19 Both psychosocial and biological factors are responsible for these associations. Smoking may suppress prolactin secretion and thereby reduce breast milk volume.20 Further, formula-fed infants have higher protein intake than breastfed infants, which may stimulate the secretion of insulin. Therefore, these infants may gain weight more rapidly due to the promotion of growth by insulin.21,22Results of pathway analyses showed that lower birth weight and non-exclusive breastfeeding were both mediators of the link between maternal smoking during pregnancy and increased growth in infancy. Primiparity was also related to the pathway. These findings are consistent with previous studies, which indicate that maternal smoking during pregnancy is associated with constrained fetal growth23 but was significantly associated with offspring being overweight and obese at 5 years of age.11 Therefore, we speculated that the characteristics of skipping breakfast and smoking during pregnancy may share similar mechanisms. On the other hand, differences in lifestyle and child rearing customs might also explain the differences in infant outcomes between mothers who smoke during pregnancy and those who do not. A study by Rittmueller et al indicated that smokers had higher caloric intake and lower dietary quality than non-smokers.24 Another study by Papoz et al showed that infants with mothers who smoke had higher caloric intake than those with non-smoking or formerly smoking mothers.25In addition to these indirect pathways through birth weight and breastfeeding status, maternal smoking during pregnancy also contributed directly to infant growth in our model. However, the mechanism for the direct effect was unclear. One possible explanation for this pathway is that maternal smoking during pregnancy results in a fetal biological change that has a relatively small effect on fetal size during pregnancy and a larger effect on growth after birth. Two other analyses using the same Project Koshu data demonstrated that the maternal habit of skipping breakfast during pregnancy did not influence the birth weight of the offspring26 and a higher prevalence of rapid growth in early infancy.27 Therefore, low SES might account for the effect of maternal smoking on growth in infancy, at least in part. To clarify this, further studies are needed. Second, when discussing the influence of maternal age on infant growth, we used age categories based on 25th and 75th percentiles. In practice, teenage mothers are more likely to smoke and more likely to give birth to low-birth-weight infants. However, few mothers in this study were teenage mothers. Therefore, it was not possible to clarify the effect of teenage pregnancy in this study. Third, the pathway analyses were conducted using the complete-case dataset, and the proportion of participants with complete data was relatively low (66%). Nevertheless, we compared the characteristics of the participants in the complete dataset to those of the dropped cases. Smoking prevalence was slightly higher among dropped cases than among included cases, but the difference was not significant. Finally, the number of smoking mothers was relatively small because the sample size of this study was not very large and the prevalence of maternal smoking during pregnancy was low. Therefore, findings from this study need further confirmation from larger studies.This study has certain limitations. First, this model may not contain all of the possible pathways between maternal smoking during pregnancy and infant growth. Some factors that were not included in the model might influence the association between maternal smoking during pregnancy and infant growth. Women who smoke during pregnancy may have other biological or personality characteristics that may influence infant growth when compared to nonsmokers, such as socioeconomic status (SES). Low SES is likely to be associated with higher smoking prevalence in mothers28 In this study, however, we were unable to differentiate ‘appropriate’ from ‘inappropriate’ catch-up growth.Of note, while rapid growth in infancy is strongly associated with childhood obesity, ‘appropriate’ catch-up growth in preterm small-for-gestational-age children has certain benefits.In conclusion, maternal smoking during pregnancy may both indirectly, through birth weight and breastfeeding status, and directly influence growth during infancy; however, there may be other implicated pathways that have not been identified yet.Abstract in Japanese.


However, numerous circulating cytokines were raised such that their combined, but not individual, elevation was significantly associated with severity of disease, and these may be further indicators of, and contributors to, the systemic inflammatory manifestations of COPD. The coelevation of numerous circulating cytokines in COPD is consistent with the insidious development, chronic nature, and systemic comorbidities of the disease.Inflammatory biomarkers, including cytokines, are associated with COPD, but the association of particular circulating cytokines with systemic pathology remains equivocal. To investigate this, we developed a protein microarray system to detect multiple cytokines in small volumes of serum. Fourteen cytokines were measured in serum from never-smokers, ex-smokers, current smokers, and COPD patients (GOLD stages 1–3). Certain individual circulating cytokines (particularly TNF Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. It is induced mainly by tobacco smoking, which leads to chronic, irreversible lung inflammation; both cellular and humoral immunological processes are involved in the pathology –6. A sigCytokines are not disease-specific biomarkers, as they may be raised for many different reasons , which may be subclinical; therefore, some never-smokers with no overt clinical disease may have raised cytokines, as well as smokers and COPD patients. This is one of the reasons why it is difficult to identify particular individual circulating cytokines as disease-specific markers for COPD.Some previous studies have investigated a broad range of systemic cytokines in COPD –14, but Serum was obtained from 57 patients with confirmed COPD, 25 ex-smokers without COPD (ExS), 9 current smokers without COPD (CS), and 11 never-smoker (NS) healthy controls. Subjects were recruited from volunteer databases and respiratory out-patient clinics and by advertisement. All subjects were over 40 years of age, of European descent, had a significant smoking history of greater than 10 pack years, and were studied at clinical stability . All subjects gave written informed consent and the study was approved by the National Research Ethics Committee . No subject recruited had active or suspected malignancy, terminal disease, or known alpha-1-antitrypsin deficiency. Samples were collected and stored at −70°C until used. The demographics of the groups are shown in μL of appropriately diluted DuoSet biotinylated detection antibody  were added, followed by streptavidin-HRP . The slides were washed and 50 μL of Bio-Rad Amplification Reagent (Opti-4CN amplification kit) was added for 10 minutes in the dark. The slides were washed extensively with 20% DMSO-PBST and with wash buffer, followed by the addition of streptavidin-conjugated cy5 . The slides were scanned with a 4200 AL microarray scanner at 635 nm (Axon GenePix®). Fluorescence was quantified using the GenePix Pro Software (Axon GenePix). The median fluorescence of each spot was measured (minus background) and the corrected fluorescence was used to calculate the average fluorescence signal across the standard detectable ranges.Serum cytokine concentrations were determined by protein microarray, as described in detail previously in this journal . BrieflyThe data were not normally distributed and therefore multiple comparisons between groups were made using the Kruskal-Wallis test for nonpaired data (5 comparisons) and the Friedman test for paired data (13 or 14 comparisons); in both cases Dunn's post hoc test for multiple comparisons was applied. Statistical analyses were performed using GraphPad Prism 6 .The demographics of the subjects who donated serum for this study are shown in Fourteen cytokines were measured in serum from never-smokers , ex-smokers without COPD , current smokers without COPD , and COPD patients at GOLD stages 1–3  by protein microarray . [One NS∗), ExS (§), and CS (†) groups (Friedman test with Dunn's post hoc test) ( per se (in the CS subjects), as well as the effects of the severity of COPD (in the S1–S3 subjects), on serum levels of the 14 cytokines studied.The majority of the COPD patients at all GOLD stages were ex-smokers (COPD-Ex) and a minority were current smokers (COPD-C). However, as shown in oc test) . This inα  and CS (†) (Friedman test with Dunn's post hoc test).The solid bars on the graphs in γ, IL-6 and TNFα in S2 and S3; and for IL-17 in S3 only.This generalised increase in serum cytokines was further investigated by identifying subjects with serum cytokine concentrations higher than the 50th percentile  concentrations seen in the NS control group, which are shown for each cytokine by the dotted lines in the graphs in α, and VEGF [α and IL-1β) to be significantly elevated in concentration in the serum of some COPD patients (and some current smokers not diagnosed with COPD), and these may serve as markers of particularly significant systemic inflammation. However, we also found a broad increase in the serum concentration of numerous circulating cytokines that was collectively significant , but without a sufficiently great increase in concentration across all patients being statistically significant for individual cytokines. Hence, this indicates the value of detecting numerous cytokines rather than just a few. The serum levels of this collection of cytokines correlated with the severity of COPD (GOLD stages 1–3), and they may be further indicators of, and contributors to, the systemic inflammatory manifestations of COPD.Our findings are consistent with those of others who have shown elevated levels of serum cytokines in COPD, particularly with regard to IL-6, IL-8, TNFand VEGF , 18, 20.The broad, relatively subtle, elevation of multiple circulating cytokines is also consistent with the insidious development and chronic nature of COPD. Indeed, this pattern of moderate, but persistent cytokine elevation may also occur in other chronic conditions, including metabolic syndrome and cardiovascular disease, which are also associated with COPD , 27. per se, particularly, IFN-γ, TGFβ, IL-8, and MCP-1. Secondly, most of the cytokines studied are elevated in COPD patients, particularly in S2 and S3; this applies to all except IL-4, eotaxin-1, and eotaxin-2 . Thirdly, only VEGF is elevated in all three stages of COPD (S1–3) relative to CS. This is consistent with other reports of the particular importance of VEGF in COPD and the fact that hypoxia is a known inducer of VEGF [β appears to be suppressed in CS and S1. At first site, this appears surprising given that IL-1β (together with TNFα) was the best discriminator of patients with cytokine levels >95th percentile of the NS group. However, this is because CS and COPD subjects tend to have either low levels of IL-1β or high levels (above 95th percentile of NS), rather than an even spread  in certain individual subjects, were not generally raised in the CS or COPD groups. This might be because these cytokines are associated with particular disease manifestations rather than the generalised, systemic, low-grade inflammation associated with COPD. Our study suggests that the latter phenotype is more associated with IFN-γ, IL-17, MCP-1, IL-8, VEGF, IL-6, and TNFα from amongst the cytokines we measured.A more detailed analysis of the trends in cytokine expression represented in Figures  of VEGF –30. Proi of VEGF . FinallyThe findings of the present study can be put into the context of “systems biology” of disease processes, in which a combination of technological and analytical advances has facilitated understanding of the complexity of factors that contribute to pathogenesis and phenotype of diseases. This, in turn, has led to the realisation that multiple, interacting factors can have a significant impact on disease development in combination, but not individually. Examples in other types of disease include the subtle, constitutive activation of multiple proinflammatory signalling pathways in certain autoinflammatory disorders  and pattIn conclusion, numerous cytokines are raised in the circulation of COPD patients such that the combined, but not individual, elevation of these cytokines is significantly associated with severity of disease. Furthermore, these cytokines may be indicators of, and contributors to, the systemic inflammatory manifestations of COPD and the pattern of multiple serum cytokines may be more informative of systemic aspects of the disease than individual cytokines. The coelevation of numerous circulating cytokines in COPD is consistent with the insidious development, chronic nature, and systemic comorbidities of this disease.


