Adult cardiorespiratory fitness and muscle strength are related to all-cause and cardiovascular mortality. Both are possibly related to birth weight, but it is unclear what the importance is of genetic, maternal and placental factors in these associations.Peak oxygen uptake and measures of strength, flexibility and balance were obtained yearly during adolescence (10–18 years) in 114 twin pairs in the Leuven Longitudinal Twin Study. Their birth weights had been collected prospectively within the East Flanders Prospective Twin Survey.P = 0.02), arm pull (b = 1.85 kg per kg birth weight P = 0.03) and flamingo balance . Maximum oxygen uptake appeared to have a U-shaped association with birth weight , but this association was no longer significant after adjustment for parental BMI. Using the individual twin’s deviation from his own twin pair’s average birth weight, we found positive associations between birth weight and adolescent vertical jump  and arm pull . Δ scores were calculated within the twin pairs as first born twin minus second born twin. Δ birth weight was associated with Δ vertical jump within MZ twin pairs only , which indicates importance of placental factors.We identified linear associations between birth weight and adolescent vertical jump  and birth weight. The associations with vertical jump and arm pull were likely based on individual, more specifically placental  factors. Our results should be viewed as hypothesis-generating and need confirmation, but potentially support preventive strategies to optimize birth weight, for example via placental function, to target later fitness and health. Adult cardiorespiratory fitness is related to all-cause mortality and, more specifically, to cardiovascular mortality. A number of studies suggest a positive association between birth weight and later cardiorespiratory fitness, which indicates that low birth weight babies may be programmed to have impaired cardiorespiratory fitness in adulthood. To address the latter issue and to further explore the developmental origins of cardiorespiratory fitness, twin studies may be a useful tool, as they provide the possibility to unravel genetic and environmental  influences on the possible associations. We define maternal factors as all non-genetic maternal influences on both members of a twin pair, such as body mass index (BMI) and smoking. In dizygotic (DZ) twins, maternal factors are identical, but genetic factors are not, while in monozygotic (MZ) twins, both maternal and genetic factors are identical. In contrast to the maternal environment, the fetoplacental environment can be different for both members of a twin pair. A previous study used a twin sample to disentangle genetic and environmental factors in the association between birth weight and adult hand grip strength  we used a twin sample from the East Flanders Prospective Twin Survey (EFPTS), in which we measured cardiorespiratory fitness, as well as muscle strength, flexibility and balance, up to eight times during adolescence (10–18 years). We studied the associations between these variables and birth weight. Additionally, the importance of individual and shared factors was disentangled. Finally, we analyzed to what extent genetic, maternal or fetoplacental factors were involved.In a longitudinal study carried out from 1985–1999  114 twin pairs and their parents were recruited from the East Flanders Prospective Twin Survey (EFPTS), which is a population-based register of all twins born in the Belgian province of East Flanders since 1964. Both parents and twins gave written informed consent/assent. Ethical approval was given by the Medical Ethics Committee of the Fund for Medical Research and of the former Institute of Physical Education and Physiotherapy, KU Leuven.2peak, measured in ml/min/kg) as obtained by a maximal exercise test on a treadmill , according to the Bruce protocol. Our primary outcome measure was peak oxygen uptake , explosive strength , muscle endurance/functional strength , muscle endurance/trunk strength , running speed and agility , speed of limb movement , flexibility  and balance . Details of these tests have been described previously Body mass was measured to the nearest 0.1 kg using a balance scale  with subjects in underwear. Standing height was measured on bare feet using a Harpenden stadiometer  to the nearest millimeter.Birth weight, gestational age, sex, zygosity and chorionicity were collected prospectively. Zygosity was determined using sequential analysis based on sex, fetal membranes, blood groups, placental alkaline phosphatase and DNA marker analysis. Zygosity and chorionicity were analyzed as three groups: dizygotic , monozygotic-dichorionic (MZDC) and monozygotic-monochorionic (MZMC) twins.2).As potential confounders we recorded smoking, physical activity and parental BMI. As a measure of physical activity, the average number of hours the children participated in sport activities expressed per week during the last year was recorded. At each visit smoking status was recorded and analyzed in three groups: current smoker, former smoker or never smoker. Parental weight and height were collected using the same methods as used in the twins during the first visit, and  BMI was calculated as  or the Wilcoxon rank sum test , when appropriate. The P-values. Therefore, a three level random intercept model was used, taking into account the fact that the measurements were taken longitudinally in the same individuals (level 2), which were in turn clustered in twin pairs . The variance-covariance structure of DZ, MZDC and MZMC twins was allowed to differ. Three models were constructed: 1) basic model, adjusted only for sex, age (as time point) and gestational age, 2) as model 1, but adjusted also for child-specific factors: zygosity-chorionicity, smoking, physical activity, height and weight at the time of assessment, and 3) as model 2, but with additional inclusion of parental factors: maternal and paternal BMI. Since VO2peak was already calculated relative to weight, weight was not included in VO2peak models 2 and 3. The data used in We used multivariable multilevel regression analysis to study the association between birth weight and the outcome variables. Failure to take the correlated nature of the data into account would lead to biased estimates and incorrect standard errors and To evaluate the importance of individual  and shared factors  in the relationship between birth weight and the outcome parameters, separate within-twin-pair  and between-pair regression coefficients (using the average birth weight of the pair as a determinant) were calculated.To unravel genetic, maternal and placental influences on the associations, the same-sex twins were studied as pairs using a two level random intercept model, which takes the longitudinal measurements within the same individuals into account. We analyzed the association between intra-pair difference in birth weight and intra-pair difference in physical fitness for MZ and DZ twins separately. Since MZ twins share approximately 100% of their genes, genetic factors cannot be responsible for any differences observed within the pair and any association between birth weight and adult fitness observed within MZ pairs hence indicates environmental influences on the association. Since maternal factors are identical, but fetoplacental factors are never identical, these environmental influences can be pinpointed to fetoplacental factors. DZ twins share on average 50% of their genes and therefore intra-pair analysis in DZ twins is still partly confounded by genetic effects. The difference scores were calculated as first-born twin minus second-born twin (this yields the same results as subtracting the fastest from the slowest growing twin). P-value <0.05 was considered statistically significant.The analyses were performed in SAS 9.2 using SAS Enterprise Guide 4 . A . As expected, MZ twins were lighter than DZ twins at birth, but they were also of shorter gestational age. The MZ twins were slightly older and taller when the first measurement was performed and they smoked less at the last measurement. MZ twins performed slightly better on the sit-and-reach test and on the shuttle run at the age of 10.5 years. Approximately two thirds of the MZ twins were monochorionic (MC), while the percentage of boys and girls in the MZ and DZ groups was approximately equal.A total of 114 twin pairs were analyzed P = 0.04) and b = −2.74 (P = 0.04), respectively. Peak oxygen uptake was not related to birth weight in the fully adjusted model (model 3). To further explore the unanticipated negative associations in models 1 and 2, we extended the analysis to study birth weight quartiles. The highest birth weight quarter was used as the reference category. We identified significant differences between the birth weight quartiles in models 1 and 2, and a similar, though non-significant, data structure in model 3 . The data in P = 0.36 from largest quarter), second quarter 50.1 ml/min/kg (P = 0.01), third quarter 50.2 ml/min/kg (P = 0.004) and the largest quarter: 46.8 ml/min/kg, overall P = 0.01. For model 3, the corresponding values were: smallest quarter 49.0 ml/min/kg (P = 0.52 from largest quarter), second quarter 50.5 ml/min/kg (P = 0.06), third quarter 50.3 ml/min/kg (P = 0.07) and the largest quarter: 47.8 ml/min/kg, overall P = 0.16. There were no other non-linear associations identified in models 2 or 3.P = 0.02), arm pull (b = 1.85 P = 0.03) and flamingo balance  were related to birth weight. In all three cases, higher birth weight was associated with better performance, since performance on the flamingo balance is better when fewer attempts are needed to complete the test .In the fully adjusted models using birth weight as a continuous variable adolescent vertical jump  and between birth weight and adolescent arm pull .When the twin pair’s average birth weight was used as the independent variable in the fully adjusted model, no associations were identified . One association remained significant in the fully adjusted model: Δ birth weight was associated with Δ vertical jump, in MZ twins only .When MZ and DZ twins were analyzed separately, no associations were found within DZ twins. Several associations were observed in MZ twins in the basic model: birth weight was positively related to vertical jump, arm pull, standing long jump, and plate tapping, while it was inversely related to flamingo balance and shuttle run In the present prospective, longitudinal, twin study we found that the lowest and highest birth weight groups had the lowest peak oxygen uptake in adolescence (i.e. a U-shaped association). However, after adjustment for parental BMI the effect size was attenuated and the association was no longer significant. We hypothesized a lesser performance by children small at birth, but apparently also high birth weight children are at risk. It is not clear what the exact mechanistic meaning of adjustment for parental BMI is. We speculate that parental BMI may be a surrogate for a sedentary lifestyle, poor nutritional habits, genetic predisposition for obesity, low educational level, or a combination of the above. It is most likely also related to birth weight, given the confounding characteristics. Given the similar data structure in models 2 and 3 and the accompanying loss of 15 twin pairs, model 3 was potentially influenced by a statistical power problem.From the other variables studied not only static (arm pull) and explosive  strength were associated with birth weight, but also a more complex measure as balance appeared to be associated with birth weight. In all cases, low birth weight was associated with a lesser performance in adolescence, thereby supporting developmental programming by low birth weight. Importantly, muscle strength, at least as measured by grip strength, is inversely related to cardiovascular and all-cause mortality. Furthermore, our study shows that the association between birth weight and static and explosive strength is most likely due to individual  factors, as the association was observed using the twin’s individual deviation of the pair’s mean birth weight. This means that having a lower birth weight than one’s co-twin results in lower adult strength than one’s co-twin and hence this cannot be the result of a shared factor. This result is contrary to the only other study in this field, which concluded that genetic factors were most likely to be important in the association between birth weight and, in this case, adult hand grip strength. As a limitation, extrapolation of birth weight studies from twins to singletons should be done with caution. Twins have lower birth weights and shorter gestational ages than singletons, and their prenatal environment is different from that of singletons. As important advantages, we present a study in which we repeatedly measured the same broad spectrum of outcome parameters in the same individuals. This yields increased precision in the measurements and provides a rather complete overview of different fitness, strength and performance parameters. Birth weight was thereby collected prospectively.In summary, we found evidence for an association between adolescent physical performance  and birth weight. The associations with vertical jump and arm pull were most likely based on individual, more specifically placental , factors. Our results should be viewed as hypothesis-generating, but potentially support early preventive strategies to optimize birth weight to target later physical performance and probably cardiovascular health. This might be possible by prevention of smoking and by promotion of healthy diets for pregnant women to improve placental function. These possible developmental strategies should be applied together with attempts to improve cardiorespiratory fitness through life style changes in adult patients at risk. With regard to cardiorespiratory fitness, more research is needed, especially on the exact meaning of adjusting these types of data for parental BMI.


Healthy infants typically regain their birth weight by 21 days of age; however, failure to do so may be due to medical, nutritional or environmental factors. Globally, the incidence of low birth weight deliveries is high, but few studies have assessed the postnatal weight changes in this category of infants, especially in Africa. The aim was to determine what proportion of LBW infants had not regained their birth weight by 21 days of age after discharge from the Special Care Unit of Mulago hospital, Kampala.A cross sectional study was conducted assessing weight recovery of 235 LBW infants attending the Kangaroo Clinic in the Special Care Unit of Mulago Hospital between January and April 2010. Infants aged 21 days with a documented birth weight and whose mothers gave consent to participate were included in the study. Baseline information was collected on demographic characteristics, history on pregnancy, delivery and postnatal outcome through interviews. Pertinent infant information like gestation age, diagnosis and management was obtained from the medical records and summarized in the case report forms.Of the 235 LBW infants, 113 (48.1%) had not regained their birth weight by 21 days. Duration of hospitalization for more than 7 days  and initiation of the first feed after 48 hours  were independently associated with failure to regain birth weight. Maternal factors and the infant's physical examination findings were not significantly associated with failure to regain birth weight by 21 days of age.Failure to regain birth weight among LBW infants by 21 days of age is a common problem in Mulago Hospital occurring in almost half of the neonates attending the Kangaroo clinic. Currently, the burden of morbidity in this group of high-risk infants is undetected and unaddressed in many developing countries. Measures for consideration to improve care of these infants would include; discharge after regaining birth weight and use of total parenteral nutrition. However, due to the pressure of space, keeping the baby and mother is not feasible at the moment hence the need for a strong community system to boost care of the infant. Close networking with support groups within the child's environment could help alleviate this problem. The prevalence of low birth weight (LBW) deliveries is higher in developing countries than developed countries and is associated with increased risks of poor health outcomes. The WorMore than 20 million infants worldwide are born with LBW with the majority 95.6%) born in developing countries, mainly South Asia and Sub Saharan Africa,2. Globa.6% born Growth assessment in the neonatal period is determined by changes in anthropometric measurements and consistent weight gain is a valuable guide indicating adequate growth. The chaIn Uganda, one out of seven newborns is LBW and requires specialized care. In the Special Care Unit (SCU) of Mulago Hospital in Kampala, the high prevalence of LBW infants poses constraints on available resources. Infants are often discharged early and followed up in the Kangaroo Clinic. Unfortunately, many mothers do not adhere to the recommended follow-up schedule due to poverty-associated reasons. This lack of timely follow-up frequently results in delayed identification of neonatal problems such as poor weight gain.Maternal factors that have been reported to affect postnatal outcome include maternal age, level of education, socio-economic status and illness. NeonataThis cross sectional study was conducted in the Neonatal SCU of Mulago Hospital between January and April 2010. Mulago hospital is the National Referral Hospital for Uganda and teaching hospital for Makerere University. The hospital has a total bed capacity of 1,500 and an annual inpatient turnover of 120,000. Special Care Unit receives babies from throughout Uganda, but primarily serves a poor urban population. At full capacity, SCU has 59 incubators/cots, with a provision for rooming-in of 6 adult beds. The mothers stay on the postnatal ward and check on their infants at two hourly intervals both day and night. On average, the unit admits three LBW infants daily. The majority of infant's are fed on breast milk, however, complementary feeding is sometimes done for the mothers who fail to produce breast milk, typically using Nan 1 infant formula (Nestle) and cow's milk.The SCU runs a cost-free Kangaroo clinic for LBW/preterm infants following discharge. Infants are seen twice a week, every Tuesday and Friday, during which their wellbeing is assessed and parents' concerns on feeding or other medical issues are addressed. Infants are followed up regularly until they attain a weight of 2,500 grams and then are subsequently followed in the preterm clinic, which they attend at 1-3 months' intervals until the age of 2 years. On average, 30 to 45 neonates are seen in the Kangaroo clinic each day and the interval between subsequent visits depends on how well the baby is thriving post-discharge with consideration for readmission when necessary.The study instrument was a structured, pre-coded case report form, written in English, and administered by the Principal investigator (FN) or the research assistant. The case report was administered in the language best understood by the mothers of the study participants. We used both qualitative and quantitative methods of collecting data. Interviews were semi-structured, using both open-ended and closed-ended questions. Pertinent patient information such as birth weight, gestational age, and interventions done during admission were obtained from the medical records and summarized in the case report forms. The infant's gestational age was estimated by the admitting doctor (the majority were pediatric residents) in the first 24 hours of age using the New Ballard Score. All LBWMothers were advised at the time of discharge to attend the Kangaroo clinic on the day closest to the 21st day of age for each infant. However, based on the baby's stability at the time of discharge, the physician on duty determined the follow-up dates. Discharge criteria from the SCU required that the baby had to be tolerating at least 10 mls per feed, the mother had to demonstrate knowledge and competency with feeding, especially if the baby was to go home with the nasal gastric tube in situ, and the baby had to be clinically stable . Upon discharge, routine information was provided by SCU staff to all mothers on proper feeding, thermoregulation , immunizations, and need for follow-up after discharge.All low birth weight neonates seen in the clinic between January and April 2010 with a documented birth weight and aged 21 ± 2 days were eligible. Relevant details regarding gestational age, discharge weight, diagnoses and management practices performed during admission were obtained from the medical records. Infants were excluded from the study if they were small for gestation age, defined as birth weight below the 10th percentile for the gestational age . The prevalence of failure to regain birth weight among LBW infants was calculated by dividing the number of infants who had not regained their birth weight at 21 days of age by the total number of infants enrolled in the study. Categorical data to test for presence of association between failure to regain birth weight and the different variables mentioned above was analyzed using Fischers exact test. Students t test was used for continuous variables. The strength of association between the factors and failure to regain birth weight among the infants studied was determined using odds ratios and confidence intervals. All factors considered at bivariate analysis were entered into a logistic regression model. Factors with p value ≤ 0.2 at bivariate analysis were subjected to the backward and forward stepwise methods at multivariate analysis to determine the factors associated with failure to regain birth weight. P values of < 0.05 were considered significant and confidence interval of 95% was used.A total of 671 infants were seen in the Kangaroo clinic during the study period: 235 infants met the eligibility criteria and were enrolled consecutively on each clinic day. One hundred and twenty eight infants were term, 75 were small for gestation age, 34 had no documented birth weights and 199 infants were older than 23 days. The characteristics of the study participants at birth and at 21 days of age are summarized in Table 1Although 37.4% (88/235) of infants had a birth weight of ≤ 1500 grams, the number of infants with measured weights of ≤ 1500 grams increased at 21 days to 42.6% (100/235). Only four infants weighed less than 1000 grams and they had not regained weight by 21 days of age. The mean gestation age for the participants was 32.0 weeks.Sixty-four infants in the study were multiples with 2 sets of triplets and the rest twins. Death occurred in 39% (24/64) of the multiples by the time of enrolment in the study. One set of triplets had a gestation age of 35 weeks and all three weighed between 1500 grams and 2000 grams, with 2 regaining their birth weight by 21 days of age. Two of the second set of triplets died and only one lived to 21 days of age, with a gestational age of 32 weeks, weighing 1260 grams at birth and 1340 grams at 21 days of age.Table The maternal factors and physical examination were not found significantly associated with failure to regain birth weight in the first 21 days of age as shown in table From the logistic regression model Table , hospitaThe current study, reflective of a predominantly low-income urban maternal and neonatal population in Uganda, provides important insights about neonatal risk factors contributing to post-discharge growth failure in LBW infants. Almost half of the LBW infants seen in the Kangaroo Clinic at Mulago Hospital, Kampala, had not regained their birth weight by 21 days of age. This is surprising since LBW infants should typically regain their birth weight within 10 to 21 days of age. Due to The large number of LBW infants in the SCU poses constraints on the available resources, which in turn may hinder optimal care encouraging good growth. The limited resources endorse early discharge of infants from the SCU with follow-up in the Kangaroo clinic. The potential success of this system is contingent on adherence to the scheduled appointments. Lack of follow-up precludes to failure to identify neonatal complications in a timely manner, which may have consequences on the infant's wellbeing, both short- and long-term. A limitation of our study is the absence of information on those who did not return to the Kangaroo clinic for follow-up. We were not able to differentiate non-participation versus non-attendance at the Kangaroo Clinic because the number of neonates who were expected to return to the clinic during the study period was not recorded. It is not clear if our data are more reflective of a follow-up bias population skewed toward infants with poor post-discharge weight gain or whether the infants in our study represent the typical weight gain pattern in LBW infants in similar low-income settings.It appears that early discharge was detrimental to proper post-natal growth in many of the study participants. Although a more prolonged hospital stay offers an appealing, albeit costly, alternative for supporting proper weight gain and neonatal care in low-income countries, this option is not currently feasible in settings similar to Uganda. The factors that significantly contributed to failure to regain birth weight among the LBW infants were hospital stay of more than 7 days and initiation of first feed more than 48 hours. We did not explore in detail the reason for long hospital stays among our study infants, however prolonged stay due to medical illness may be fatal to growth as opposed to stay for nutritional support. Feeding in the first one hour of life (WHO recommendation) may not be possible in many of the LBW infants and other institutional setups, like the initial separation of the mother and baby may contribute to the delay. Although there have been documented cost-effective interventions in neonatal care, few reach preterm infants in low-income countries,18. FurtAlthough it was not one of our study objectives, we found that among the multiple infants, 39% (25/64) had lost their siblings by 21 days of age. This is a high prevalence and concurs with other findings of risks, morbidity and mortality associated with preterm multiple births. SpecialThis study had a number of limitations. It did not include a matched control group to which outcomes could have been compared. We were not able to use dry weight since it was not feasible to withhold feeds from infants for more than 2 hours and their reporting time to the clinic varied. The estimated gestational ages may have not always been accurate since they were based off maternal history of last menstrual period and Ballard scores. In the antenatal care clinic, fetal ultrasonography, the most accurate technique for estimating gestational age, is not routinely done and costs a fee, which most mothers cannot afford. We had no control over timing of discharge and scheduling of follow-up dates. Ideally, all infants would have been seen at 21 days of age: however, this could not be done due partly to logistic reasons, post-discharge state of the infant, and caretaker's decision on when to return to the clinic. Many infants were excluded due to post-dates and this could have affected our results. Since all recorded information was obtained from the mother and the available medical records, recall bias and incomplete documentation respectively, may have affected our results. Additionally, maternal nutritional status and health was not assessed, which may have impacted the growth of the infants in the study.Failure to regain birth weight among low birth weight infants by 21 days of age is a common problem in Mulago Hospital occurring in almost half of the neonates attending the Kangaroo clinic. Currently, the burden of morbidity in this group of high-risk infants is undetected and unaddressed in many developing countries. Measures for consideration to improve care of these infants would include; discharge after regaining birth weight and use of total parenteral nutrition. However, due to the pressure for space, keeping the baby and mother is not feasible at the moment hence the need for a strong community system to boost care of the infant. Close networking with support groups within the child's environment could help improve growth. There is a need for evaluation of simple, cost effective interventions in the community settings prior to discharge of the LBW infants.The authors declare that they have no competing interests.FN, JM & GN contributed to the design of the study and assisted with data analysis. FN coordinated the study and supervised the enrolment of patients. All authors contributed to the interpretation of data, preparation of the manuscript and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/12/1/prepub


Childhood asthma may have a fetal origin through fetal growth and development of the immunocompetence or respiratory organs.We examined to which extent short gestational age, low birth weight and fetal growth restriction were associated with an increased risk of asthma hospitalization in childhood.We undertook a cohort study based on several national registers in Denmark, Sweden and Finland. We included all live singleton born children in Denmark during 1979-2005 , in Sweden during 1973-2004 , and a 90% random sample of singleton children born in Finland during 1987-2004 . The children were followed from three years of age to first hospitalization for asthma, emigration, death, their 18th birthday, or the end of study , whichever came first. We computed the pseudo-values for each observation and used them in a generalized estimating equation to estimate relative risks (RR) for asthma hospitalization.A total of 131,783 children were hospitalized for asthma during follow-up. The risk for asthma hospitalization consistently increased with lower birth weight and shorter gestational age. A 1000-g decrease in birth weight corresponded to a RR of 1.17  1.15-1.18). A one-week decrease in gestational age corresponded to a RR of 1.05 (95% CI 1.04-1.06). Small for gestational age was associated with an increased risk of asthma hospitalization in term but not in preterm born children.Fetal growth and gestational age may play a direct or indirect causal role in the development of childhood asthma. Asthma is a significant health problem for families and society. More thPreterm birth, low birth weight and fetal growth restriction may be associated with disturbed immunocompetence and restrict normal lung growth and development-6, thereLarge population-based studies using the register systems in three Nordic countries allowed a detailed analysis of the association between gestational age, birth weight, fetal growth and risk of childhood asthma. We included information on a number of social demographic variables and explored the risk for asthma hospitalization as a function of gestational age, birth weight, and fetal growth in a cohort of children followed until they reached 18 years of age.We used a cohort study based on linkage between several national registers in Denmark, Sweden and Finland. All live-born children and new residents in the three Nordic countries are assigned a unique personal identification number, which can be used to link information at the individual level in all national registers. We first identified all singleton live births during 1979-2005 recorded in the Danish Medical Birth Registry (DMBR) and during 1973-2004 in the Swedish Medical Birth Register (SMBR). In Finland, we were only allowed to include a 90% random sample of singleton children born during 1987-2004 in the Finnish Medical Birth Registry (FMBR). The DMBR has been computerized since 1973 and includes data on gestational age from 1978. The SMBR was established in 1973, and the FMBR was established in 1987. The DMBR was linked to the Danish Civil Registration System, the Danish National Patient Register, and theWe used three different indicators of fetal development and growth: gestational age, birth weight, and SGA. Information on birth weight and gestational age were obtained from the DMBR, SMBR and FMBR. Gestational age in the DMBR was previously based on the date of last menstrual period (LMP), but in the recent 20 years, ultrasound measurements have been increasingly used to correct LMP if needed. In Sweden, early second trimester ultrasonography to estimate gestational age is routinely offered since 1990, and 95% of women accept this offer, otherwise the date of the LMP is used. Gestational age in the FMBR was estimated from the date of the LMP, unless there was a discrepancy with the first-or second-trimester ultrasonography measurements of more than seven or 14 days, respectively, in which case the latter measurements were used. We categorized gestational age at birth into six groups: 22-28 weeks, 29-32 weeks, 33-36 weeks, 37-38 weeks, 39-41 weeks, and 42-45 weeks. We categorized birth weight into 10 groups: <1000 g, 1000-1499 g, 1500-1999 g, 2000-2499 g, 2500-2999 g, 3000-3499 g, 3500-3999 g, 4000-4499 g, 4500-4999 g, and ≥5000 g. Expected birth weight was calculated using the sex-specific fetal growth curves for gestational age by Marsal et al.. SGA wasDiagnosis of asthma in young children aged 0-3 years is difficult because asthma symptoms are often non-specific. Self-reThe children were followed from three years of age to hospitalization for asthma, emigration, death, their 18th birthday, or the end of follow up , whichever came first.We measured the cumulative risk for asthma hospitalization, using STATA (version 11.2). A method based on pseudo-values has been proposed for direct regression modeling of cumulative incidence function with right censored data at a fixed point in time, which wWe explored the association between gestational age, birth weight, SGA and asthma hospitalization separately. When we focused on predictors as recorded at birth, we did the analysis by including gestational age and birth weight in the same model. In order to explore whether the association between fetal growth and asthma hospitalization changed with gestational age, we also performed the analysis stratified by gestational age. The association between birth weight and asthma risk was explored using 3500-3999 g as a reference. We used full term (39-41 weeks) children as the reference group when analyzing the association between gestational age and hospitalization for asthma. The association between estimated fetal growth and asthma risk was explored using AGA children as reference.As cesarean section has been proposed to affect immune system development, we did 2-agonists , inhaled glucocorticoids , fixed-dose combination of inhaled β2-agonists and glucocorticoids (R03AK06 and R03AK07), and leukotriene receptor antagonists (R03DC03). We defined asthma according to at least two prescriptions of asthma drugs after three years of age. Medications prescribed on the same day were considered to represent one prescription. The first asthma occurrence was defined as the date of first anti-asthmatic drugs redeemed.To examine whether associations between fetal development and onset of asthma were dependent on the asthma definition, we also replicated our analyses by identifying asthma on the basis of asthma medications in live singleton births during 1993-2005 recorded in the DMBR. Information on asthma medication was obtained from the Danish National Prescription Registry. The anaThe study was approved by Danish Data Protection Agency and Scientific Ethics Committee of Central Region Jutland in Denmark and Research Ethics Committee (EPN) at Karolinska Institute in Sweden, Statistics Finland and National Institute for Health and Welfare (THL) in Finland. No informed consent is needed for register-based study based on encrypted data according to the legislation in Denmark, Sweden, and Finland.7 person-years. A total of 131,783 children were hospitalized at least once for asthma during the study period. Table The mean follow-up time was 10.6 years (95% CI 1.1-15.0), altogether 6.0 × 10The risk of hospitalization for asthma increased with lower birth weight and shorter gestational age. The highest cumulative incidence was found in children born with a birth weight of < 1000 g and extremely preterm births (children born at 22-28 weeks)  children. However, with the exception of extremely preterm born children (22–28 weeks) and children born after 42 gestational weeks, the risk of asthma increased with decreasing Z-sore in all preterm and term gestational age groups. In extremely and moderately preterm born children (22-32 weeks), LGA children had a decreased risk of asthma hospitalization . The associations between low birth weight, short gestational age, SGA, and risk of asthma hospitalization attenuated slightly by further adjustment for complications of preterm birth but remained statistically significant (data not shown). In sub-analysis, after further adjustment for weight before pregnancy, maternal asthma during pregnancy, maternal smoking during pregnancy, breastfeeding duration, ETS exposure, contact with pets, mother’s marital status, and cohabit status, the estimated RRs were similar to those obtained without adjustment for these potential confounders (data not shown).When the outcome was asthma medication, the aforementioned associations remained statistically significant although the magnitude of associations decreased slightly Table .Our results indicated that risk for asthma hospitalization increased with lower birth weight and shorter gestational age. Even early term (37-38 weeks) children had a higher risk of hospitalization for asthma than full term (39-41 weeks) children. SGA was associated with a slightly increased risk of hospitalization for asthma in term but not in preterm born children. LGA was associated with a decreased risk of asthma hospitalization in extremely and moderately preterm born children.The inverse association we found between birth weight and asthma hospitalization concurs with most previous cohort studies,21,23-26In the present study, an increased risk of hospitalization for asthma was associated with decreasing gestational age. The finding is consistent with most-17, althWe also found that early term children had a higher risk of hospitalization for asthma compared with full term children. AlthougEvidence linking fetal growth restriction to childhood asthma is limited. In the present study, SGA was associated with a slightly increased risk of hospitalization for asthma. Our findings are in concordance with two of three cohort studies,24, but A novel finding in our study was LGA was associated with a deceased risk for asthma hospitalization in extremely and moderately preterm born children. Our result is consistent with one previous study showing the association of high birth weight for gestational age and a decreased risk of asthma in preterm children. Our datOur study is the largest study so far on this topic. The large sample size allowed us to analyze the association between birth characteristics and child hospitalization for asthma with almost complete follow-up.Our study also has limitations. We did not investigate confounding from familial lifestyle and environmental factors, including housing status, early exposure to allergens, diet and nutrition. We used first hospital discharge diagnosis after three years of age as the outcome, and therefore, our findings addressed severe asthma or factors that may lower the threshold for asthma hospitalization. Although we did a sub-analysis with asthma medication and got similar findings, we did not include all patients with mild symptoms who did not seek medical help. Therefore, our findings cannot be generalized to those children as well as children with transient asthma. Second, children born with low gestational age or fetal growth restriction may have more opportunities to have asthma diagnosed because they are hospitalized more frequently due to co-morbidity. Our finFetal growth and gestational age may play a direct or indirect causal role in the development of childhood asthma.AGA: Appropriate for gestational age; CI: Confidence interval; DMBR: The Danish Medical Birth Registry; ETS: Environmental tobacco smoke; FMBR: The Finnish Medical Birth Register; GA: Gestational age; ICD: The International Classification of Diseases; LGA: Large for gestational age; LMP: Last menstrual period; RR: Relative risk; SGA: Small for gestational age; SMBR: The Swedish Medical Birth Register.All authors declare that they have no competing interests.XL contributed to data preparation, analysis and interpretation of data, and drafted the manuscript. JO contributed to study design, data analysis, interpretation of the results, and revised the manuscript. EA contributed to study design, data analysis, interpretation of the results, and revised the manuscript. WY contributed to study design, data analysis, the interpretation of the results, and revised the manuscript. SC contributed to study design, data analysis, the interpretation of the results, and revised the manuscript. MG contributed to study design, data analysis, the interpretation of the results, and revised the manuscript. JL contributed to the conception and the design of the study, to data acquisition, data analysis, the interpretation of the results, and revised the manuscript. All authors approved the final manuscript as submitted.


The IGF system is recognised to be important for fetal growth. We previously described increased Insulin-like growth factor binding protein (IGFBP)-2 cord serum concentrations in intra-uterine growth retardation (IUGR) compared with appropriate for gestational age (AGA) newborns, and a positive relationship of IGFBP-2 with Interleukin (IL)-6. The role of cortisol in the fetus at birth is largely unknown, and interactions among peptides are their real effect on birth size is unknown. Furthermore, almost all studies have previously assayed peptides in serum several years after birth, and follow-up data from pregnancy are always lacking. This study aimed at establishing and clarifying the effect of cord serum insulin, IGF-II, IGFBP-2, cortisol and IL-6 concentrations on birth length and weight.23 IUGR and 37 AGA subjects were followed up from the beginning of pregnancy, and were of comparable gestational age. Insulin, IGF-II, IGFBP-2, cortisol and IL-6 concentrations were assayed in cord serum at birth, and a multiple regression model was designed and applied to assess which were the significant biochemical determinants of birth size.:0.546; p: 0.001) and weight . IGFBP-2 had a near significant negative effect on both birth weight  and length .Insulin, cortisol, and IL-6, showed similar concentrations in IUGR and AGA as previously described, whereas IGF-II was lower, and IGFBP-2 increased in IUGR compared with AGA. IGF-II serum concentration was found to have a significant positive effect on both birth length (r:IGF-II cord serum concentration was shown to have a significant positive effect on both birth length and weight, whereas IGFBP-2 had a significant negative effect. Insulin, cortisol, and IL-6 cord serum concentrations had no significant effect on birth size. Fetal growth is driven mainly by the IGF system as experiments in knockout mice have shown We described increased IGFBP-2 cord serum concentrations in IUGR compared with appropriate for gestational age (AGA) newborns, and a positive relationship of serum IL-6 with IGFBP-2 Very little data has been published on the fetus to date. Certainly it is of interest to consider the described molecular mechanisms by which IL-6 can induce insulin-resistance Furthermore, animal and human studies showed a marked activation of the hypothalamic-pituitary-adrenal axis by IL-6 infusion We previously described similar cortisol and insulin concentrations in cord serum of newborns, both IUGR, and AGA.The role of cortisol in the fetus at birth is largely unknown. Interestingly, Cianfarani et al. reported a positive correlation of cortisol serum concentrations with IGFBP-1, and a negative correlation with IGF-I and length gain in the first 3 months of life, in normal neonates IUGR and appropriate for gestational age (AGA) newborns show differences but are subjected overall to the same biological laws.We followed-up a unique series of newborns, both IUGR and AGA, from pregnancy to birth Twenty-three IUGR  and 37 AGA , births of comparable gestational age  were followed during pregnancy. All pregnancies were dated correctly by ultrasound during the first trimester of gestation.AGA newborns were defined on the basis of a normal birth weight (<80th and >10th centiles) with respect to the Italian standards The diagnosis of IUGR was made within the 32nd week of gestation and was ascribed to a probable placental cause after excluding other causes as infections, chromosomal abnormalities, genetic syndromes, maternal malnutrition, substance abuse including smoking, gross placental abnormalities and multiple fetuses. No cases with hypertension, gestational diabetes or reduced amount of amniotic fluid were included in the study. Mothers were all normal-weight subjects and data on parity, were collected also.The IUGR pregnancies were diagnosed by ultrasound according to the following criteria: abdominal circumference <10th centile and/or shift of fetal growth with a reduction of abdominal circumference with respect to the measure taken during the 20th week of gestation. In IUGR subjects, doppler velocimetry was altered in almost all cases in the placenta and/or fetus site. All neonates, both IUGR and controls, were delivered by elective caesarean section (CS).The causes of preterm birth (<38 weeks of gestation) in the AGA newborns were: intra-hepatic cholestasis in a woman who had underwent a previous CS, premature rupture of membranes with breech presentation and delivery within 4 hours with no signs of infection. Elective CS in these subjects was performed because of refusal of vaginal delivery for psychological reasons, or because of a previous CS.We could obtain a complete data-set on which this study was performed in 34 subjects. The main clinical features of these newborns are shown in Length (Lt) and weight (Wt) at birth was measured by trained midwives at our hospital using exact measuring equipment for length measurements. At birth auxological data were plotted according on to the Italian standards At birth the following information was also collected: age of the mother, weight at birth of both parents, body mass index (BMI) of the mother before pregnancy, previous gynaecological history, medical history during pregnancy, fetal biophysical data , clinical data at delivery , weight and macroscopic aspect of the placenta.Umbilical cord serum from IUGR and AGA births were collected at birth and treated as previously described Peptides were assayed as previously described, prior to this study Briefly, IGF-II was measured using an IRMA method .IGFBP-2 was assayed using a RIA method .Insulin was assayed using a specific IRMA assay .IL-6 was measured using an ultrasensitive ELISA method .Cortisol was assayed using a specific RIA assay .Informed consent was obtained from the mothers. The study was approved by the Ethics Committee of the University of Parma Medical School.Statistical analysis was performed using SPSS 18.0 for Windows .A complete data-set with both growth parameters at birth, and all peptide concentrations in placenta could be obtained for 34 subjects.Results for males and females were combined as in this series we did not detect any significant difference between the two sexes.Prior to the study we compared concentrations between cord serum peptide concentrations to check whether previous reported differences were confirmed in this population using an unpaired T test. Results are reported in The normal distribution of the data was determined using the Kolmogorov-Smirnov test.Multiple linear regression analysis was used, with stepwise eliminations of the independent variables, to study the effect of individual peptides on parameters of birth size of both IUGR and AGA newborns, as both populations are subjected to the same general growth rules, and to obtain acceptable statistical power. No significant effect of gestational age was found as expected, considering that gestational ages were similar.Parity was ruled out also as was similar in the two groups.We used Bonferroni correction to correct for multiple linear regression modeling. Statistical significance was set at a p-value less than 0.007 .For the analysis of the variables influencing both length and weight at birth, the independent variables were insulin, IGF-II, IGFBP-2, cortisol, and IL-6 concentrations in cord serum at birth.2 was 0.570 (p:0.0001).In the multiple regression model the overall RIGF-II cord serum concentration was shown to have a significant positive effect on birth length (p: 0.001) .IGFBP-2 cord serum concentration had a negative effect on birth length (p:0.03) .No significant effect of insulin, cortisol, and IL-6 was shown.2 was 0.461 (p:0.0001).In the multiple regression model the overall RIGF-II cord serum concentration was shown to have a significant positive effect on birth weight ( p: 0.0001) .IGFBP-2 cord serum concentration had a negative effect on birth weight (p:0.05) .Among the cord serum peptides analysed, we described IGF-II and IGFBP-2 as the peptides having the most important effect on both birth length and weight. In this pilot study, insulin, cortisol, and IL-6 cord serum concentrations did not seem to have a major effect on birth size.Data in the Literature to date, concerning the relationship of IGF-II with fetal growth are still controversial Interestingly, IGF-II has been shown to have also a critical role for neural and cardiovascular development in zebrafish embryos Our finding is in agreement with previous studies also, in humans, showing that IGF-II gene polymorphisms are associated with birth weight and fetal growth, as some gene polymorphisms through IGF-II gene expression clearly associate with birth weight SDS We previously described increased IGFBP-2 cord serum concentrations in IUGR compared with AGA newborns, however, the biological meaning of that finding with respect to other changes detected in serum in IUGR at that time was unknown, as well as clear relationships with birth length and/or weight This pilot study now suggests a major negative effect of IGFBP-2 on both birth length and weight, altogether a novel and interesting finding, in particular, in the light of recent publications. IGFBP-2 has been found in fact to be possibly the most useful, among other peptides of the IGF system, in assessing nutritional status in preterm neonates during the early postnatal period Interestingly, in recent years independent effects of IGFBP-2 on glucose metabolism have been discovered also Altogether, our findings related to IGF-II and IGFBP-2 still suggest an important role of insulin, although our analyses suggested an indirect and not a major effect. Cortisol and IL-6 in the fetus at birth, do not seem to have a major role in determining birth size, however, the model did not exclude completely an effect which could also be indirect. Interestingly, our recent data show a positive effect of insulin placental concentration on both birth length and weight (unpublished), although the major effect would be mediated through cortisol placental concentration.In conclusion, the findings of this pilot study evidenced a relationship, in particular, of IGF-II and IGFBP-2 cord serum concentrations with birth size, and also could suggest a possible relationship of fetal size with insulin sensitivity in later life, as previous clinical observations have hypothesized


The prenatal ultrasonographic diagnosis of ectopia cordis associated with a complex intra-cardiac defect  is illustrated in a 32-week gestation fetus. The fetus showed associated features of amniotic band disruption sequence. The cardiac autopsy findings correlated with the antenatal diagnosis. The association of ectopia cordis with amniotic band disruption is rare and infrequently reported in literature. The association of amniotic band disruption sequence with ecopia cordis is very rare. The antenatal ultrasonographic diagnosis of this condition is reported here. The complex cardiac anomalies were correlated with fetal autopsy.A primigravida with 32 weeks of amenorrhea and no risk factors for congenital heart defects was referred to us for a routine cardiac screening. Fetal echocardiography performed using Aloka 5000 equipment showed features suggestive of ectopia cordis . The intThe parents opted for therapeutic abortion. The autopsy of the fetus showed ectopia cordis and multiple anomalies consistent with amniotic band disruption sequence . The lowPrenatal diagnosis of ectopia cordis has been reported rarely in the literature. The condition has a prevalence of 5.5–7.9 per 1 million live births.[Ectopia cordis results from the failure of migration of lateral mesoderm into the midline. The most common associations include sternal defect, pericardial defect, cardiac anomalies and abdominal wall defect, commonly referred to as Pentology of Cantrell. In this fetus, there were no other features of Pentology of Cantrell. Ectopia cordis may be associated with major chromosomal anomalies, especially trisomy 18. The extrThe natural course of this condition together with amniotic band disruption sequence is invariably lethal.


Low Birth Weight is a major public health problem in developing countries. The causes of LBW are multifactorial including complication during pregnancy, genetic, environmental, social-cultural, demographic and nutritional variables. Comparison of anthropometric risk factors for neonatal size of adolescent mothers are lacking from West Bengal. Therefore, this study was undertaken to identify maternal anthropometric characteristics, which most strongly influence neonate weight and length among Bengalee mothers.A hospital based cross-sectional study was undertaken during 2004 in a Government general hospital in South Kolkata, India. A total of 76 adolescent (age<20years) pregnant women were enrolled from obstetric ward who were admitted for delivery. