Poly (ADP-ribose) polymerase (PARP) participates in inflammation by cellular necrosis and the nuclear factor-kappa-B (NF-κB)-dependent transcription. The purpose of this study was to examine the roles of PARP in ventilator-induced lung injury (VILI) in normal mice lung.2O + positive end-expiratory pressure (PEEP) 3 cm H2O + 90 breaths per minute for the LPV group and PIP 40 cm H2O + PEEP 0 cm H2O + 90 breaths per minute for the VILI and PJ34+VILI groups. After 2 hours of MV, acute lung injury (ALI) score, wet-to-dry (W/D) weight ratio, PARP activity, and dynamic compliance (CD) were recorded. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), myeloperoxidase (MPO) activity, and nitrite/nitrate (NOX) in the bronchoalveolar lavage fluid and NF-κB DNA-binding activity in tissue homogenates were measured.Male C57BL/6 mice were divided into four groups: sham tracheostomized (sham), lung-protective ventilation (LPV), VILI, and VILI with PARP inhibitor PJ34 pretreatment (PJ34+VILI) groups. Mechanical ventilation (MV) settings were peak inspiratory pressure (PIP) 15 cm HX, and concentrations of TNF-α and IL-6 along with lower CD than the sham and LPV groups (P < 0.05). In the PJ34+VILI group, PJ34 pretreatment improved all histopathologic ALI, inflammatory profiles, and pulmonary dynamics (P < 0.05). NF-κB activity was increased in the VILI group as compared with the sham and LPV groups (P < 0.05) and was decreased in the PJ34+VILI group as compared with the VILI group (P = 0.009). Changes in all parameters were closely correlated with the PARP activity (P < 0.05).The VILI group showed higher ALI score, W/D weight ratio, MPO activity, NOOveractivation of PARP plays an important role in the inflammatory and transcriptional pathogenesis of VILI, and PARP inhibition has potentially beneficial effects on the prevention and treatment of VILI. Ventilator-induced lung injury (VILI) has been established as a significant risk in patients receiving mechanical ventilation (MV). The spectrum of VILI includes not only air leaks and increases in endothelial and epithelial permeability but also increases in pulmonary and systemic inflammatory mediators ,2. Altho+) into nicotinamide and ADP-ribose and then uses the latter to synthesize polymers of ADP-ribose in DNA repair  , which is not antioxidant and does not directly interfere with the reactivity of peroxynitrite [The mice in the PJ34+VILI group were intraperitoneally pretreated with 20 mg/kg of PJ34 [ynitrite . To deteX), the stable end products of NO metabolism, in the BALF using an NO (NO2-/NO3-) assay kit . Nuclear proteins from the tissue homogenate subgroup mice were prepared with a nuclear extract kit . Activation of the NF-κB p65 subunit in 5 μg of nuclear extracts was measured using an NF-κB p65 ELISA-based transcription factor assay kit  [As an indicator of activated neutrophil accumulation, a major source of reactive oxygen species (ROS), the activity of MPO was determined directly in cell-free BALF according to the method described previously , with mie Motif) ,23.® . Intergroup differences were determined by nonparametric Mann-Whitney U and Kruskal-Wallis tests. Statistical significance was defined as a P value of less than 0.05. Spearman rank correlation coefficient was used to determine the correlations between PARP activity in the tissues and the other parameters examined.All data are expressed as mean ± standard error of the mean. Statistical analysis was performed using SPSS for WindowsP < 0.05). The score of the PJ34+VILI group (2.67 ± 0.67) was significantly lower than that of the VILI group (P = 0.001) and was not different from those of the sham and LPV groups (P > 0.05). W/D weight ratio . In the PJ34+VILI group, the ratio (5.05 ± 0.32) was significantly decreased relative to that of the VILI group (P = 0.012) and was similar to those of the sham and LPV groups (P > 0.05). There were no differences in CDs  . After 2 hours of MV, however, there were statistically significant differences between the three groups ; the CD of the PJ34+VILI group (0.0284 ± 0.0006 mL/cm H2O) was significantly higher than that of the VILI group (0.0244 ± 0.0004 mL/cm H2O) (P = 0.021) and lower than that of the LPV group (0.0316 ± 0.0004 mL/cm H2O) (P = 0.020).Histopathologic examination of the VILI group indicated high levels of ALI parameters Figure . These fo Figure  was alsos Figure  at the bP < 0.05). The number of cells in the PJ34+VILI group (23.50 ± 3.704) was lower than in the VILI group (P = 0.002), but there were no statistically significant differences among the sham, LPV, and PJ34+VILI groups (P > 0.05). In Spearman correlation analysis, PARP activity was positively correlated with the ALI score  and W/D weight ratio  in significance. The CD showed negative correlation with PARP activity .The PARP activity assay showed large numbers of positively stained cells in the VILI group Figure . HoweverP < 0.05). The PJ34+VILI group showed lower activity than the VILI group (P = 0.035) but higher activity than those of the sham and LPV groups (P < 0.05). Spearman correlation analysis showed this activity to be significantly correlated with PARP activity . The concentrations of NO metabolites nitrate and nitrite (NOX) in BALF . The PJ34+VILI group (3.76 ± 0.76 ìM) showed lower levels than the VILI group (P = 0.017) and higher levels than the sham and LPV groups  (P < 0.05). NOX level was also closely correlated with PARP activity .The optical densities (ODs) of the MPO activities in the BALF Figure  were sigF Figure  were alsP < 0.05). IL-6 concentration . The concentrations of inflammatory cytokines were correlated with PARP activity . NF-κB DNA-binding activity measured in lung tissue homogenates . However, NF-κB DNA binding in the PJ34+VILI group (0.91 ± 0.189 OD) was lower than that in the VILI group (P = 0.009) and higher than those in the sham and LPV groups (P < 0.05). NF-κB activity was positively correlated with those of PARP  and the inflammatory cytokines .TNF-α was not detected in BALF of the sham group, and IL-6 was not detected in the sham and LPV groups. TNF-α concentration Figure  in the Vn Figure  in the Ps Figure  was highAlthough the LPV strategy is useful in reducing VILI in patients with ARDS , it is nT [X due to injurious MV could yield peroxynitrite by reaction with increased ROS, along with PARP activity, and inhibition of PARP reduced MPO activity and NOX level. Injurious MV upregulates pulmonary cytokine production, which may result in an inflammatory reaction that aggravates lung injury. Most alveolar cells are capable of producing proinflammatory mediators when stretched in vitro or when ventilated with a large VT in ex vivo and in vivo experiments [ROS, a major cause of lung injury, is an important trigger of DNA damage and PARP activation . AlthougT . Despiteeriments . On the eriments -44. NF-κeriments . Recent eriments ,10,45. Teriments -48. SimiOveractivation of PARP plays an important role in the inflammatory and transcriptional mechanisms of the pathogenesis of VILI. A clearer understanding of the action mechanisms of PARP and modulation of its effects may be clinically useful in the prevention and treatment of VILI in ARDS patients.• The lung-protective ventilation strategy in acute lung injury and acute respiratory distress syndrome (ARDS) may be limited because of severe spatial heterogeneity of lung involvement. Alternative therapeutic strategies based on a precise understanding of the pathophysiology of ventilator-induced lung injury (VILI) are necessary.• In the VILI model of normal mice lung, injurious mechanical ventilation strategy overactivated poly (ADP-ribose) polymerase (PARP), which is known to have important roles in inflammation and nuclear factor-kappa-B (NF-κB)-dependent transcription. The PARP activity was closely related with histopathologic lung injury, inflammatory cytokines, myeloperoxidase activity, nitric oxide, dynamic compliance, and NF-κB activity in the development of VILI.• PARP inhibitor pretreatment protected the development of VILI in relation to the decrease in PARP activity.• Understanding the roles of PARP and modulation of its effects may be clinically useful in the prevention and treatment of VILI in ARDS patients.D: dynamic compliance; ELISA: enzyme-linked immunosorbent assay; HPF: high-power field; IL-6: interleukin-6; LPS: lipopolysaccharide; LPV: lung-protective ventilation; MPO: myeloperoxidase; MV: mechanical ventilation; NAD: nicotinamide adenine dinucleotide; NF-κB: nuclear factor-kappa-B; NO: nitric oxide; NOX: nitric oxide metabolites nitrate and nitrite; OD: optical density; PARP: poly (ADP-ribose) polymerase; PBS: phosphate-buffered saline; PEEP: positive end-expiratory pressure; PIP: peak inspiratory pressure; ROS: reactive oxygen species; TNF-α: tumor necrosis factor-alpha; VILI: ventilator-induced lung injury; VT: tidal volume; W/D: wet-to-dry.ALI: acute lung injury; ARDS: acute respiratory distress syndrome; BAL: bronchoalveolar lavage; BALF: bronchoalveolar lavage fluid; CThe authors declare that they have no competing interests.JHK designed and performed the entire experiment, analyzed the data, and wrote the manuscript. MHS performed the statistical analysis and the interpretation of data. DWY and KHJ participated in the experiments and drafted the manuscript. HYK contributed to the revision of the literature search and to the drafting of the manuscript. EHK, SYL, and SYL performed the literature search. IBS advised about experimental methods. CS, JJS, KHI, and SHY reviewed the final manuscript. KHK conceived of and designed the entire study. All authors read and approved the final manuscript.


Cervus elaphus) on the Isle of Rum, Scotland. Significant genetic variation for both ageing rates in a key maternal performance measure (offspring birth weight) and ELF was present in this population. We found some evidence for a negative genetic covariance between the rate of ageing in offspring birth weight and ELF, and also for a negative environmental covariance. Our results suggest rare support for the AP theory of ageing from a wild population.The antagonistic pleiotropy (AP) theory of ageing predicts genetically based trade-offs between investment in reproduction in early life and survival and performance in later life. Laboratory-based research has shown that such genetic trade-offs exist, but little is currently known about their prevalence in natural populations. We used random regression ‘animal model’ techniques to test the genetic basis of trade-offs between early-life fecundity (ELF) and maternal performance in late life in a wild population of red deer ( Drosophila melanogaster  on the Isle of Rum, Scotland. A key indicator of maternal performance in this population is offspring birth weight  to be associated with increasingly poor maternal performance with age. A previous study of wild mute swans was able to demonstrate genetic correlations between age at first and last reproduction that supported AP theory of ageing .We used random regression ‘animal model’ techniques . Female'y)of female (i) at occasion (j) asμ is the population mean and β1 and β2 are the population mean age slope and its quadratic, respectively. Other fixed-effect terms included were offspring sex, female's reproduction status, offspring birth date, female's age at first reproduction and female's age at last reproduction and its quadratic  relating a focal trait to a covariate . These mai and pei represent the intercept of the additive genetic and permanent environment effect, respectively. The terms aiage agej and peiage agej are the linear slopes with age at additive genetic and permanent environment levels, respectively. This model therefore estimates the variance in intercept and slope and their covariance as random effects at both additive genetic and permanent environment levels. By including the fixed effects for age and its quadratic , the variances in elevation and slope are estimated as deviations from this population mean ageing curve. We tested the significance of the slope terms using model comparison based on likelihood-ratio tests (LRTs). We ran models including additional additive genetic and permanent environment quadratic functions with age, but these did not significantly improve the model fit  or caused the model to fail to converge (permanent environment effect).Between-individual phenotypic variance in covariance functions estimated in a random regression can be further decomposed within an animal model framework into additive genetic and permanent environmental components , using aIn addition, we ran an animal model of ELF with only additive genetic and residual environmental effects and no fixed-effect terms. ELF is measured once across an individual's lifetime and so permanent environment and annual effects cannot be measured. Maternal and birth cohort effects on ELF were tested by fitting mother's identity and year of birth as random effects, but LRTs revealed that these were not significant. We tested the significance of the additive genetic variance component for ELF by comparing models with and without this effect fitted using LRTs.We found evidence for significant additive genetic variation for both ELF and ageing rates in offspring birth weight see . TherefoΧ12=6.00; p=0.015). In the univariate models of offspring birth weight, the additive genetic term for individual slope was significant , as was covariance between genetic elevation and slope terms for offspring birth weight . The permanent environment slope term was not significant  and was dropped from all subsequent models. The genetic covariance between elevation and slope was positive, suggesting an outward fanning pattern of genetic reaction norms . There was also a marginally non-significant, negative additive genetic covariance between ELF and the slope term for offspring birth weight . The genetic covariance between ELF and the intercept of offspring birth weight was weak and non-significant . The negative genetic correlation between offspring birth weight slope and ELF (rA=−0.54) reveals that genes for increased ELF are associated with more rapid declines in offspring birth weight with age. The absence of genetic covariation between offspring birth weight and ELF implies that genes affecting ELF did not influence offspring birth weights in prime age.In the bivariate random regression animal model, additive genetic and residual environmental components of variation in offspring birth weight were negatively correlated with ELF. The genetic and residual environment (co)variances estimated in the bivariate model are presented in table 1b). In all the cases, the estimated variance and covariance terms were in the same direction and of similar magnitude, but had larger standard errors due to the restricted sample size . Additive genetic variation for ageing rates in reproductive performance remained significant among these older females . The genetic correlation between ELF and the age slope for offspring birth weight remained negative and was almost identical in magnitude to correlation in the full model (rA=−0.55) but, presumably owing to the substantially reduced sample size and therefore power in the restricted model, no longer approached significance . The consistency of direction and magnitude of the estimates in table 1a,b suggests that the patterns observed here were not due to effects specific to early adulthood.Re-running the above bivariate random regression model having excluded offspring birth weights from females in early adulthood (aged below 9 years), yielded qualitatively similar results b. In alla were back-transformed to generate a G matrix of age-specific predictions of additive genetic variance for ELF and offspring birth weight and of covariance between the traits. Age-specific predictions of additive genetic variance in offspring birth weight increased with age  between these two traits changed from +0.40 at age 3 to −0.17 at age 15.The additive genetic covariance functions in table 1with age . Althougwith age . The estCygnus olor) in a fitness-correlated trait , suggesting that both environmental and genetic factors could underlie the observed trade-off at the phenotypic level. However, our analyses do assume that residual environmental variance in offspring birth weight is constant with age and so this finding should be interpreted with caution. Models estimating age-specific residual variance components have been applied to univariate analyses in wild pedigreed populations variance components without the loss of power suffered by splitting data into separate age classes (


Several obesity risk alleles affecting adult adiposity have been identified by the recent wave of genome wide association studies. We aimed to examine the potential effect of these variants on fetal body composition by investigating the variants in relation to birth weight and ponderal index of the newborn.th percentile), normal (10th–90th percentile) and high birth weight (≥90th percentile) to allow for non-linear associations. There was no difference in the number of risk alleles between the groups (p = 0.57). No interactions between each risk allele and birth weight in the prediction of adult BMI were observed. An obesity risk score was created by summing up risk alleles. The risk score did not associate with fetal body composition. Moreover there was no interaction between the risk score and birth weight/ponderal index in the prediction of adult BMI.Midwife records from the Danish State Archives provided information on mother's age, parity, as well as birth weight, birth length and prematurity of the newborn in 4,744 individuals of the population-based Inter99 study. Twenty-four risk alleles showing genome-wide associations with adult BMI and/or waist circumference were genotyped. None of the 24 risk variants tested showed an association with birth weight or ponderal index after correction for multiple testing. Birth weight was divided into three categories low (≤1024 common variants associated with adult adiposity did not affect or interact with birth weight among Danes suggesting that the effects of these variants predominantly arise in the post-natal life. Obesity has become a worldwide health epidemic with increasing prevalence. Several studies have demonstrated that birth weight is associated with Body Mass index (BMI) in later life Although the mechanisms behind the association are far from clarified, both genetic and environmental factors seem to play a role. From an environmental point of view the association between high birth weight and adult obesity may in part be explained by alterations in the intrauterine environment mediated by maternal obesity or maternal hyperglycaemia during pregnancy, as it has been shown that newborns of diabetic or overweight mothers are heavier TMEM18, ETV5 (SFRS10), BDNF, FTO and MC4R loci have been examined for associations with birth weight previously. No association with birth weight has been observed for variants in or near TMEM18, ETV5 (SFRS10), BDNF and MC4R loci FTO locus and birth weight FTO obesity risk allele to be associated with higher BMI and ponderal index at birth Few studies have investigated the genetic contribution of the association between birth weight and adult obesity. A total of 24 gene variants, influencing adult adiposity, have been identified by the recent wave of genome-wide association studies (GWAS) FTO, MC4R, TMEM18, GNPDA2, KCTD15, NEGR1, BDNF and ETV5 (SFRS10) loci has been tested for an association with birth weight in 7,146 individuals Moreover, an obesity-risk-allele score, comprising variants in or near the These previous studies do not indicate that adult obesity loci may contribute strongly to the variation in birth weight, however, the effect of many obesity risk variants recently identified have not yet been investigated.We hypothesized that some of the genetic variants associated with adult adiposity may have an impact on fetal growth and therefore explain part of the link between birth weight and subsequent risk of obesity. Therefore, we aimed to test the single effect of each variant as well as the combined effect of the 24 variants on birth weight and ponderal index in the Inter99 population. Since previous studies have reported both high and low birth weight to associate with BMI, it is possible that these phenotypes and the genetic risk variants may have an interactive effect on adult BMI, e.g. the risk variants may have a stronger effect in those born with birth weights lying near the ends of the distribution. For that reason, we also aimed to test for an interactive effect between high/low birth weight and the genetic variants in the prediction of adult BMI.www.inter99.dk) 3). Information about the mother's diabetes status was obtained by a questionnaire during the baseline visit in 1999–2001. The age of onset of maternal diabetes was not registered.Ethics statement: All participants gave written informed consent and the protocol was in accordance with the Helsinki Declaration, approved by Copenhagen County ethic committee and registered with ClinicalTrials.gov (NCT00289237). Individuals examined in the present study were from the Danish Inter99 Study, which at baseline comprised 6,784 individuals living in the suburb of Copenhagen. The Inter99 is a population-based randomised non-pharmacological intervention study aimed to prevent ischemic heart disease. The study was conducted at the Research Centre for Prevention and Health in Glostrup, Denmark (Pregnancies were considered at term when gestation attained 36 completed weeks and did not exceed 41 completed weeks. Participants born preterm (n = 446) or as part of a multiple pregnancy (n = 85) were excluded, since these newborns have a lower birth weight presumably due to non-genetic factors. Preterm birth was a clinical assessment by the midwife based on the pregnancy due date together with clinical signs of prematurity. The final number of individuals included in the study was 4,213. All individuals were Danes by self report.MC4R, NRXN3, TFAP2B and MSRA were initially identified to associate with waist circumference MC4R locus in strong linkage disequilibrium with rs12970134 (HapMap CEU: r2 = 0.81) was simultaneously found to associate strongly with BMI and risk of obesity NRXN3 rs10146997 was also associated with BMI and the association with waist circumference was completely attenuated when adjusting for BMI TFAP2B rs987237 was at genome-wide significance level associated with BMI in addition to waist circumference in the main paper MSRA rs545854 was only weakly associated with BMI but the signal for waist circumference was abolished when adjusting for BMI In order to include variants affecting overall adiposity, SNPs were selected based on their previous genome-wide association with adult BMI/obesity and waist circumference. BMI and waist circumference are highly correlated measures of adiposity and although variants in or near Genotyping was performed as described previously for 19 of the variants http://www.r-project.org). Z-scores were calculated as z-score  =  (x-mean)/sd. The effect of the genetic variants on birth weight and ponderal index was calculated using linear regression models adjusted for sex, maternal diabetes (yes vs. no/NA) and parity . No transformation of data was performed. Only additive genetic models were considered assuming a constant change in birth weight per risk allele and p<0.05 was considered statistically significant. The Bonferroni corrected threshold for 24 tests was p<0.0021. Individuals were classified into groups of birth weight; newborns born at or below the sex-specific 10th percentile in the Inter99 population were defined as having low birth weight, whereas those born at or above the 90th percentile were defined as having high birth weight. Age and sex-adjusted interaction analyses between each risk allele and low-normal-high birth weight were performed in relation to adult BMI. An un-weighted genetic risk score was calculated by summing up risk alleles assuming similar effects of all alleles. A total of 2,926 individuals had a genotype call for all variants and could be included in the analyses. The association between the risk score and adult BMI was performed by using linear regression adjusted for age and sex. The association between the risk score and birth weight/ponderal index was tested by linear regression adjusted for sex, maternal diabetes status and parity. The association between the risk score and groups of birth weight was calculated by a one-way anova. The interaction between the risk score and birth weight were tested in relation to adult BMI adjusted for age and sex using linear regression. Statistical power was estimated using 1000 simulations. We used the empirical variance of the observed traits adjusted for sex, maternal diabetes and parity to simulate phenotypes from a normal distribution, so that variance across genotypes is drawn from the estimated variance.All statistical analyses were performed using RGui version 2.8.1 .The 24 genetic variants were then investigated for an association with birth weight and ponderal index in the Inter99 population –S2. Noneth percentile), normal (10th–90th percentile) and high birth weight (≥90th percentile). Each risk variant was tested for an interaction with these groups of birth weight in the prediction of adult BMI. No interactions were observed between each risk allele and groups of birth weight in the prediction of adult BMI (Supplementary Birth weight was divided into three categories low (≤10−13),  , . The ris−13), . Moreove−13), . We also−13), . Moreove−13), .MTCH2-rs10838738 obesity risk variant was associated with a decreased ponderal index, however this association did not remain significant after accounting for multiple testing and it is therefore likely to be a chance finding. Moreover, an obesity risk score was strongly associated with adult BMI in the subsample of Inter99 with birth weight data available, but it did not associate with birth weight and ponderal index. We did not observe any interactions between risk variants and birth weight in the prediction of adult BMI.We found that none of the 24 genotypes which have a confirmed association to adult adiposity are associated with birth weight. The et al. found a modest association between an obesity risk score comprising alleles from FTO, MC4R, TMEM18, GNPDA2, KCTD15, NEGR1, BDNF and ETV5 (SFRS10) loci and BMI at birth in 7,146 individuals. When constructing a similar risk score in 3,496 individuals from Inter99 with genotype and birth weight data available no significant association was found (data not shown). This may be due to a lower statistical power in the present study, but as discussed by Elks and colleagues the obesity risk variants seem to have a larger effect on weight gain during infancy and childhood.Our results therefore suggest that the risk variants affecting adult adiposity do not have a strong impact on size at birth and therefore are not likely to explain the association between birth weight and adult BMI. Supporting this notion, previous studies have not found altered birth weight in carriers of mutations causing monogenic forms of obesity It is well known that the central nervous system and especially areas of hypothalamus play key roles in whole body energy regulation. The majority of the genes, in which mutations cause monogenic obesity, are involved in the control of appetite and food intake through the hypothalamus We hypothesized that the examined gene variants known to affect adult adiposity could be affecting the individual from as early as pre-natal life by facilitating energy uptake and storage and thus weight regulation of the fetus. In the present study we did, however, not find any genetic support for this hypothesis, suggesting that the genetic components of fetal and adult weight gain are largely independent. During pregnancy, multiple factors including nutrient supply, placental function and available space are determinants of fetal growth, indicating a major importance of external factors on the growth of the fetus. Thus, it is likely that the central appetite regulation is mainly activated or only matures in post natal life, when the external environment becomes a less marked limiting factor for the intake of nutrients and subsequent growth. Moreover, the lack of an interactive effect between genetic risk and birth weight suggests that these variables influence adult BMI and risk of obesity independently in this cohort.We have recently reported that specific type 2 diabetes risk alleles influence birth weight in the Inter99 population A total of 11 of the 24 investigated genes were significantly associated with adult BMI in the subsample of Inter99 with birth weight data available. For these variants, the effects of the risk alleles are therefore most likely to arise during post-natal life. The statistical power to detect minor changes in quantitative variables such as birth weight may be low for the remaining variants. In the Danish Inter99 study, we have more than 80% statistical power to detect effects in birth weight of 30 g, 35 g and 45 g assuming minor allele frequencies of 10%, 20% and 40%, respectively, and we can thus exclude effects on birth weight within this range.It could have been of interest if the alleles with the strongest effect on adult BMI also had the strongest effect on birth weight although not reaching statistical significance in this study sample. We have illustrated this in A recent study comprising 20 of the variants investigated in the present study, found that the 20 variants explained only 4.5% of the obesity status in the Inter99 cohort Lack of exact information regarding gestational age between week 37 and 42 is a limitation of this study. However, none of the investigated variants have to our knowledge been associated with gestational age and thus an even genotype distribution can be anticipated. Moreover, in a recent meta-analysis three studies without information of gestational age did not introduce heterogeneity in to the results It is known from previous studies that the maternal genotype can interact with the fetal genotype in the determination of birth weight suggesting a complex pattern for the regulation of fetal growth In conclusion, 24 common variants associated with adult adiposity did not have a strong effect on birth weight in the Danish Inter99 population. Also no interactions between the genetic variants and birth weight in the prediction of adult BMI was observed.Table S1Fetal genotype and birth weight in 4,213 individuals from the Danish Inter99 study population. Bonferroni threshold for 24 test is p<0.0021. Data are means +/− standard deviation of birth weight (g). Effects and p-values are calculated assuming an additive genetic model adjusted for sex, maternal diabetes status and parity. Pint, p-value for the interaction between each variant and groups of birth weight in relation to adult BMI. CI, confidence interval.(0.15 MB DOC)Click here for additional data file.Table S2Fetal genotype and ponderal index in 4,213 individuals from the Danish Inter99 study population. Bonferroni threshold for 24 test is p<0.0021. Data are means +/− standard deviation of ponderal index (kg/m3). Effects and p-values are calculated assuming an additive genetic model adjusted for sex, maternal diabetes status and parity. CI, confidence interval.(0.08 MB DOC)Click here for additional data file.


Please cite this paper as: Urquia M, Frank J, Moineddin R, Glazier R. Immigrants’ duration of residence and adverse birth outcomes: a population-based study. BJOG 2010;117:591–601.This study aimed to examine preterm and small-for-gestational-age (SGA) births among immigrants, by duration of residence, and to compare them with the Canadian-born population.Population-based cross-sectional study with retrospective assessment of immigration.Metropolitan areas of Ontario, Canada.A total of 83 233 singleton newborns born to immigrant mothers and 314 237 newborns born to non-immigrant mothers.We linked a database of immigrants acquiring permanent residence in Ontario, Canada, in the period 1985–2000 with mother–infant hospital records (2002–2007). Duration of residence was measured as completed years from arrival to Canada to delivery/birth. Logistic regression models were used to estimate the effects of duration of residence with adjusted odds ratios and 95% confidence intervals. In analyses restricted to immigrants only, hierarchical models were used to account for the clustering of births into maternal countries of birth.Preterm birth (PTB) and SGA birth.Recent immigrants (<5 years) had a lower risk of PTB (4.7%) than non-immigrants (6.2%), but those with ≥15 years of stay were at higher risk (7.4%). Among immigrants, a 5-year increase in Canadian residence was associated with an increase in PTB , but not in SGA birth .Time since migration was associated with increases in the risk of PTB, but was not associated with an increase in SGA births. Ignoring duration of residence may mask important disparities in preterm delivery between immigrants and non-immigrants, and between immigrant subgroups categorised by their duration of residence. The reduction of preterm birth (PTB) represents one of the most important challenges in perinatal health. Preterm birth is a major predictor of perinatal morbidity and mortality, and is associated with childhood disabilities and adult onset of diseases.3Immigrant women contribute to more than one-fifth of all live births in the United States,20The assessment of health disparities by immigrants’ duration of residence involves two relevant comparisons. First, birth outcomes of immigrant subgroups defined by time in the receiving country can be compared with those of native-born women. Such a comparison allows the assessment of whether any disparities observed between newly arrived immigrants and non-immigrants change with increasing time spent in the new country. One common assumption in the literature is provided by the ‘convergence hypothesis’, inspired by the studies on coronary heart disease and stroke gradients of Japanese men living in Japan, Hawaii, and California.21The second comparison is among immigrants, and assesses whether health outcomes of recent immigrants differ from those with a longer time of residence. This comparison may provide clues about environmental influences, and may be relevant from a surveillance standpoint.Comparisons by duration of residence may also help clarify other hypotheses regarding immigration and health, such as the so-called ‘epidemiologic paradox’ and the ‘healthy migrant effect’. The epidemiologic or ‘Hispanic paradox’ refers to the findings that Mexicans in the USA tend to have lower rates of low birthweight than USA-born women, despite their lower socioeconomic status.Our first objective was to test the convergence hypothesis for PTB and small-for-gestational-age (SGA) birth in urban Ontario: that is, to compare immigrant subgroups defined by their duration of residence versus non-immigrants.Our second objective, restricted to immigrants only, was to examine changes in the occurrence of PTB and SGA birth according to maternal duration of residence after arrival.Urban Ontario is an appropriate setting to study the health of immigrants as Ontario is Canada’s most populous province, and annually receives about half of all immigrants arriving in Canada (approximately 120 000 each year), with more than 90% of them concentrated in urban areas.28This is a population-based, data-linkage, cross-sectional study with retrospective assessment of immigration characteristics. Outcomes, and some maternal and obstetric characteristics, were obtained from hospital records within the most recent 5-year period (April 2002–March 2007), in order to ensure that their measurement was the same for all of the study subjects, and not affected by secular trends in the outcomes or by changes in coding schemes and reporting practices over time. Duration of residence and other immigration characteristics were retrospectively assessed via a data linkage with an immigration database (January 1985–December 2000), mostly built on legal documentation provided by the immigrants during their application processes.The Discharge Abstract Database (DAD) of the Canadian Institute for Health Information (CIHI) compiles information on admissions, services, and discharges of all acute Ontario hospitals, where approximately 99% of provincial deliveries take place, excepting home births. The DAD is an excellent source for population-based estimates of perinatal outcomes.n = 360), because of their high likelihood of being data errors; births with missing information on birthweight or gestational age (n = 125); births with gestational ages of less than 22 and more than 43 completed weeks (n= 72); births with missing information on infant sex, parity, or maternal age (n= 54); births with missing information on maternal sociodemographic and immigration characteristics (n= 576); and births to immigrants classified as ‘other’ (n= 487). After excluding 509 records to which census information could not be assigned, our study population for analyses comprised 397 470 singleton live births (with 83 233 born to immigrant mothers).We extracted all 474 614 singleton live births born to mothers having a valid provincial health-card number (to ensure that they were Ontario residents) and living in any of the 11 Ontario Census Metropolitan Areas All outcomes were categorised into binary variables. Preterm birth was defined as a delivery before 37 weeks of gestation completed, and was divided into preterm subgroups . In Ontario, gestational age is largely estimated by ultrasound dating, and since 2002 hospital medical records departments have recorded gestational age based on the attending physician’s best interpretation of all clinical data, backed up by documentation from nursing staff as a secondary source.34Mothers were categorised as foreign-born if listed in the immigration database. Non-immigrants included Canadian-born women and some immigrant women with a residency of longer than 17 years (i.e. permanent residence obtained before 1985). As information on sociodemographic characteristics was only available from the immigration data, we could not measure these characteristics among the non-immigrant group. Information from hospital records available for the entire study population was measured at the time of delivery/birth, and included infant sex , maternal age at delivery , and parity (primiparae versus multiparae).Information specific to immigrants was obtained from their landing records, last updated at the port of entry on the date of their arrival to Canada. With the exception of language knowledge, which was self-reported, the remaining information was ascertained based on legal documentation provided by the immigrants during their application processes. We assessed the level of exposure to the Canadian setting by duration of residence in Canada as completed years from arrival to delivery. We also modelled duration in approximately 5-year duration groups .Relevant covariates included maternal country of birth, high school graduation (no versus yes), marital status , immigrant class , and knowledge of either of the official Canadian languages (none versus English or French). Countries of birth were grouped into world regions using a modified version of the UNICEF classification, with industrialised countries as the reference group .35 We ex37We inspected the shape of the relationship between duration of residence and adverse birth outcomes using bivariate plots and logistic models with linear, quadratic, and cubic terms, as well as with dummy variables. A linear specification of duration resulted in a better fit, as assessed by the likelihood ratio test.To compare outcomes of all immigrants and immigrant subgroups versus non-immigrants we used logistic regression to compute odds ratios with 95% confidence intervals, adjusted for maternal age and parity at delivery.sas 9.1  were used to estimate the effects of duration of residence on the outcomes, with 5-year adjusted odds ratios with 95% confidence intervals. We also calculated the predicted probabilities of PTB according to length of residence in Canada. Finally, to assess whether the association between duration of residence and birth outcomes varied according to the maternal region of birth, we tested for interaction by including a product term (duration × region of birth) in the adjusted models.Analyses restricted to immigrants only were more complex because of the availability of richer information. To account for the clustering of births within maternal countries of birth among immigrants, we modelled countries as two-level random intercepts. Multilevel logistic regression analyses using GLIMMIX in We repeated multivariate analyses with the excluded variables infant sex, maternal morbidity during pregnancy, induction of labour, caesarean section, and neighbourhood deprivation. We further adjusted for year of arrival to evaluate the possibility that the observed associations between duration of residence and adverse birth outcomes were confounded by cohort effects. We also adjusted for the infant’s year of birth to eliminate any residual confounding resulting from trends in PTB within the study period. Finally, we restricted analyses to immigrant women who arrived at age 20 or older to reduce the misclassification of maternal education and marital status.Immigrants differed from non-immigrants in the outcomes and in most covariates . ImmigraCompared with non-immigrants, all immigrants, as a single category, had similar odds of PTB (<37 weeks) . When imDuration of residence was independently associated with PTB and all preterm subgroups in multilevel models further adjusted for maternal sociodemographic and immigration characteristics, although duration did not reach statistical significance for infants born within 28–31 weeks of gestation . AlthougFinally, an interaction between duration and maternal region of birth was not significant for all outcomes. Stratified analyses by maternal region of birth were consistent, and showed increases in PTB with increasing duration of stay for all regions of the world.post hoc analyses restricted to preterm infants we did not find statistically significant trends in induction of labour (P= 0.187) or caesarean sections (P= 0.074) with duration of residence.Although we based our covariate adjustment on theoretical considerations, we also fitted the models with the excluded variables maternal morbidity during pregnancy, induction of labour, caesarean section, neighbourhood deprivation, and infant sex, but the results of Our findings indicate that the duration of residence of immigrants in urban Ontario was independently associated with increases in PTB, but not in SGA. Recent immigrants were at lower risk of PTB compared with a mostly Canadian-born population, but immigrants became at higher risk after 10 years of stay in Canada. There was no strong evidence indicating that the influence of duration of residence on birth outcomes varied with the maternal region of birth or with preterm subgroups.Our study had some limitations. Non-matched immigrants who stayed in Ontario after arrival were counted as non-immigrants, thus diluting the differences in outcomes between these two groups. Because data collection on immigrants started in 1985, we could not identify those immigrants who obtained their permanent residency before 1985. This misclassification, however, would bias our results against significance, because false non-immigrants were immigrants with at least 17 years of residence, and so were at a higher risk of PTB. Because immigration data were linked until 31 December 2000, we had to exclude all newcomers to Ontario after that date to avoid misclassification by immigrant status. This resulted in some under-representation of recent immigrants in our study population. Although the immigration data were of good quality, some variables were measured on arrival and not at delivery, which is a concern for time-dependent variables. Marital status and maternal education may have changed for some women, especially for young women on arrival, who may have got married and become more educated with a longer duration of residence. As educational attainment is not reversible, the bias resulting from adjustment for an underestimate of the true educational attainment of long-term immigrants would have reduced the significance of our results, because higher education protects against adverse birth outcomes. To reduce this bias, we repeated analyses restricted to mothers aged 20 years or older on arrival, but the results did not substantially change.The use of a Canadian standard for SGAFinally, we had no data on some important predictors of the outcomes, such as tobacco smoking, alcohol consumption, and maternal height, weight, or body mass index (BMI). However, these are mediators rather than confounders of the association between duration of residence and birth outcomes.Despite these limitations, the association between duration of residence and PTB was quite robust, remaining unaltered across a range of sensitivity analyses. Our main finding regarding a linear increase in PTB with time spent in Canada is consistent with previous studies on Mexicans showing increases in PTB after 2 years of residence in Washington,The increase in PTB rates observed in the USA and Canada during the last two decades has been mainly attributed to increased obstetric interventions near term to prevent neonatal complications.Our findings do not support the ‘convergence hypothesis’, which seems to hold for increased BMI/obesityRegarding the frequently debated epidemiologic paradox of low birthweight and PTB and the healthy migrant effect, it is noteworthy that these two hypotheses have been largely discussed in the literature in the absence of information on duration of residence. Our findings on PTB suggest that these two phenomena may apply only to recent immigrants. Although studies reporting the epidemiologic paradox in PTB could not distinguish between recent and long-term immigrants, it is likely that the results were driven by the healthier recent immigrants, as most immigrant women have their babies within 10 years of migration. Recent immigrants may enjoy better health than the native-born women soon after arrival, but lose the short-term benefits of selective migration with cumulative exposure to the new physical and social environment, experiencing a sort of ‘regression to the mean’.42The lack of an association between duration and SGA births in our study suggests that the influence of duration of residence on perinatal health may be outcome-specific. Although our data were not detailed enough to elucidate mechanisms explaining the deterioration of PTB among immigrants, we can advance some hypotheses. One candidate explanation is acculturation, which has already been linked with adverse birth outcomes.Another potential pathway leading to an association between duration of residence and PTB, but not SGA, albeit unexplored among immigrant women, may involve psychosocial factors. A few studies found that different measures of maternal stress were associated with PTB, but not with intrauterine growth restriction.65Previous studies have described increases in PTB rates among Mexicans with prolonged residency in the USA. This study was conducted on an ethnically diverse immigrant population, and the association between duration and PTB did not vary according to the world region of maternal origin. This suggests that not only Hispanics but all immigrants were equally affected by prolonged residency in urban Ontario. However, our findings cannot be generalised to rural areas, urban settings with distinctive immigrant groups, or to other health outcomes, without additional empirical research.Immigrants’ duration of residence was associated with increases in the risk of preterm delivery, but not SGA births. This suggests the presence of outcome-specific processes related to duration of residence. The fact that important changes in preterm delivery take place within relatively short periods of time after migration suggests that their causes are mainly environmental, and therefore at least theoretically preventable. Ignoring duration of residence may mask important disparities in preterm delivery between immigrants and non-immigrants, and between immigrant subgroups. The clarification of the mechanisms behind the association between duration of residence and PTB merits further investigation.The authors declare that they have no competing interests.MU conceived the study, performed the analyses, and led the writing of the manuscript. JF and RG supervised the study, helped to interpret the results, and revised the manuscript for important intellectual content. RM helped with the statistical methods and helped interpret the results. All authors read and approved the final version of the manuscript.The use of the data was approved by the Research Ethics Boards of the Sunnybrook Health Sciences Centre and the University of Toronto (protocol reference #23550), Toronto, Ontario.No funding was sought or obtained to undertake this specific study. MU was supported by a personal research grant (CIHR IOP-44972) from JF, Canadian Institutes of Health Research.


In an analysis of a cohort of pregnant women, Radek Bukowski and colleagues describe an association between taking folic acid supplements and a reduction in the risk of preterm birth. Low plasma folate concentrations in pregnancy are associated with preterm birth. Here we show an association between preconceptional folate supplementation and the risk of spontaneous preterm birth.p = 0.004) and a 50% decrease in the risk of spontaneous preterm delivery between 28 and 32 wk . Adjustment for maternal characteristics age, race, body mass index, education, marital status, smoking, parity, and history of prior preterm birth did not have a material effect on the association between folate supplementation for 1 y or longer and spontaneous preterm birth between 20 and 28, and 28 to 32 wk . Preconceptional folate supplementation was not significantly associated with the risk of spontaneous preterm birth beyond 32 wk. The association between shorter duration (<1 y) of preconceptional folate supplementation and the risk of spontaneous preterm birth was not significant after adjustment for maternal characteristics. However, the risk of spontaneous preterm birth decreased with the duration of preconceptional folate supplementation  and was the lowest in women who used folate supplementation for 1 y or longer. There was also no significant association with other complications of pregnancy studied after adjustment for maternal characteristics.In a cohort of 34,480 low-risk singleton pregnancies enrolled in a study of aneuploidy risk, preconceptional folate supplementation was prospectively recorded in the first trimester of pregnancy. Duration of pregnancy was estimated based on first trimester ultrasound examination. Natural length of pregnancy was defined as gestational age at delivery in pregnancies with no medical or obstetrical complications that may have constituted an indication for delivery. Spontaneous preterm birth was defined as duration of pregnancy between 20 and 37 wk without those complications. The association between preconceptional folate supplementation and the risk of spontaneous preterm birth was evaluated using survival analysis. Comparing to no supplementation, preconceptional folate supplementation for 1 y or longer was associated with a 70% decrease in the risk of spontaneous preterm delivery between 20 and 28 wk  are born prematurely (before 37 completed weeks of pregnancy). Sadly, premature babies are more likely to die than full-term babies and many have short- and/or long-term health problems. Premature babies often have breathing problems, they are susceptible to life-threatening infections, and they are more likely to have learning and developmental disabilities than those born on time. The severity of these health problems depends on the degree of prematurity—preterm babies born between 34 and 36 weeks of pregnancy rarely develop severe disabilities, but a quarter of babies born before 28 weeks of pregnancy develop serious lasting disabilities and half have learning and behavioral problems. Although doctors have identified some risk factors for early delivery , it is impossible to predict who will have an early birth and there is no effective way to prevent preterm births.Some researchers think that folate supplements may prevent preterm births. Folate (folic acid), a vitamin found in leafy green vegetables, fruits, and dried beans, helps to prevent neural tube birth defects. Consequently, women are encouraged to take folic acid supplements throughout (and preferably before) pregnancy and many governments now mandate that bread, pasta, and other grain products be fortified with folic acid to help women get sufficient folate. There is some evidence that women who deliver early have less folate in their blood than women who deliver at term. Furthermore, folate supplementation during pregnancy has increased the length of pregnancy in some but not all clinical trials. A possible explanation for these mixed results is that the duration of pregnancy reflects conditions in the earliest stages of pregnancy or before conception and that folate supplementation needs to start before conception to reduce the risk of preterm birth. In this study, the researchers test this idea by analyzing data collected from nearly 35,000 pregnant women enrolled in a study that was originally designed to investigate screening for Down's syndrome.During the first three months of their pregnancy, the women were asked whether they had taken folate supplements before conception. The duration of each pregnancy was estimated from ultrasound measurements taken early in the pregnancy and from the time of delivery. During the study, 1,658 women had spontaneous preterm deliveries before 37 weeks and 160 delivered before 32 weeks. After allowing for other maternal characteristics that might have affected the likelihood of preterm delivery, the risk of spontaneous preterm delivery between 20 and 28 weeks was 70% lower in women who took folate supplements for more than a year before becoming pregnant than in women who didn't take a supplement. Long-term folate supplementation also reduced the risk of preterm delivery between 28 and 32 weeks by 50% but did not affect the risk of preterm birth beyond 32 weeks. Folate supplementation for less than a year before conception did not reduce the risk of preterm birth, and folate supplementation was not associated with any other complications of pregnancy.These findings show that folate supplementation for a year or more before conception is associated with a 50%–70% decrease in early (but not late) spontaneous preterm births and that the longer a woman takes folate supplements before becoming pregnant, the lower her risk of a preterm birth. Although the researchers allowed for maternal characteristics that might have affected the duration of pregnancy, it is possible that folate supplementation may not be responsible for the reduction in preterm birth risk seen in this study. For example, taking folate supplements may be a marker of healthy behavior and the women taking the supplements might have been doing something else that was reducing their risk of preterm birth. However, despite this and other limitations of this study, these findings suggest that long-term folate supplementation before conception is worth investigating further as a potential way to prevent preterm births.http://dx.doi.org/10.1371/journal.pmed.1000061.Please access these Web sites via the online version of this summary at PLoS MedicinePerspective by Nicholas FiskThis study is further discussed in a premature babies (in English and Spanish); MedlinePlus provides links to other information on premature babies (in English and Spanish)The MedlinePlus encyclopedia contains a page on preterm labor and birthThe US National Institute of Child Health and Human Development provides information on preterm birth and on folic acid (in English and Spanish)The March of Dimes, a nonprofit organization for pregnancy and baby health, provides information on premature babies (in English and Spanish)The Nemours Foundation, another nonprofit organization for child health, also provides information on folateThe US Office of Dietary Supplements has a fact sheet on  Preterm birth is the main cause of neonatal mortality and short- and long-term morbidity. Neonatal mortality and morbidity are inversely correlated with gestational age at delivery, with the majority of those adverse outcomes associated with early preterm birth  Unfortunately there are no effective methods of prevention or treatment of preterm birth. The most promising intervention—progesterone supplementation—to date has been shown to be effective only in a small subset of women at risk for preterm birth Several studies demonstrated an association between lower concentrations of folate and shorter duration of pregnancy. Observational studies have showed that women delivering preterm have lower concentrations of folate in plasma or red blood cells However, a growing body of evidence of human studies and animal models suggests that duration of pregnancy may be the ultimate consequence of conditions in the very earliest stages of pregnancy or before conception In a secondary analysis of a previously described prospective cohort study Extensive information on patients' demographics, medical and obstetrical history, socioeconomic status measures, and exposures during pregnancy were recorded. Copies of medical records were reviewed in all cases where parents reported a possible neonatal medical problem, in all Down syndrome screen–positive cases without karyotype results, and in a 10% random sample of all other enrolled cases by a single abstractor. Institutional review board approval was obtained in all participating centers, and all participants gave written informed consent.Out of 42,367 patients approached, 38,033 patients were enrolled. Of those enrolled 189 (0.5%) had missing data on folate supplementation, 430 (1.1%) missing confounder data, 529 (1.4%) gestational age at delivery >42 wk, 2,103 (5.5%) missing gestational age at delivery, and 302 (0.8%) miscarried or aborted pregnancy between enrollment and 20 wk. Complete data on maternal characteristics, exposures, pregnancy complications, and outcomes were available for 34,480 (91.4%) of the remaining pregnancies.Duration of consistent preconceptional folate supplementation, with or without multivitamins, was self-reported in the first trimester of pregnancy at the time of enrollment and patients were followed until delivery. Duration of preconceptional folate supplementation was reported as: <1, 1–2, 3–4, 5–6, 7–11, and >12 mo and was categorized into three groups: ≥1 y, <1 y, and no preconceptional supplementation.Maternal age was defined as maternal age at the time of birth and categorized into <35 and ≥35 y to reflect advanced maternal age associated with increased risk of adverse pregnancy outcomes. Race and ethnicity were self-reported by mother in the first trimester of pregnancy. Maternal height and weight obtained in the first trimester of pregnancy, respectively in cm and kg, were included in calculation of body mass index. Body mass index was categorized into three groups: <20, 20–30, and >30. Maternal education level was self-reported in total completed years of education and categorized into ≤12 or >12 y. Marital status was defined as married or not married. Both education and marital status were used as measures of socioeconomic status. Smoking was defined as a self-reported smoking status of the woman in the first trimester. Prior preterm birth was defined as prior pregnancy lasting less than 37 wk 0 d.Duration of pregnancy in all women was based on ultrasound measurement of crown–rump length in the first trimester. Preterm birth was defined as duration of pregnancy between 20 wk 0 d and 36 wk and 6 d Spontaneous preterm birth was defined as preterm birth not associated with medical or obstetrical complications constituting indications for preterm delivery. Those indications included: chromosomal abnormalities, structural malformations, stillbirth, termination of pregnancy, pregnancy induced hypertension, preeclampsia, chronic hypertension, gestational or pregestational diabetes, placenta previa, and placental abruption.Small for gestational age was an infant with birth weight below the 10th percentile for gestational age Duration of preconceptional folate supplementation, categorized ≥1 y, <1 y, and no supplementation, was compared across maternal and obstetric characteristics. Univariate comparison of continuous variables was performed using the Kruskal-Wallis test and of categorical data using the Chi-square or Fisher exact tests.p-values are two-sided. Statistical significance is assumed at p<0.05.All The risk of spontaneous preterm birth between the categories of duration of preconceptional folate supplementation was compared using time-to-event analysis. The gestational age from 20 wk onward was used as the time scale. Gestational age at delivery between 20 wk 0 d and 36 wk 6 d was taken as time of the event in the absence of indications for delivery or as the time of censoring in either their presence or if gestational age at delivery was 37 wk 0 d or later. This approach allows assessment of the variation in relative risk of spontaneous preterm birth with gestational age.http://www.stata.com/).The time to event data are presented as a cumulative percentage of the event, as recommended for rare events In the study population of 34,480 women, preconceptional folate supplementation was used by 6,777 (19.6%) for ≥1 y, by 12,444 (36.1%) for <1 y, and not at all by 15,259 (44.3%) . Preconcp = 0.01; A total of 2,660 women (7.7%) delivered prematurely before 37 wk, and 473 (1.4%) before 32 wk. Spontaneous preterm birth occurred in 1,658 women (4.8%) before 37 wk and in 160 (0.5%) before 32 wk . The risThe protective effect of folate supplementation decreased with advancing gestational age . Comparep<0.0001 and p = 0.006 for duration of folate supplementation for more or less than a year, respectively). However, for gestational age intervals 20 to 28 (20 wk 0 d to 27 wk 6 d), 28 to 32 (28 wk 0 d to 31 wk 6 d) and 32 to 37 wk (32 wk 0 d to 36 wk 6 d) risks of spontaneous preterm birth were constant and proportional hazard assumption was met . Hence, the preterm birth gestational age intervals evaluated in this study were identified by testing proportional hazard assumption, rather than selected arbitrarily and are the gestational age intervals with constant hazard ratios for spontaneous preterm birth.The proportional hazard assumption was violated for the gestational age interval from 20 to 37 wk, indicating that the risk of spontaneous preterm birth in relation to duration of folate supplementation varies during this interval . Thus there was a significant association between duration of preconceptional folate supplementation and the risk of preterm birth before 32 wk when pregnancies with complications constituting indication for delivery were censored or not. However, there was no significant association between duration of preconceptional folate supplementation and the risk of non-spontaneous, complicated by obstetrical or medical conditions, preterm birth.Duration of preconceptional folate supplementation was also associated with significantly reduced risk of all preterm birth, spontaneous or not, in a similar manner (test for trend of survivor functions p = 0.042) and proportion of spontaneous preterm birth before 32 wk (test for trend p<0.0001) . A gradient is evident with less than 1 year of supplementation in the trend of survivor functions (test for trend of survivor functions <0.0001) .Adjustment for the range of characteristics  had only a minimal effect on the association between preconceptional folate supplementation for year or longer and spontaneous preterm birth at 20 to 28 wk and 28 to 32 wk. The adjustment for those characteristics eliminated significant association between shorter preconceptional folate supplementation for less than a year and spontaneous preterm birth .p = 0.021 and 0.72, 0.54–0.96, p = 0.027, respectively). There was no significant association with the risk of spontaneous preterm birth at 32 to 37 wk .There were no statistically significant interactions between duration of preconceptional folate supplementation and any of the maternal characteristics or risks factors for preterm birth in predicting the risk of spontaneous preterm birth . AdditioBody mass index (BMI), being married, and more than 12 y of education were associated with lower risk, while Hispanic ethnicity, black or Asian race, nulliparity, and prior preterm birth were associated with higher risk of spontaneous preterm birth . The effp>0.05 for all) or globally for the whole model (p = 0.7). The goodness of fit test showed no evidence of poor fit (p = 0.5)There was no evidence of violation of the proportional hazard assumption for each variable .The proportion of the PPROM among folate supplementation groups in women with spontaneous preterm birth before 32 wk was not significantly different  for 1 y or longer and by 1,018 (28.6%) for less than 1 y, and was not used by 1,827 (51.4%).We have shown that preconceptional folate supplementation for a year or longer is associated with a decreased risk of early spontaneous preterm birth. The association is characterized by strength, temporal relationship, biological gradient, nonsignificant effect of confounders, consistency with other studies, and biological plausibility.Preconceptional folate supplementation for a year or longer was associated with over a 70% reduction in the risk of spontaneous preterm birth before 28 wk and 50% reduction of the risk of spontaneous preterm birth between 28 and 32 wk.The strength of the association was significantly weaker if folate supplementation was shorter than a year. There was also a significant trend of decreased risk of spontaneous preterm birth across the original seven categories of duration of folate supplementation. This indicates existence of a biological gradient in the association between preconceptional folate supplementation and the risk of spontaneous preterm birth.The relationship between preconceptional folate supplementation and risk of spontaneous preterm birth could potentially be confounded by risk factors for spontaneous preterm birth and indications for preterm delivery. However, adjustment for the risk factors for spontaneous preterm birth had a minimal effect on the association. The change in the coefficients observed with adjustment was 32% for spontaneous preterm birth between 20 and 28 wk and 26% for spontaneous preterm birth between 28 and 32 wk. The resulting reduction in the risk of spontaneous preterm birth, due to adjustment, of 78% versus 69% or of 55% versus 47%, respectively, is clinically of no material importance.Pregnancies with indications for preterm delivery were censored, contributing to the denominator, the number of pregnancies at risk, but not included in the nominator, the number of spontaneous preterm births at each gestational age time point. Analysis without censoring showed a significant association between preconceptional folate supplementation and lower risk of all early preterm births. However, there was no significant association between duration of folate supplementation and the risk of nonspontaneous preterm birth.There were no significant interactions between folate supplementation and maternal charactersitics and risk factors for preterm birth, indicating that this association is observed regardless of risk factors for preterm birth. Although the confidence intervals of some of the strata were large and crossed unity due to a small size of the strata, the interaction terms of these covariates and duration of folate supplementation, which do not depend on the size of the strata, are not significant.The findings of this study are consistent with prior observational studies showing that women delivering prematurely have lower folate concentrations in plasma or red blood cells than women delivering at term A substantial body of evidence suggests that the association between folate supplementation and spontaneous preterm birth is biologically plausible. Spontaneous preterm birth, especially at early gestation, is strongly associated with intrauterine infection Early spontaneous preterm birth is a devastating but rare outcome. Thus the findings of this study are based on small number of cases, and additional cases may change substantially those estimates in either direction. This is reflected in their wide confidence intervals. However, the estimates remain statistically significant also using exact logistic and Poisson regressions methods, which are accurate with rare outcomes .A limitation of observational study design is a possibility that relevant confounders have not been accounted for. Thus it is conceivable that folate supplementation could be a marker of healthy behavior. Such an alternative hypothesis is unlikely because the association between folate supplementation and the risk of spontaneous preterm birth is specific to spontaneous preterm birth, is not affected by other markers of healthy behavior, and depends on the length of supplementation in a similar fashion to the effect of folate supplementation on other disorders observed in randomized trials.Preconceptional folate supplementation was associated specifically with the risk of early spontaneous preterm birth and not with the risk of other complications of pregnancy, preeclampsia, small for gestational age infant, placental abruption, or nonspontaneous preterm birth. Markers of healthy behavior, maternal characteristics associated with healthy behavior, maternal age, BMI, education, marital status, and smoking minimally affected the association between folate supplementation and the risk of spontaneous preterm birth. If preconceptional folate supplementation were a mere marker of healthy behavior, adjustment for other markers of healthy behavior would either eliminate or significantly attenuate the observed association with the risk of spontaneous preterm birth. In fact, in our study, adjustment for markers of healthy behavior did not have a substantial effect.Thus it is unlikely that unaccounted confounding could eliminate observed associations because of the specificity of this association with only early spontaneous preterm birth and because two unaccounted confounders—markers of socioeconomic status or healthy behavior—are strongly correlated with measured confounders Short interpregnancy interval and unintended pregnancy are unlikely to confound the findings, because the association between preconceptional folate supplementation and risk of spontaneous preterm birth was significant also in nulliparous women and largely independent of maternal age, race/ethnicity, marital status, and education, known risk factors for unintended pregnancy None of the above-mentioned observations is sufficient by itself to exclude the existence of unaccounted confounders, such as healthy behavior of women who use folate preconceptionally. However, collectively they strongly support a nonspurious association of preconceptional folate supplementation and the risk of spontaneous preterm birth. Definitive support for this hypothesis can be provided only by a randomized trial.The importance of duration rather than dosage of folate supplementation has been observed previously. Meta-analyses of folate for prevention of neural tube defects or stroke did not show a significant association between dosage of folate supplementation and the strength of its effect Finally, women who used folate supplementation preconceptionally had a lower risk of spontaneous preterm birth than women who did not use it, regardless of the unknown dosage and frequency of their use.Use of folate in a combination with other micronutrients could potentially confound the findings. However, a systematic review and individual clinical trials have shown that supplementation with multiple micronutrients in pregnancy did not offer additional benefit over folate and iron supplementation alone Supplementation with zinc has been associated with lower risk of preterm birth Postconceptional folate supplementation also could not confound our findings. In the US 93% of women use postconceptional folate supplementation and in a population with early onset of prenatal care (as in this study) the rate is likely much higher Another limitation of this study is that preconceptional folate supplementation was self-reported at enrollment in the first trimester of pregnancy. However, self-reported folate intake from supplements has been shown to correlate with serum folate concentrations during pregnancy and was a reliable measure of folate supplementation The validity and generalizability of these findings are supported by a large number and proportion of patients included in the analysis. Less than 10% of patients were excluded from the multivariable analysis. Distribution of the length of folate supplementation in the excluded pregnancies was similar to that observed in the study population. Moreover, the study population is a good representation of a general US population We have shown that preconceptional folate supplementation is associated with a 50%–70% reduction in the incidence of early spontaneous preterm birth. These findings, despite their limitations, provide a basis for further inquiry into preconceptional folate supplementation for prevention of spontaneous preterm birth in clinical trials.Table S1Risk of spontaneous preterm birth and preconceptional folate supplementation: Results of maximum likelihood and exact models. Definitions: No folate, no preconceptional folate supplementation; folate <1 y, preconceptional folate supplementation for <1 year; folate ≥1 yr, preconceptional folate supplementation for ≥1 y. HR 95% CI is from Cox regression with Breslow assumption maximum likelihood model; OR 95% CI, from exact logistic regression; IRR (incidence rate ratio) 95% CI, from exact Poisson regression.(0.04 MB DOC)Click here for additional data file.Table S2Participating centers.(0.02 MB DOC)Click here for additional data file.


To analyze apolipoprotein gene polymorphisms in the Tunisian population and to check the relation of these polymorphisms and homocysteine, lipid and apolipoprotein levels to the coronary artery disease (CAD).In healthy blood donors and in patients with CAD complicated by myocardial infarction (MI) four apolipoprotein gene polymorphisms  were determined and plasma levels of total homocysteine, total cholesterol (TC), triglycerides (TG), HDL-cholesterol (HLD-C) and apolipoproteins  were measured.Analysis of the four apolipoprotein gene polymorphisms shows a relative genetic homogeneity between Tunisian population and those on the other side of Mediterranean basin. Compared to controls, CAD patients have significantly higher main concentrations of TC, TG, LDL-C, apo B and homocysteine, and significantly lower ones of HDL-C, apo A-I and apo E. The four apolipoprotein gene polymorphisms have not showed any significant differences between patients and controls. However, the APO E4 allele appears to be associated to the severity of CAD and to high levels of atherogenic parameters and low level of apo E, which has very likely an anti-atherogenic role.Although APO (a) PNR, APO CI and APO CII genes are analyzed in only few populations, they show a frequency distribution, which is not at variance with that of APO E gene and other widely studied genetic markers. In the Tunisian population the APO E 4 appears to be only indirectly involved in the severity of CAD. In the routine practice, in addition of classic parameters, it will be useful to measure the concentration of apo E and that of Homocysteine and if possible to determine the APO E gene polymorphism. Coronary artery disease (CAD) is a multifactorial disease caused by genetic and environmental factors. Apolipoprotein genes involved in lipoprotein synthesis and metabolism are considered excellent candidates for studying the susceptibility to CAD and myocardial infarction (MI) . FunctioAmong apolipoproteins, apolipoprotein (a) is a highly glycosylated protein component of lipoprotein (a) . It is aAPO E gene, the most extensively studied polymorphism among apolipoproteins, has three common alleles designated E2, E3 and E4 coding for three protein isoforms   that ser13,2). The apo CI protein inhibits the bindings of beta very low-density and intermediary density lipoproteins to lipoproteins' receptors. Apo CII is required as a cofactor in the hydrolysis of triacylglycerides of chylomicrons and VLDL by lipoprotein lipase. Two restriction polymorphisms, the HincII site in the promoter region of the APO CI gene and the AvaII site in the intron 3 of the APO CII gene  in the Tunisian population and we check the relation of these polymorphisms and homocysteine, lipid and apolipoprotein levels to CAD.We conducted a hospital-based case study in 80 patients (80% men) with CAD complicated by MI (confirmed by ECG and coronary angiography) that were diagnosed and treated at the Department of Cardiology of the University Hospital Fattouma Bourguiba . The mean age at diagnosis was 57.42 ± 8.37 years. Among these patients, 56 were smokers; 51 had diabetes; 35 had hypertension and 22 were hyperlipidemic. As a control group, 100 individuals (76% men with age range close to that of the patient group), free of any CAD or any related disorders, were randomly selected from the same large geographical area to which belonged the patients (the center of Tunisia). Appropriate informed consent was obtained from patients and controls in accordance with the Ethical Committee Guidelines of the participating Hospital and University.Quantitative analyses of homocysteine, lipids and apolipoproteins were performed on only 50 controls and 50 patients within 24 h from collection. Plasma levels of total cholesterol (TC), triglycerides (TG) and HDL-cholesterol (HDL-C) were measured by an enzymatic colorimetric assay . LDL-cholesterol concentrations were calculated by using the Friedewald formula . ApolipoLeukocyte DNA was extracted from blood samples by phenol-chloroform method and amplified by PCR using oligonucleotide primers and amplification conditions described previously in  for the Allelic and genotypic frequencies were calculated by direct gene counting method. The chi-square test was used to test Hardy-Weinberg equilibrium of all gene variants. Differences in frequencies between cases and controls were analyzed by Fisher's exact test. Haplotype calculations for the APO E-CI-CII gene cluster were done by means of the Arlequin v 2.0 package . The samAllele and genotype frequencies of the APO (a) PNR, APO E, APO CI and APO CII polymorphisms analyzed in controls and patients are presented in Table APO E allele frequencies of the present control sample from a wide area of the center of Tunisia are very close to those previously described in a Tunisian sample representative of the whole country  . The lacAllele frequencies of APO (a), APO CI, and APO CII polymorphisms in the control Tunisian sample are compared with those found in other populations Table . ConcernThe pattern of allele variation in North Africa Table  is repreLipid, lipoprotein and apolipoprotein mean concentrations are indicated in Table No significant differences have been detected between patients and controls neither for apolipoprotein gene frequencies Table  nor for In spite of the lack of significant differences between patients and controls, several features are worth noting particularly at the level of APO E polymorphism. In the case of APO (a) PNR polymorphism, whereas the 8 repeats allele is found with similar frequencies in both groups, the frequency of the 9 repeats allele is higher in the control group. On the other hand, the 7 repeats allele frequency is higher in the CAD group.As for APO E polymorphism, when the CAD sample is divided in two groups: hyperlipidemic (TC> 5.70 mmol/l) and non-hyperlipidemic subjects, the APO E4 allele is significantly (p < 0.05) higher in the hyperlipidemic patients . The same is true for the controls, although they all have normal TC concentrations (between 3 and 5 mmol/l) when controls are divided in two groups, a group with TC< 4 mmol/l and a second with TC> 4 mmol/l, the APO E4 allele is significantly (p < 0.05) higher in the second group .Given this relation between the APO E polymorphism and the TC concentration, we have analyzed the influence of APO E genotypes on the distribution of all studied lipid, lipoprotein and apolipoprotein concentrations Table . In the The severity of CAD in our sample has been estimated by the number  of affected coronary vessels. Allele distributions of apolipoprotein gene polymorphisms in patients with 1, 2 and 3 affected vessels are shown in Table In this study we have analyzed for the first time the polymorphisms of APO (a) PNR, APO E, APO CI and APO CII genes together with several lipid and apolipoprotein levels in Tunisian CAD patients and in controls. This constitutes an approach to the genetic characterization of the lipid profiles of Tunisian cardiovascular patients and supplies anthropological data on the Tunisian population.Although APO (a) PNR, APO CI and APO CII gene polymorphisms have been analyzed in only few populations from South Europe Table , they shComparative analyses between control and CAD groups from Tunisia have shown important observations, particularly in comparison with those found in other populations. Concerning the analyses of plasma lipid mean concentrations, the significant high values of TC, LDL-C, apo B and TG, and the significant low values of HDL-C, apo A-I, and apo E shown by Tunisian patients with CAD have been also observed in other populations ,30. The In the case of the APO CI and APO CII polymorphisms, no association was noted with CAD. In fact, only few studies have analyzed the association of these polymorphisms with the disease. In a study on the Chinese population  the authRegarding the APO (a) PNR polymorphism, we ascertained a global negative association between the number of TTTTA repeats and CAD. This agrees with conclusions relating to Russian, French and Irish populations ,34. HoweThe differences in results on CAD risk noted between populations probably reflect the complexity of the involvement of many genetic and environmental factors in CAD appearance. In fact, these risk factors are probably candidate factors and the appearance of the CAD does not need the presence of all of them. Namely, in a given population the presence of only some (effecter factor group) of these candidate factors, are enough for favoring the CAD appearance without or with slight secondary influence of the other factors. The effecter factor group is very likely the result of a complex gene-environmental interaction, which can vary from a population to another. Our observations relating to APO E polymorphism may illustrate this proposal.In some populations  a significantly higher frequency of APO E4 allele was observed in CAD patients compared with healthy subjects ,11,30,37The study of the impact of APO E genotype variations on serum lipid parameters shows that CAD patients and healthy subjects with APO E4 allele have higher levels of atherogenic parameters , whereas subjects with APO E2 allele have low levels of these parameters. Such APO E allele effects have been also noted in some other populations ,30. The The effect of APO E4 on CAD severity could be the consequence of its association to significant high levels of atherogenic parameters and its association to low levels of apo E. In fact, the significant low mean concentration of apo E in CAD patients, observed also in the Italian population ,30 suggeThe effect of APO E genotypes on HDL-C concentration has not demonstrated the same consistency across populations. In the present study, no association has been found between APO E polymorphism and HDL-C concentrations in both patient and control groups. The relation of APO E polymorphism and HDL-C has been reported in some populations ,37,42 buThe authors declare that they have no competing interests.RB performed all the experiments and data analyses and contributed in the interpretation of results and in the editing of the manuscript, EE performed a direct help and supervision of all DNA analyses and contributed in data analyses and in the review of the manuscript, PM performed a general supervision of all DNA analyses and data analyses and participated in the review of the manuscript, MH participated in control's group collection, KBH participated in coronary artery disease patient collection, HC supervised all the work and participated in the result interpretations and in the editing and the review of the manuscript. All authors read and approved the final manuscript.


Evidence for protection of preterm born infants from invasive pneumococcal disease (IPD) by 7-valent pneumococcal conjugate vaccination (PCV7) is relatively sparse. Data from randomized trials is based on relatively small numbers of preterm born children.We report data from active prospective surveillance of IPD in children in Germany. The cohorts of preterm born children in 2000 and 2007 and the respective whole birth cohorts are compared regarding occurrence of IPD.P < .001). The impact among the preterm birth cohort was comparable: A reduction in notification rate from 26.1 to 16.7 per 100,000 comparing the 2000 with the 2007 preterm birth cohort (P = .39). Preterm born infants with IPD were either unvaccinated or vaccinated delayed or incomplete.After introduction of PCV7 we observed a reduction in the rate of IPD in preterm born infants comparing the 2000 and 2007 birth cohort. The rate of IPD among the whole birth cohorts was reduced from 15.0 to 8.5 notifications per 100,000 (This adds to evidence that PCV7 also protects preterm born infants effectively from IPD. Preterm born infants should receive pneumococcal vaccination according to their chronological age. Streptococcus pneumoniae is a major source of morbidity and mortality worldwide. It is estimated that S. pneumoniae causes around 11% of all deaths in children aged 1-59 months [9 months -6. In Fe9 months -11. Seve9 months  includin9 months .Preterm born infants are at higher risk for infectious diseases like invasive pneumococcal disease (IPD), with the highest risk in early infancy . VaccinaStreptococcus pneumoniae from at least one culture of blood, cerebrospinal fluid, or a sample from any other normally sterile body site. Cases were excluded if they did not match the case definition, were older than 15 years, or double reports. Physicians filled comprehensive questionnaires on disease manifestation, outcome, vaccinations, and underlying conditions. There was a check box specifically asking for preterm birth and week of gestation of preterm births. Details of the study have been described elsewhere [We report data from active prospective surveillance of IPD in all German pediatric hospitals. Hospitals were contacted on a monthly basis via postcards to ask if a case of invasive pneumococcal disease had been observed the last month. The case definition of IPD was isolation of lsewhere ,21,22. Tlsewhere .Microbiologic laboratories were invited to send pneumococcal strains isolated from a normally sterile body site to the German National Reference Center for Streptococci, where the species identification was confirmed and serotyping, using Neufeld's Quellung method , was perIn order to account for a population denominator, the number of preterm born children (before 37 completed weeks gestation) was estimated based on the assumption that about 7% of children in Germany are born preterm. Based on the hospital surveillance, we compared data from two preterm birth cohorts: those born in 2000 and those born in 2007. Infants from both birth cohorts were under surveillance for at least one and two years at maximum: 2000-2001 and 2007-2008 respectively. Also based on the hospital surveillance we compared IPD notifications for all children (irrespective whether preterm born or not) for the birth cohorts of the years 2000 and 2007 using the same approach. Numbers on all births in Germany were provided by the Federal Statistical Office Germany. Incidence estimates were calculated based on IPD notifications from the respective (preterm) birth cohort reported in the year of birth and the subsequent year. These total numbers were divided by the respective number of (preterm) born children. Incidence estimates from 2000-2001 and 2007-2008 were compared using Fisher's exact test for difference in proportions.Because this study is based on retrospective case analysis, without the possibility of identifying individual subjects, only data protection issues are of concern. Consent of the Bavarian data protection office was given.P < .001). The reported number of preterm born children with IPD was 14 in 2000-2001 and 8 in 2007-2008. This corresponds to a reduction in notification rate from 26.1 to 16.7 per 100,000 comparing the 2000 with the 2007 preterm birth cohort (P = .39). Among the 14 cases of IPD among preterm born children, 9 had information on serotype and for 7 of these the serotype was included in PCV7 (14 (3 cases), 18C (2 cases), 19A, 23F, 3, 9V).Between January 2007 and December 2008, 58 cases of IPD in children born in 2007 were reported from Germany pediatric hospitals compared to 115 cases in children born in 2000 reported between 2000 and 2001 . Another limitation is the lack of trend data, e.g. for the birth cohorts between 2001 and 2006. The hospital surveillance system was confined to vaccinated cases between 2003 and 2006. Furthermore, a recommendation to vaccinate preterm born children with PCV7 was issued in 2001. Therefore the 2000 birth cohort represents the only birth cohort for which data on unvaccinated preterm born children could be analysed.In summary this study adds to evidence that PCV7 also protects preterm born infants from IPD and points to the need for timely vaccination of preterm children according to their chronological age as recommended e.g. by the German Standing Vaccination Committee (STIKO) and the American Academy of Pediatrics Committee on Infectious Diseases .RvK and MvdL have received grant support from WyethSR suggested the idea for the article, performed the statistical analysis and wrote the manuscript. MvdL contributed to the writing of the manuscript and revised the final version for important intellectual content. RvK supervised the writing of the manuscript and revised the final version of the manuscript for important intellectual content.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/12/prepub


Colobomatous cyst of the orbit is a rare congenital cystic malformation associated with ocular maldevelopment. Usually, the cyst is associated with a microphthalmic globe. We present a rare case of a unilateral large colobomatous cyst associated with a normal-sized globe, giving the appearance of a double globe on imaging. A colobomatous cyst is defined as a neuroectoderm-lined mass that protrudes through a coloboma in the wall of a microphthalmic eye. It is usA 2-year-old child presented with a swelling under the right lower lid and corneal opacities in both eyes since birth . The sweAn external ocular examination of the right eye showed a formed globe with a corneal opacity. The left eye had a central and inferomedial corneal opacity with a normal anterior segment . B-scan The patient was diagnosed as a case of bilateral corneal opacities with a colobomatous cyst communicating with the globe in the right eye. He underwent an inferior transconjunctival orbitotomy, with aspiration of the contents of the cyst to reduce its volume, followed by excision of the cyst and suturing of the defect in the globe.Histopathology of the cyst revealed the outer wall consisting of dense fibrocollagenous tissue. The cyst wall was lined with round to oval cells of neuroectodermal origin.A majority of the cases of colobomatous cyst are associated with microphthalmos. There is no predilection for any particular sex. The condition is usually unilateral. The etioShields and associates reviewed 645 orbital biopsies and reported that microphthalmos and colobomatous cyst accounted for 2% of 193 orbital cystic lesions and <1% of 645 biopsies.[The presentation of microphthalmos with a cyst can be as a protruding mass in the inferior orbit associated with a malformed microphthalmic eye; the cyst may be so small that it cannot be detected clinically or it may be so large that it obscures the globe. These eyImaging is of primary importance in the evaluation of cystic lesions of the orbit. It helps to differentiate colobomatous cyst from optic nerve sheath cysts, cephalocele, and optic nerve meningocele as well as from solid tumors of the orbit. Imaging Management of these cysts depends on the age of the patient, the size of the cyst, the presence of communication between the globe and the cyst, and the visual prognosis. Surgical