BCG immunogenicity in infants differs between populations and these differences have been attributed to various factors. In this study, the influence of geographical location, season of birth, timing of vaccination, micronutrient status (zinc) and inflammatory status  were assessed.M. tuberculosis PPD, using a multiplex bead assay. Results were correlated with the plasma zinc and CRP concentrations at the time of sampling, and with interview and household data. BCG vaccinated infants were recruited in Malawi, The Gambia and the UK.Immunogenicity was assessed by cytokine signature in culture supernatants from diluted whole blood samples stimulated with In Malawi, infants vaccinated within the first week after birth showed lower production of most cytokines measured than those vaccinated later. The number of cytokines showing significant differences between Malawian and Gambian infants decreased after adjusting for season of birth. In Malawi, a proportion of infants had zinc deficiency and elevated plasma CRP (>10 mg/L), but neither zinc deficiency nor high CRP was associated with production of any of the cytokines measured.M. tuberculosis PPD in infants at 3 months post BCG vaccination were affected by geographical location, season of birth, and timing of vaccination but not associated with the concentration of plasma zinc or inflammatory status. These factors should be considered in future trials of new TB vaccines.The cytokine/chemokine signatures observed in response to  Mycobacterium tuberculosis (M. tb) and is one of the major global health challenges with 8.6 million incident cases worldwide [Tuberculosis (TB) is caused by orldwide . Despiteorldwide , the varorldwide -4 highliMicronutrient deficiencies may impact on this immune response. Zinc has been classified by WHO as a “problem” micronutrient which requires supplementary provision to breast-milk fed infants from about 6 months of age . Zinc deInflammation, as a result of infection, may also influence the immune response following vaccination. C-reactive protein (CRP) is an acute phase protein which is released from the liver. It exists normally in trace levels in serum and rapidly increases in response to a variety of infections and inflammatory conditions . QuantitM. tb purified protein derivative (PPD) in Malawian infants compared with UK infants [M. tb PPD were analysed.Previous immunology studies undertaken at the Karonga Prevention Study (KPS) to compare the immune responses between Malawi and the UK, following BCG vaccination, showed different IFN-γ responses to mycobacterial antigens in adolescents and infants in these two populations. This was regardless of the extent of exposure to environmental mycobacteria, which has been the dominant hypothesis for interpreting variability in BCG vaccine-induced protection ,14,15. T infants -8. In adSamples from studies assessing immune responses following BCG vaccination in infants from Malawi ,16, The M. tb PPD (5 μg/mL), PHA (5 μg/mL) and the control (RPMI medium only) to examine the IFN-γ response to mycobacterial antigens [M. tb PPD, the cytokine response data from UK infants from a previous study of post BCG immune responses were used [Infants were vaccinated with BCG within the first week of life in Malawi  ,16 and Tantigens . For theere used . The BCGere used ,17.Haemophilus influenzae b (Hib), hepatitis B (HBV) and inactive polio vaccines (IPV) [In Malawi, 63 plasma samples from the infants at 3 months post BCG vaccination were tested for plasma zinc and CRP. Among the 403 infants vaccinated within the first week of life , those wes (IPV) , infantsEthical approval for the studies was obtained from the London School of Hygiene & Tropical Medicine Ethics Committee, the National Health Sciences Research Committee in Malawi and the Gambian Government/Medical Research Council (MRC) Joint Ethics Committee; permission to export samples was granted by the National Health Sciences Research Committee in Malawi and by the Gambian Government/MRC Joint Ethics Committee in The Gambia.MAP Kit, Millipore, Billerica, MA, USA) as described previously [R 100 System .Concentrations of 42 cytokines and chemokines: IL-1α, IL-1ra, IL-1β, IL-2, sIL-2Rα, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IFN-α2, IFN-γ, TNF-α, TNF-β, sCD40L, MIP-1α (CCL3), MIP-1β (CCL4), Gro-α (CXCL1), IL-8 (CXCL8), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MDC (CCL22), TGF-α, G-CSF, GM-CSF, IL-3, IL-7, Eotaxin, FGF-2, Flt-3 L, Fractalkine (CX3CL1), EGF, VEGF, PDGF-AA, PDGF-AB/BB and RANTES (CCL5) were determined in culture supernatant samples obtained from 30 Malawian and 24 Gambian infants. Multiplex beads were diluted 1 in 2 in bead diluent and the 42plex bead assay was performed according to the manufacturer’s protocol  were derived from the same clones. Samples from both Malawi and The Gambia were transported to LSHTM for testing.The same protocol for the 42 multiplex bead assays was used for testing all samples, although the assays were performed at different times. The antibodies for all 42 cytokines in all the kits used , 50 μL of diluted plasma samples (1:5) and sample blanks (50 μL sample + 2 μL EDTA) were transferred into the wells of a clear flat-bottom 96 well plate and 200 μL of working solution (200 μl of “reagent A” and 4 μl of each “reagent B” and “reagent C”) was added. After incubation for 30 minutes at room temperature, optical density was measured at 425 nm using a Spectramax M3 plate reader .Zinc concentration was measured in 63 Malawian infant plasma samples by a zinc colorimetric method. A QuantiChrom™ zinc assay kit  was used according to the manufacturer’s protocol. Briefly, 100 μL of 5 standards . Plates were read at 450 nm with a reference wavelength of 490 nm using a Dynal plate reader .Plasma CRP concentrations were measured by sandwich ELISA using human anti-CRP IgG , HRP-conjugated anti-human CRP protein  and human CRP standard serum . Plasma samples were diluted 1:200. Substrate solution was prepared by adding 500 μL of 10 mg 3,3′,5,5′-tetramethylbenzidine dissolved in DMSO (Sigma-Aldrich) into the 50 mL of substrate buffer  values.M. tb PPD was examined in Malawian and Gambian infants. The Gambian infants vaccinated at birth showed more than twice the median concentration of IFN-γ, IL-1α, IL-1ra, IL-6, TNF-β, TGF-α, IL-12p70 and RANTES in response to M. tb PPD compared to Malawian infants vaccinated at birth (P < 0.05)   Figure B. The meM. tb PPD, the cytokine/chemokine signatures were compared between Malawian infants with early vaccination (within 1 week of birth) and a group of Malawian infants from the same cohort, studied previously, who had received BCG vaccination late (between 3 and 11 weeks of life) [To determine the effect of age at vaccination on immune responses to of life) . IFN-γ pM. tb PPD was higher in infants born in the dry season (P < 0.001) while the plasma concentrations of zinc and CRP were unaffected by season of birth in Malawi (P > 0.05) . Looking at the correlation between plasma CRP concentration and the cytokine/chemokine signatures observed in 17 Malawian infants who had samples for testing of both culture supernatant and plasma, there was no evidence that any cytokine or chemokine induced by M. tb PPD was associated with plasma CRP concentration .Plasma concentrations of zinc and CRP were measured to determine zinc deficiency and the degree of inflammation in Malawian infants, and to investigate possible associations with season of birth and cytokine/chemokine signatures. Zinc deficiency (< 65 μg/dL) was shown in 9.5% (6/63) of Malawian children. Eight of 57 Malawian infants showed plasma CRP levels greater than 10 mg/L, while 2 infants had CRP levels greater than 100 mg/L Figure . The IFN) Figure  althoughin vitro stimulation, and not with PPD or MTB antigens [M. tb PPD between Malawi and The Gambia may be due to season of birth rather than to the BCG vaccine strains.This study focused on the evaluation of infant immune responses at 3 months post BCG vaccination, investigating potential factors that may affect cytokine/chemokine signatures in infants from different countries and vaccinated at different ages. It has been reported that variation in resistance to TB and efficacy of BCG may be influenced by latitude dependent factors such as climate, exposure to nontuberculous mycobacteria and genetics ,21. Immuantigens . In addiantigens . In our M. tb PPD for most cytokines and chemokines measured but not for IFN-γ. A similar finding of varying BCG immunogenicity by age at vaccination was reported in South African infants, showing that enhanced BCG-specific T cell responses were observed in infants when vaccination was delayed from birth to 10 weeks [+ T cells were detected in the group vaccinated at birth compared with those vaccinated at 6 weeks of age in Uganda [The cytokine/chemokine signatures observed were influenced by the timing of BCG vaccination. In Malawi, when compared with infants vaccinated early, the infants vaccinated later (a median age of 5 weeks) showed higher responses to 10 weeks . However10 weeks  and Ugan10 weeks  showed c10 weeks , whereasn Uganda .M. tb PPD than infants vaccinated at birth. This observation suggests that different immune signatures develop according to timing of vaccination which might be an important factor in relation to the induction of protective immune responses following vaccination. The samples obtained from early and late BCG vaccinated infants in Malawi were derived from the same large cohort study [In this study, there was no evidence of a difference in median IFN-γ production between Malawian infants vaccinated early compared with those vaccinated late, but there was a trend of higher responses in those vaccinated late. The Malawian infants vaccinated late showed higher median (p < 0.001) Th2  recall responses to rt study ,25 unlikrt study .M. tb PPD and CRP concentration (data not shown). Most of the Malawian infants had a normal range of plasma zinc concentrations and none of the cytokines tested were associated with the concentration of zinc (data not shown), indicating that zinc status might not be a major factor affecting the differences in infant immune responses in Malawi, at least when measured at 3 months post BCG vaccination.In terms of the proportion of infants who showed greater than 10 mg/L of CRP in their plasma, the majority (7/8) were born in the rainy season. In the whole infant cohort no infants showed helminth infections at 3–6 months of age. No infants were bled if unwell or with symptoms of malaria or other infection. We excluded the infants whose mothers had HIV, malaria, or helminth infections but we did not test for other diseases which could induce inflammation in the infants and might affect the CRP concentration in the infants and their mothers. Therefore higher plasma CRP concentration, reflecting level of inflammation, most likely indicates a higher burden of infections, but there was no association between the IFN-γ responses to To examine if cytokine activity had been lost over time in storage, we tested culture supernatant samples which had been collected from 4 infants at 3 years post BCG vaccination and stored in the same place as the archived samples we used for this study, since 2006. There was no significant difference in IFN-γ concentrations of 19 culture supernatant samples from each of 4 subjects when measured in 2006 and again in 2010, and a strong correlation was found between IFN-γ values obtained in 2006 and 2010  .M. tb PPD following BCG vaccination are documented at different latitudes and in different populations. The extent to which these influence BCG efficacy remains to be evaluated alongside studies looking for correlates of protection against TB [Differences in the infant immune responses to ainst TB . Season M. tb PPD, in infants at 3 months post BCG vaccination, while the concentration of plasma zinc or CRP did not correlate with the cytokine/chemokine signature to M. tb PPD. We therefore recommend that the season of birth and timing of priming vaccines, as well as geographical setting, should be considered in the design of clinical trials of new TB vaccines.In conclusion, variation in geographical location, season of birth, and age at vaccination may alter the cytokine/chemokine signature to Haemophilus influenzae b; IPV: Inactive polio vaccine; KPS: Karonga prevention study; M. tb: Mycobacterium tuberculosis; PPD: Purified protein derivative; TB: Tuberculosis.CRP: C-reactive protein; DPT: Diphtheria pertussis tetanus; HBV: Hepatitis B vaccine; Hib: The authors declare that they have no competing interests.YGH, PGS, HMD conceived the study. ABS, HM, JRF, KLF, SB, MOO, ACC participated in enrolment of study subjects in Malawi and The Gambia. YGH, MKL carried out experiments. YGH, SF performed statistical analysis. JR, SGS, HMD contributed reagents for the multiplex bead assay and CRP test. YGH drafted the manuscript and PGS, HMD helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/184/prepubM. tb PPD between Gambian and Malawian infants, Gambian and UK infants. The cytokine responses to M. tb PPD were compared among Malawian and Gambian infants who were vaccinated at 1 week after birth, and UK infants vaccinated between 3-13 weeks of life [d,f The fold differences of each cytokine response to M. tb PPD were calculated by dividing the median values of Gambian infants by Malawian infants with early vaccination and by dividing the median values of UK infants by those of Gambian infants, respectively. eP values for the median differences of each cytokine response to M. tb PPD between the two groups (Mann Whitney test).The fold differences of each cytokine response to  of life . d,f TheClick here for file


Short birth intervals are independently associated with increased risk of adverse maternal, perinatal, infant and child outcomes. Anemia in children, which is highly prevalent in Africa, is associated with an increased risk of morbidity and mortality. Birth spacing is advocated as a tool to reduce anemia in preschool African children, but the role of gender differences and contextual factors has been neglected. The present study aims to determine to what extent the length of preceding birth interval influences the hemoglobin levels of African preschool children in general, as well as for boys and girls separately, and which contextual factors thereby play a crucial role.This cross-sectional study uses data from Demographic and Health Surveys (DHS) conducted between 2003 and 2011 in 20 African countries. All preschool children aged 6–59 months with a valid hemoglobin measurement and a preceding birth interval of 7–72 months as well as their corresponding multigravida mothers aged 21–49 years were included in the study. Hemoglobin levels of children and mothers were measured in g/l, while birth intervals were calculated as months difference between consecutive births. Multivariate analyses were done to examine the relationship between length of preceding birth interval and child hemoglobin levels, adjusted for factors at the individual, household, community, district, and country level. A positive linear relationship was observed between birth interval and the 49,260 included children’s hemoglobin level, whereby age and sex of the child, hemoglobin level of the mother, household wealth, mother’s education and urbanization of place of residence also showed positive associations. In the interaction models, the effect of a month increase in birth interval is associated with an average increase of 0.025 g/l in hemoglobin level (P = 0.001) in girls, while for boys the effect was not significant. In addition, for girls, the effect of length of preceding birth interval was highest in young mothers and mothers with higher hemoglobin levels, while for boys, the highest effect was noticed for those living in more highly educated regions. Finally, significantly higher hemoglobin levels of girls compared to boys were observed at birth but with increasing age, the sex difference in hemoglobin level gradually becomes smaller.A longer birth interval has a modest positive effect on early childhood hemoglobin levels of girls, and this effect is strongest when their mothers are in their early twenties and have a high hemoglobin level. Remarkably, although the physiological iron requirement is higher for boys than girls, birth spacing has little influence on hemoglobin levels of preschool boys. We speculate that the preference for male offspring in large parts of Africa significantly influences nutritional patterns of African preschool boys and girls, and as such also determines the different effect of birth spacing. Finally, gender aspects should be considered in intervention programs that aim to improve anemia in African children. Anemia is a major public health problem, affecting 1.62 billion people worldwide. The prevalence of anemia in low-income countries is around 43% in contrast to 9% in high-income countries [The etiology of childhood anemia in Africa is multifactorial, including nutritional iron and folate deficiencies , 6, and Delaying pregnancy until the mother’s current child reaches at least its second birthday increases the likelihood that the child gets adequate care during its first vulnerable years. It also may lengthen the lactating period, which is favorable as breast milk continues to be an important source of nutrition during the second year of life , 16.Despite the general consensus about the positive effects of birth spacing, the optimal duration of the interpregnancy interval is not clear. Some studies suggest that a birth interval of 2–3 years reduces child mortality while on the other hand recent studies from the United States Agency for International Development (USAID) recommend a birth interval of 3–5 years , 18. An Socioeconomic status, lifestyle, stress, and adequacy of prenatal care, which are all associated with birth interval length, may also influence maternal, prenatal, infant, and child outcomes . HoweverAlthough there is some circumstantial evidence suggesting a positive association, large-scale studies exploring the relation between birth spacing and anemia in pre-school children are still lacking, despite the circumstantial evidence suggesting a positive relation. The aim of the present study is therefore to determine to what extent the length of preceding birth interval influences the hemoglobin levels of African pre-school children in general, as well as for boys and girls separately and which contextual factors thereby play a crucial role.This study used cross-sectional data derived from the Demographic and Health Surveys (DHS) , 24. DHSHemoglobin levels of children were available for children of up to 59 months. Within the 20 countries, 142 subnational regions , and 1999 clusters were distinguished. Information at district and cluster level was used to study effects of context factors. In all analyses, the household weights provided by DHS were used to obtain national representative samples. These weights were recoded to a mean of one, so that their application did not increase the sample sizes. For descriptive purposes, also national weights were constructed that made the sample representative of all countries together. To construct these national weights, the household weight factors were adjusted based on the World Bank population estimates for children aged 0–14 years within their respective country and year of survey . InformaInclusion criteria were valid hemoglobin measurement, preceding birth interval of more than six months and less than six years and having a mother aged 21 and over. Mothers aged under 21 were excluded to avoid potential bias that might arise due to adverse outcomes of under-age pregnancies. A preceding birth interval of less than 7 months was considered implausible and of over six years as an indication of unusual household situation. The total number of children satisfying these criteria was 50,264. Of these children, 197 had to be removed because mothers BMI was missing, 147 because mother’s education was missing, 659 because the child’s age was missing and one because mother’s work was missing. The final study population therefore constituted of 49,260 children.To measure hemoglobin levels, the blood of the women and children, obtained through a finger or heel prick, was tested for hemoglobin level. This testing was voluntary . ConsentPreceding birth interval was expressed in months and defined as the period between the previous childbirth and the index child birth. The difference in months between two consecutive live births was considered a birth interval.Control factors were included at the level of the children, the mother, the household and the context in which the household lived. Characteristics of the children that are associated with hemoglobin level include age (months), sex, birth order and twin status (singleton or multiple birth) –29. CharContextual factors were included at the cluster as well as at the district level. At cluster level, we included mean years of education of adults; preventive health measures (proportion of children having received vaccination against measles) and cluster altitude (in meters) , 34. ForBivariate and multivariate regression analyses were performed to determine the associations between the selected independent variables and the children’s hemoglobin level. All regression models contained fixed-effects dummy variables at the district level, with separate dummy variables for the urban and rural parts of the districts, to control for clustering and achieve complete control for direct effects of confounders at those levels. Given that all variation at the district level was controlled by these dummy variables, no direct effects of the context factors at the district level could be estimated. These context factors are therefore only used in the interaction analysis. We tested for nonlinearities by including quadratic terms for all interval variables, but only for the altitude variable the quadratic effect was significant. For altitude, this term was therefore included in the models.Interaction analysis was used to examine the influence of characteristics of the children, their mothers, the households and the context on the strength of the preceding birth interval effect. We tested for significant interactions between the birth interval effect and the children’s age, birth order, their mother’s age, mother’s hemoglobin level, mother’s BMI, and mother’s age difference with her partner, household wealth, cluster educational level, district level of development and availability of health facilities. To adjust the significance level for multiple interaction testing, the Bonferroni correction was applied . Given tThe baseline characteristics of children and mothers are described in Coefficients of the bivariate regression analyses are presented in the second column of The outcomes of the multivariate regression analysis are shown in the third column of The results again show that the hemoglobin level of girls is significantly higher than that of boys. We also observe that mother’s age, maternal BMI, maternal hemoglobin level, maternal educational level and her age difference with her partner all have significant positive effects on hemoglobin level of the index children. Household wealth has also a strong effect. Children living on higher altitude had significantly higher hemoglobin levels but the effect is nonlinear, with the effect starting to stabilize when the residence of the child was above 4,000 meters.To analyze whether the effect of length of preceding birth interval affected hemoglobin levels of boys and girls differently, an interaction term was added to the multivariate model. This term turned out to be significant and positive (effect size 0.023 P = 0.019), which means that the preceding birth interval effect was substantially stronger for girls than for boys. Separate multivariate analyses for boys and girls made clear that the effect was only significantly present in girls  and not in boys .To analyze whether the effect of length of preceding birth interval depended on the circumstances in which the children were living, interactions between this effect and the other variables in the model were studied. Given the substantial sex difference in the birth interval effect, the interaction analysis was performed separately for boys and girls. Because little knowledge of such interactions was available, this analysis is largely explorative in nature. We tested all interactions between preceding birth interval and the major other variables in the model  and added the significant interactions to the model.The interaction effects are visualized in Several important conclusions can be drawn from the present study with regard to the birth interval effect. First, we found that the length of the preceding birth interval has a modest positive effect on hemoglobin levels of African preschool girls, but not of that of boys. Second, having a long preceding birth interval is particularly important for African girls with a young mother. Third, girls were also found to profit more if their mother has a higher hemoglobin level. Fourth, boys were found to profit from a longer birth interval only under specific circumstances, namely if they lived in an area where the educational level of mothers is high.Positive effects of increasing birth intervals on health outcomes of children and their mothers have been noticed in several studies , 15, 36,The focus on gender differences turned out to be important too. When studying differences between boys and girls, while controlling for confounding factors, a significant effect of longer birth interval was particularly found for girls but turned out to be insignificant for boys. The positive effect observed in girls was in the order of 0.025 g/l for a one month increase in the birth interval, or 0.3 g/l for an increase of a year. In addition, we found that the highest hemoglobin increments were noticed in girls with young mothers and in girls with mothers with a high hemoglobin levels. Compared to girls with the oldest mothers in our data (late forties) and the mothers with the lowest hemoglobin levels (under 60), for those with the youngest mothers (early twenties) and with the mothers with the highest hemoglobin levels (over 160) the effect of a year increase in birth interval is associated with an approximate increase of 1.4 g/l in hemoglobin level. Given that the effects are additive, for girls with a young mother with a high hemoglobin level the birth interval effect is in the order of 2.8 g/l higher than for those with an old mother with a low hemoglobin level.We did not find a significant overall effect of birth interval on hemoglobin level among boys. However, the interaction analysis made clear that in areas where mothers have relatively high educational levels, boys profit more from a longer birth interval than in areas where mothers have relatively low educational levels. This difference is in the order of 1 g/l for an increase of a year.Childhood anemia is very prevalent in Sub-Sahara Africa but gender differences are thereby rarely considered. Our results suggest that increasing hemoglobin levels in mothers may positively affect hemoglobin levels in their daughters. Furthermore, it seems important to target the interventions especially to young mothers, as their female offspring profits most from longer birth intervals.Our data were derived from the DHS, including 49,260 children in 20 African countries whereby many biological, maternal, socioeconomic and contextual factors were collected as well. Because of the large sample size, internal validity was secured and variance of estimates reduced. Adjustment for covariates was performed using multivariate analysis with country and region fixed effects. In these analyses, it was noticed that biological, maternal, socioeconomic, environmental and contextual factors were related to hemoglobin levels of African preschool children and that most of the factors affect hemoglobin levels of African boys and girls rather similarly.Apart from the separate effect of the various factors, a common denominator may play a significant role in the different sex response to birth spacing. We hypothesize that the preference for male offspring in many African countries leads to more maternal investments in boys compared to girls, making them less susceptible for the effect of birth spacing. Studies from Ethiopia   and PeruThe maternal depletion syndrome could be another factor that contributes to hemoglobin levels of their offspring. Maternal depletion syndrome is characterized by a deterioration of the maternal nutritional status during the reproductive cycle, especially when the period of depletion is long while the time for repletion is short and the mother has a marginally inadequate food intake , 15. ForGiven that no significant association between preceding birth interval and the children’s age was found, our study also indicates that the positive effects of a longer birth interval persist across the first 6 years of life. This may be explained by the fact that the age difference between the two succeeding children remains, which may create the opportunities for more maternal investments over the whole period. In addition, also biological factors, like epigenetic changes may contribute to the constant positive effect of a longer birth interval. The clinical relevance of our finding is difficult to give, as the effects on hemoglobin levels are only modest and mostly seen in girls that have already a greater chance of survival while boys are biologically weaker and more susceptible to disease and premature death , 46.Apart from the birth interval effect, our analyses made clear that hemoglobin levels of African children steadily increase during the first 59 months of life  and that hemoglobin levels of girls were significantly higher than those of boys . An additional test for sex differences in the age effect revealed a significant interaction , whereby the increment with age is significantly stronger in boys  than in girls . Hence, with increasing age, the sex difference in hemoglobin level becomes smaller. In Various other studies have also reported higher hemoglobin levels of girls , 29. FurOur study is limited by the lack of information on major co-morbidities that affect hemoglobin status, such as malaria, tuberculosis and Human Immunodeficiency Virus (HIV) infections as well as non-communicable diseases, like nutritional deficiencies, including those for folate, vitamin B12 and vitamin A. The same applies to inherited or acquired disorders that affect hemoglobin synthesis, red blood cell production or red blood cell survival. The overestimation effect of miscarriage and underestimation effect of stillbirth in the length of birth interval cannot be discussed upon due to restricted information of maternal pregnancy in DHS data. Further, the study uses a cross-sectional design, which prevents the inference of cause and effect relationship.In conclusion, increasing birth interval has a moderate positive effect on hemoglobin levels of preschool African girls. Remarkably, although iron requirement of boys seem higher than girls, birth spacing did not influence hemoglobin levels of preschool boys. The preference for male offspring in large parts of Africa may not only significantly contribute to the sex differences in hemoglobin levels but may also determine the different effect of birth spacing on hemoglobin levels of African boys and girls. Finally, gender aspects should be considered in interventions programs that aim to improve anemia in African children.S1 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Mitochondria are sensitive to environmental toxicants due to their lack of repair capacity. Changes in mitochondrial DNA (mtDNA) content may represent a biologically relevant intermediate outcome in mechanisms linking air pollution and fetal growth restriction.We investigated whether placental mtDNA content is a possible mediator of the association between prenatal nitrogen dioxide (NO2) exposure and birth weight.We used data from two independent European cohorts: INMA  and ENVIRONAGE . Relative placental mtDNA content was determined as the ratio of two mitochondrial genes (MT-ND1 and MTF3212/R3319) to two control genes (RPLP0 and ACTB). Effect estimates for individual cohorts and the pooled data set were calculated using multiple linear regression and mixed models. We also performed a mediation analysis.Pooled estimates indicated that a 10-μg/m3 increment in average NO2 exposure during pregnancy was associated with a 4.9% decrease in placental mtDNA content  and a 48-g decrease  in birth weight. However, the association with birth weight was significant for INMA  but not for ENVIRONAGE . Placental mtDNA content was associated with significantly higher mean birth weight . Mediation analysis estimates, which were derived for the INMA cohort only, suggested that 10%  of the association between prenatal NO2 and birth weight was mediated by changes in placental mtDNA content.Our results suggest that mtDNA content can be one of the potential mediators of the association between prenatal air pollution exposure and birth weight.Clemente DB, Casas M, Vilahur N, Begiristain H, Bustamante M, Carsin AE, Fernández MF, Fierens F, Gyselaers W, Iñiguez C, Janssen BG, Lefebvre W, Llop S, Olea N, Pedersen M, Pieters N, Santa Marina L, Souto A, Tardón A, Vanpoucke C, Vrijheid M, Sunyer J, Nawrot TS. 2016. Prenatal ambient air pollution, placental mitochondrial DNA content, and birth weight in the INMA (Spain) and ENVIRONAGE (Belgium) birth cohorts. Environ Health Perspect 124:659–665; http://dx.doi.org/10.1289/ehp.1408981 These “power plants of the cell” also play a critical role in signaling transduction for cell proliferation, apoptosis, calcium storage, and metabolism . SeveralThe placenta plays a unique role in the transfer of gases, nutrients, and waste between the mother and developing child. It is both a metabolic and an endocrine organ. However, the placenta has a limited capability to metabolize a large number of foreign compounds . The plaONAGE), we aimed to assess the role of mediating effects of placental mtDNA content on the association of prenatal NO2 exposure with birth weight.Recently, it has been shown that exposure to particulate air pollution during pregnancy was associated with placental mtDNA content . We hypoStudy design and population. The Spanish population-based birth cohort study INMA  recruited pregnant women in four centers , following a common protocol  did not show differences in maternal age, pregestational body mass index (BMI), parity, and ethnicity.protocol . A totalONAGE , 556 pregnant women were enrolled between 2010 and 2013 at the South-East-Limburg Hospital in Genk (Belgium) when they arrived for delivery. The inclusion criteria were ≥ 18 years of age, singleton pregnancy, and ability to fill out questionnaires in Dutch. The overall participation rate of eligible mothers was 56%. A comparison of the ENVIRONAGE birth cohort with all births in Flanders . The entire placentas were frozen after delivery at –20°C until they were transferred on dry ice to the Hospital Universitario San Cecilio (HUSC) Biobank in Granada (Spain) and stored at –86°C. No information was available about the time between delivery and storage at the different Spanish hospitals involved in the study. MtDNA content was measured in 390 out of the 502 randomly selected placentas in INMA. In the ENVIRONAGE cohort, all placentas (n = 556) were collected after delivery and were frozen within 10 min at –20°C. In both cohorts, placentas were thawed minimally to obtain tissue biopsies for DNA extraction. To minimize the impact of within-placenta variability, biopsies were all taken 1–1.5 cm below the chorioamniotic membrane at a fixed location and preserved at –80°C  following the manufacturer’s instructions. In the ENVIRONAGE cohort, DNA was extracted from placental tissue cells using the QIAamp DNA minikit  following the manufacturer’s instructions. In both cohorts, DNA samples were quantified using the Nanodrop spectrophotometer  and the Quant-iT™ PicoGreen® dsDNA Assay Kit  using the Omega Fluostar plate reader .MT-ND1) and mitochondrial forward primer for nucleotide 3212 and reverse primer from nucleotide 3319 (MTF3212/R3319)] to two nuclear control genes  using a quantitative real-time polymerase chain reaction (qPCR) assay  assay . qPCR waR) assay .Ambient air pollution assessment. In INMA, ambient concentrations of NO2 were measured with the aid of passive samplers  distributed outside across the study areas according to geographic criteria, taking into account the expected pollution gradients and the distribution of the residences of the participating women. The samplers remained exposed during various 7-day sampling periods through pregnancy. The methodology has been described in detail elsewhere  for annual mean NO2 from 0.52 to 0.75 in the four INMA subcohorts .lsewhere , and furONAGE, regional background levels of NO2 for each woman’s home address were calculated using a kriging interpolation method  for hourly averages > 0.80 and a spatial explained variance (R2) for annual mean NO2 of 0.82.In ENVIRn method  that usen method . This mo2 concentrations for the three trimesters of pregnancy using the same procedure used for the entire pregnancy: 1–13 weeks starting from date of conception (trimester 1), 14–28 weeks (trimester 2), and 29 weeks to delivery (trimester 3).To explore potentially critical exposures during pregnancy, we calculated individual NOONAGE can be found in Supplemental Material, Table S1.More details on exposure measurements for INMA and ENVIRStatistical analysis. Continuous data were checked for normality using the Shapiro–Wilk test statistic. Placental mtDNA content data were right skewed and therefore logarithmically transformed (log10). Generalized additive models (GAMs) were used to assess the linearity of the associations between a) prenatal NO2 exposure and mtDNA content, b) mtDNA content and birth weight, and c) prenatal NO2 exposure and birth weight . Multiple linear regression models were used in ENVIRONAGE. Multiple linear mixed models with a random cohort effect were used in INMA and in the pooled data set (four INMA cohorts and ENVIRONAGE). Covariates used in the model were gestational age (linear and quadratic term), newborn’s sex, maternal age, ethnicity, parity, smoking status, education, season of birth , and prepregnancy maternal BMI. For the present analysis, we excluded 14 mother–newborn pairs from INMA and 6 mother–newborn pairs from ENVIRONAGE with missing values in the outcome, exposure, and confounders. After these exclusions, the final study population consisted of 376 subjects for INMA and 550 subjects for ENVIRONAGE.2 exposure and birth weight, we performed a mediation analysis. The direct effect (DE), indirect effect (IE), and total effect (TE) were estimated using the SAS macro developed by 2 exposure on birth weight after controlling for mtDNA content, and the IE is the estimated effect of NO2 exposure during pregnancy operating through mtDNA content .Characteristics of the study population. Characteristics of the 376 and 550 mother–newborn pairs in, respectively, the INMA and the ENVIRONAGE cohort are shown in ONAGE. The mean birth weight was lower and placental mtDNA content higher in INMA newborns compared with newborns from ENVIRONAGE.2 was 25.5 ± 11.4 μg/m3 and 21.1 ± 4.2 μg/m3 in INMA and ENVIRONAGE, respectively. Similar differences in exposure levels between cohorts were observed in the mean trimester concentrations (The mean (± SD) pregnancy average exposure to NOtrations .2 exposure.Association between placental mtDNA content and NO In the INMA cohort, NO2 exposure during each trimester and the entire pregnancy was negatively and significantly associated with placental mtDNA content : –8.8, –2.1%]. In the ENVIRONAGE cohort, NO2 exposure was also negatively associated with placental mtDNA content in each trimester and in the entire pregnancy, but it was statistically significant only during the second  and third trimesters of pregnancy   . The poo, –6.4%) .2 exposure.Association between birth weight and NO The association between birth weight and prenatal NO2 exposure was significant in the INMA cohort for all three trimesters of pregnancy  decrease in birth weight . After both cohorts were pooled, the association between birth weight and NO2 was significant in all pregnancy trimesters and in the entire pregnancy   .Association between placental mtDNA content and birth weight. Placental mtDNA content was positively and significantly associated with birth weight in both cohorts and in the pooled analysis   .Mediation analysis. In INMA, the mediation analysis suggested that 10%  of the association between birth weight and pregnancy average NO2 exposure may be mediated by differences in placental mtDNA content , 11.4% , and 12.2% , respectively. When we limited the mediation analysis to boys in the INMA cohort, the analysis suggested a mediation effect of 16%  , or for NO2 exposure during the different trimesters . Mediation analysis of the pooled data for boys suggested placental mtDNA content mediated 6.4%  of the association between NO2 during trimester 3 and birth weight, but mediation was not statistically significant for average pregnancy NO2 or NO2 during trimester 1 or 2 . content . The cor7, 13.2) . BecauseSensitivity analysis. The reported associations did not change after excluding mother–newborn pairs with nonvaginal deliveries (n = 83) (data not shown). Furthermore, when we used cohort as a fixed effect, our reported estimates did not change either (data not shown). Finally, associations were stronger when the analysis was limited to 214 INMA participants who spent ≥ 15 hr/day at home, including associations between NO2 and birth weight  .2 exposure. The key findings, based on two independent European cohorts were that a) prenatal NO2 exposure is inversely associated with placental mtDNA content; b) placental mtDNA content is positively associated with birth weight; c) prenatal NO2 exposure is inversely associated with birth weight; and d) placental mtDNA content can be a potential mediator of the association between birth weight and prenatal NO2 exposure.Mitochondria, the energy producers of the cells, are particularly sensitive to environmental toxicants because they lack repair capacity . Fetuses2 is frequently used as a surrogate for traffic-related air pollution because it is considered to be a good proxy of other pollutants originating from the same sources  reported an estimated decrease in birth weight of 11 g for every 10-μg/m3 increment in NO2  than in the ENVIRONAGE cohort . This association also varied among the four INMA subcohorts, ranging from –12 g  for the Valencia subcohort to –156 g  for the Asturias subcohort . Differences in effect sizes across the studied cohorts might be attributable to different levels of exposure, population variability, and variation in meteorological conditions, but also noncausal mechanisms could explain these differences—for example, differences in study design and conduct, exposure assessment, and differences in residual confounding. Nevertheless, our GAM plots showed linear associations between NO2 and mtDNA content, NO2 and birth weight, and mtDNA content and birth weight in both cohorts .Ambient air contains a mixture of pollutants. NO sources . An appr sources . An earlt in NO2 . Data frregnancy . In our 2 during a commute, at work, and elsewhere was not taken into account. Time spent at home can influence associations between ambient air pollution measured at the mother’s residence and birth weight , environmental and dietary contaminants that have been associated with lower birth weight, as well as exposure to NOh weight ; in this2 exposure could be explained by placental mtDNA content. This finding was demonstrated in a subsample of 376 mother–child pairs of the INMA cohort. We are aware that epidemiological studies can only show associations, but cannot prove causality; nevertheless, our formal mediation analysis is based on a predefined hypothesis and is in line with experimental evidence.In contrast to the epidemiological evidence, the mechanisms responsible for fetal growth restriction due to air pollution are largely unknown. Hypotheses are that air pollutants could cause oxidative stress or inflammation, alter placental growth, decrease placental exchange of nutrients and gases, foster endocrine disruption, or cause maternal health effects—all of which could possibly lead to altered fetal growth . Oxidatiin vitro  channels which are highly expressed in placenta, and their activation has been suggested to play important roles in placental development and regulating the fetal–maternal interface in mice models  with a smaller sample size. We also added more information by performing a mediation analysis that supports our hypothesis that mitochondrial damage may be a causal intermediate in biological mechanisms linking air pollution exposure to birth outcomes, which may provide some mechanistic clues to the adverse effects of early exposure to air pollution observed in humans.The major strengths of this study are that we tested the different windows of exposure, and made use of two independent birth cohorts in Southern (Spain) and Western (Belgium) Europe. Also, we used new methods and their assumptions to study causal interference . Neverth2 exposure was inversely associated with both placental mtDNA content and birth weight. Considering the high levels of NO2 in urban areas, which are increasing worldwide, this study indicates the relevance of further exploring this biological pathway linking early air pollution exposure and complications at birth.In conclusion, we have shown that prenatal NO(398 KB) PDFClick here for additional data file.


To better understand factors that may impact infant mortality rates (IMR), we evaluated the consistency across birth hospitals in the classification of a birth event as either a fetal death or an early neonatal (infant) death using natality data from North Carolina for the years 1995–2000.A database consisting of fetal deaths and infant deaths occurring within the first 24 hours after birth was constructed. Bivariate, followed by multivariable regression, analyses were used to control for relevant maternal and infant factors. Based upon hospital variances, adjustments were made to evaluate the impact of the classification on statewide infant mortality rate.After controlling for multiple maternal and infant factors, birth hospital remained a factor related to the classification of early neonatal versus fetal death. Reporting of early neonatal deaths versus fetal deaths consistent with the lowest or highest hospital strata would have resulted in an adjusted IMR varying from 7.5 to 10.64 compared with the actual rate of 8.95.Valid comparisons of IMR among geographic regions within and between countries require consistent classification of perinatal deaths. This study demonstrates that local variation in categorization of death events as fetal death versus neonatal death within the first 24 hours after delivery may impact a state-level IMR in a meaningful magnitude. The potential impact of this issue on IMRs should be examined in other state and national populations. The definition of the infant mortality rate (IMR) as the number of deaths in the first year after birth per 1000 live births gained popular acceptance by the late 1800’s -4.Disparities in the birth rates and newborn care of infants, especially preterm infants, may lead to incongruent comparisons. Very early preterm infants have much higher neonatal mortality rates than do term and near-term live-born infants . DiffereA consistent classification of perinatal deaths is necessary if IMR-based comparisons are to be meaningful. The World Health Organization definition of a live birth is “the complete expulsion or extraction from its mother of a product of conception, irrespective of the duration of the pregnancy, which, after such separation, breathes or shows any other evidence of life ” .Even with this stringent definition, differences in reporting fetal and infant deaths continue. The landmark study, “Five Decades of Missing Females in China,” was among the first to highlight a bias in reporting of infant deaths . InaccurA recent outcomes study of births weighing less than 500 grams showed substantial variation in the proportion classified as neonatal death versus fetal death at the state level in the United States from 1999 through 2002 ,18. We htime lived ; and 2) comparing the calendar date of birth to the calendar date of death. The latter allowed 21 infant deaths to be classified as occurring within the first 24 hours after birth when data were missing in the time lived fields.Live birth, infant death, and fetal death files for North Carolina for the years of birth 1995–2000 were obtained from the North Carolina State Center for Health Statistics after approval of the university Institutional Review Board. These files are currently publicly available. This analysis used the subset of records that represented 1) fetal deaths and 2) infant deaths that occurred within the first 24 hours after live birth. The latter were identified by 1) information contained in two fields denoting Four groups (see below) were determined after an initial view of the frequency distribution of the number of events per hospital for the 135 hospitals in the database, as we could not do a meaningful comparison of all hospitals due to sample size issues. A decision was made to retain 31 hospitals with larger sample sizes (60 or more events in the 6 years of data) and then group the other hospitals and situations into the three comparison groups for the 31 individual hospitals. It seemed rational to use birth events occurring outside as a distinct group. It also seemed reasonable to break the 104 hospitals with < 60 events during the study period into two groups as follows: 1) those in counties that contained one of the 31 ‘high event number’ hospitals and 2) those in counties that did not. We posited that there could be cross-coverage or other similarities in hospital culture within counties with more than one hospital. We had no way to confirm whether this was true. This decision was made prior to the performance of other analyses of association of these groups or hospitals or other covariates with the outcome.North Carolina had 100 counties and 135 hospitals represented in the database during the study years. Individual hospitals were selected for this analysis if they had at least 60 birth events that were fetal deaths or infant deaths during the study period (Group 1). Three comparison groups were constructed from the remaining records: Group 2) fetal or infant delivery occurring outside of a hospital setting, regardless of county of occurrence; Group 3) fetal or infant delivery occurring in hospitals in counties where no hospital met the inclusion criterion of having at least 60 such events during the study period; and Group 4) fetal or infant delivery occurring in hospitals with less than 60 such events in counties with 1 or more hospitals having 60 or more such events.To allow adjustment for potential differences in numbers and types of high-risk pregnancies managed among the hospitals, birth certificate data were used to construct categorical variables for birth year, birth weight, gestational age, gender of the fetus or infant, maternal race/ethnicity, delivery method, plural birth, prenatal care visits, maternal age, maternal education, alcohol use during pregnancy, tobacco use during pregnancy, prior fetal deaths or pregnancy terminations of any type, maternal history of the death of a prior live-born child, gravidity, parity, and marital status. Dichotomous variables were constructed for 1) occurrence of an adverse event during labor or delivery ; 2) maternal medical history positive for a disease or predisposing condition ; and 3) presence of any congenital anomaly.hospital of birth is not associated with this classification. Bivariate associations were evaluated using Pearson Chi square tests. Cramer’s V was used to assess correlation between two nominal variables . Two-level logistic regression modeling (one-stage clustering sampling frame) using a general estimating equations approach was used to determine variation among individual hospitals relative to control groups while adjusting for other predictor variables and the potential cluster effect of birth hospitals . SPSS 22.0  was used for all analyses.The outcome measure used was whether a pregnancy outcome was classified as a fetal death or a live birth with infant death occurring within 24 hours after birth. The null hypothesis was that Reported infant deaths and live birth files were used to determine initial numerators and denominators for IMRs. As the ratios of infant deaths within 24 hours after birth to fetal deaths were adjusted to selected reference standards, appropriate adjustments in numerators and denominators were made .early neonatal deaths within the first 24 hours after birth consisted of 8044 such events.During the six years of 1995–2000 in North Carolina, there were a total of 649,252 live births, with 5813 infant deaths (8.95 per 1000 live births), and 5311 fetal deaths. Among the infant deaths, 2733 occurred within 24 hours after birth, with 89.7% occurring during the first six hours after birth : birth year, alcohol use during pregnancy, tobacco use during pregnancy, occurrence of an adverse event during labor or delivery, prior fetal deaths or pregnancy terminations of any type, maternal history of the death of a prior live-born child, gravidity, parity, marital status (p = .064), and gender of the fetus-infant.The following factors had neither meaningful nor statistical association with the classification outcome  and 1801–2000 grams  who were also more likely to be classified as early neonatal versus fetal deaths as birth weights > 4000 grams . Birth weight was known for all 8044 cases, while gestational age was missing for 111 (1.4%). Prenatal care visits were missing from 556 cases (6.9%). The number of prenatal visits was modestly correlated with birth weight . Given scattered missing data in other variables, inclusion of prenatal care visits in the final model would have resulted in loss of > 10% of evaluable records.Gestational age, prenatal care visits, and maternal county of residence were not used in the final model. Gestational age and birth weight were highly correlated, with a Spearman correlation coefficient of 0.78 (p < .001). A single county accounted for ≥80% of maternal residence for 14 (45%) of the 31 individually-evaluated hospitals. Two counties accounted for ≥90% of maternal residence for another 4 (13%) and for ≥75% for another 6 (19%).Maternal county of residence also was excluded from multivariable analysis as this was highly associated with birth hospital .The IMR is a key measure of population health and is widely used as a comparative measure, determinate of healthcare policy, and/or an outcomes measure. Preterm birth and its complications are well-recognized causes of infant death. Differences in preterm birth rates and interventions have been identified as explanatory factors for apparent difference in IMR between populations. Further, differences in classification and reporting of infant or fetal deaths have also been suggested as a factor for differences in IMR among various entities or regions -21. HoweOf note, the three hospitals with statistically significant odds ratios of classifying these events as early neonatal deaths that were more than 2-fold higher than the reference group were affiliated with three different academic medical centers. This could reflect greater rigor in adhering to live-birth definitions in these centers, greater availability of resources to resuscitate and care for extremely low birth weight neonates, and/or other unrecognized factors at these institutions relative to other sites of newborn care.Interventions at the limits of gestation may also vary based upon physician attitudes and parental preferences. Factors that have been implicated in interventions at the limits of viability include maternal age, parity, race, insurance status, education, prenatal care, gestational age, and birth weight . These dFor many obstetricians and neonatologists, uncertainty exists in decisions to intervene and/or resuscitate between 500–600 grams or 23–24 weeks gestation ,23. A prMuch of the relatively high IMR in the United States can be attributed to a high percentage of preterm births ,15. A reVariations in classification of fetal deaths and infant deaths on the first postnatal day could potentially misinform efforts to prevent adverse outcomes of pregnancy. Until recently, the focus in the U.S. has been more toward reducing infant mortality with less attention being given to the problem of fetal mortality. It is now clear that fetal mortality, even when limited to fetal death beyond 20 weeks gestation, is a significant problem and that it has been underreported ,27. InteOur study was limited by the inability to ascertain directly whether any of the reported fetal deaths actually showed signs of life that would have met the WHO definition of live birth. However, the variation among birth hospitals persisted in two-level logistic regression modeling to control for potential unmeasured confounding at the hospital level as well as multiple other factors that may contribute to true fetal death versus true live birth with rapid demise. Our analysis also was restricted to rapid demise after birth, with 90% of infant deaths occurring within 6 hours after birth. These “very early neonatal deaths” and many fetal deaths reasonably can be construed as a clinical continuum “ready-made” for subjectivity in classification despite the extant international definition of live birth.Additional limitations of our retrospective cohort study include lack of any data elements beyond those collected as part of the vital statistics programs for live births, fetal deaths, and infant deaths during the study period. Some of the captured data elements, such as self-reported alcohol use during pregnancy, are not always sensitive or accurate measures. We also are unable to account for any under-reporting of fetal deaths beyond 20 weeks gestation during the study period, though we believe this would have been, at most, a rare occurrence .Lastly, the age of our data is the primary limitation, but we believe the point we are able to illustrate remains important. To the extent that delivery room care of fetuses and newborns at the border of viability changed after 2000, our data conceivably might not be relevant to current practice. However, because we are aware of no efforts at a state or national level to standardize classification of deaths at the border of viability in the United State, it is likely that our study demonstrates the potential impact of a variation in practice that still exists. Additionally, there have been no changes in national regulations for registration of stillbirths or live births in the U.S. in the past 20 years. The rates of live births and still births have declined slightly in recent years, corresponding with the economic downturn in the U.S., but we do not believe these changes would influence practice variation in classification of live birth versus fetal death status in the delivery rooms of most local hospitals. Even if the local hospital-level variation we detected in this study has declined during the subsequent decade, this type of variance, which has not been previously described, still could have relevance and should be considered in future comparative analyses of infant mortality and other birth-related vital statistics between states and nations.Repeating this analysis in more current databases from other regions of the U.S. and other countries would add further insight regarding the importance of this issue on reported IMRs. Future research would be strengthened by the inclusion of a mixed-methods approach that adds qualitative data from health care providers and staff involved in delivery and newborn care to better understand origins of variation in classification by hospital or hospital type. This could lead to system-level interventions that improve adherence to the current definition of live birth and reduce variation in classification.The purpose in this analysis was to demonstrate that local hospital-level variation in classification of live birth with death in the newborn period versus fetal death may have an impact on reported IMR at the state level that is important both clinically and for policy development. Impacts at the state level could, in turn, impact national IMR. Vigilance and diligence at local and state levels are needed to ensure consistent classification of early neonatal deaths so that valid comparisons can be made between counties and states.Integrity of international or intra-national state/provincial comparisons of IMR as a measure of population health might be improved if fetal and neonatal death rates were compared by birth weight and/or gestational age strata rather than single aggregate summary statistics. Our findings further support the utility of Perinatal Mortality as a metric, whether defined as stillbirths after 22 weeks gestation plus infant deaths within seven completed days after birth ,29 or otWe have no financial or non-financial competing interests to disclose.CRW initiated the study, developed the analysis database, conducted most analyses, produced the initial draft of the manuscript, and supported the development of the final manuscript. He gives final approval for publication of the current version of the manuscript. DWD participated in interpretation of the data and development and ongoing revision of the manuscript. She gives final approval for publication of the current version of the manuscript. SDD participated in interpretation of the data and development and ongoing revision of the manuscript. He gives final approval for publication of the current version of the manuscript. JAM conducted analyses and supported development of the final manuscript. He gives final approval for publication of the current version of the manuscript. TMO participated in the study design, interpretation of data analyses, and revising the manuscript for important intellectual content. He gives final approval for publication of the current version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/14/108/prepub


To describe Type 1 retinopathy of prematurity (ROP) and its laser treatment outcomes in premature infants with birth weight > 1250 g in Eastern China.A retrospective review of 3175 ROP records was conducted at Shanghai Eye & ENT Hospital of Fudan University. The records were collected at the ROP clinic from 2006 to 2014, including their demographic and medical information such as gestational age, birth weight, supplemental oxygen therapy, systemic complications, ROP stage, location, presence of plus disease. All infants were examined by RetCam fundus camera. Those with Type 1 ROP were also examined by indirect ophthalmoscope before undergoing transpupillary laser treatment.A total of 12 infants (24 eyes) with Type 1 ROP and birth weight > 1250 g were enrolled. All infants enrolled had plus disease and ROP in zone II retina. Specifically, 16 eyes (67%) had stage 2 ROP. 8 eyes (33%) had stage 3 ROP. ROP regressed in 23 eyes (96%) following laser treatment. Partial retinal detachment developed in one eye (4%). No severe involution sequelaes or laser-related complications were recorded. Mean follow-up was 30±6 weeks.Type 1 ROP may occur in large premature infants who have undergone supplemental oxygen therapy. This Type 1 ROP is mainly located in zone II retina. Laser treatment is a safe and effective intervention for these infants. Retinopathy of prematurity (ROP), characterized by avascular retina that may potentially lead to retinal detachment and blindness, is mostly seen in small preterm infants . ScreeniStudies have demonstrated that laser photocoagulation can significantly reduce unfavorable outcomes of severe ROP in infants with low birth weight (< 1250g) , 10. HowThis study was approved by the Ethics Review Board of Shanghai Eye & ENT Hospital of Fudan University, East China. A retrospective review of ROP records from 2006 to 2014 was conducted. The records were collected at our ROP clinic, including infant’s demographic and medical information such as name, sex, date of birth, gestational age, birth weight, systemic complications, history of oxygen supplementation, ROP location, stage, presence of plus disease, laser treatment and follow-up examinations. Enrollment criteria included those with birth weight > 1250g, diagnosed with Type 1 ROP, underwent laser treatment and were followed up > 12 weeks.Written informed consents were obtained from parents before screening examinations. All infants were examined using a RetCam fundus camera  as previously described. In brieTranspupillary laser treatments were performed within 24 hours of Type 1 ROP diagnosis. The pupil was dilated and topical anesthesia was performed. The experts gently open the eye with a lid speculum and re-examined the fundus using a 28D lens and an indirect ophthalmoscope, integrated with 532 nm laser accessories . The photocoagulation was performed on a nearly confluent pattern in the avascular retina. Repeat mode was used with 0.2s exposure time and 0.2s interval time. Power was set to achieve white-grey burns, generally between 200 to 350 mW. Topical antibiotic eye drops were used during and after the procedure. Any ocular or systemic complications related to photocoagulation were recorded.Following laser treatment, infants were examined by RetCam weekly until ROP regression appeared. They were then imaged every 2–4 weeks until no active ROP existed. If ROP recurred post-treatment, additional photocoagulation or vitrectomy was applied when necessary.All data were analyzed using MS Excel and Prism. Fisher’s exact test was used to compare the difference of Type 1 ROP location, staging and regression rate following laser treatment between our large preterm infants and those with low birth weight in the ET-ROP study. Data were presented as mean ± SD.A total of 12 infants (24 eyes) were enrolled from 3175 records of our ROP clinic between the period from 2006 to 2014. All were diagnosed as Type 1 ROP and underwent laser treatment. Their mean birth weight was 1450±155g (1265–1800g). The mean gestational age was 30±2 weeks (28–34 weeks). Of all, 7 were male (58%) and 5 were female (42%). Type 1 ROP was located in zone II retina of all infants . SpecifiAll eyes underwent laser treatment at mean 6±2 weeks after birth (3–10 weeks). The mean postmenstrual age was 37±2 weeks (33–40 weeks). The infants received mean 1552±518 laser spots (1050–2515). ROP regressed spontaneously in 23 eyes 96%, . DetailsOur study described Type 1 ROP and its laser treatment outcomes in a series of large preterm infants in Eastern China. All had birth weight > 1250 g and were diagnosed with stage 2 or 3 ROP in zone II retina with plus disease. Following laser treatment, ROP generally regressed with minor involution sequelaes.We compared our results with the outcomes of the ET-ROP study to determine any difference of Type 1 ROP characteristics between low and large birth weight infants in our cohort of ROP infants. In the ET-ROP study, infants with birth weight <1251 g and high-risk prethreshold ROP were randomized to treatment or observation. Of all The difference of ROP location and stage between large and small preterm infants may well be due to the disparities in their vascular maturity and development after birth. According to the results, our large preterm infants were almost 5 weeks older than those in the ET-ROP study at gestational age (30±2 vs. 25±1 weeks). Hughes et al made a comprehensive study of retinal vascularization in human fetal retina . They foHowever, our results indicate that those large preterm infants are still susceptible to severe ROP, particularly when they have risk factors such as supplemental oxygen therapy. In the study, 92% of infants had mask oxygen supplementation and 75% had mechanical oxygen therapy. Apnea occurred in 83% of infants enrolled in the study. In small preterm infants, however, only 24% had systemic complications such as apnea and reincubation , 14, sepFortunately, our study indicates laser treatment can achieve similar favorable outcomes in large preterm infants when compared with small preterm infants. Actually, the ROP regression rate following laser treatment in our study is slightly higher than that of the ET-ROP study, although no statistic significance was found . Meanwhile, large preterm infants were 2 weeks older than those with BW < 1251 g in the ET-ROP study in terms of the postmenstrual age when laser was applied (37±3 vs. 35±2 weeks). Similar results were also reported by Chen et al . These dOverall, our study demonstrates Type 1 ROP may occur in infants with birth weight >1250 g. Laser photocoagulation is a safe and effective intervention for those infants. Additional interventions should be considered for those who do not respond well and show signs of progression following laser treatment.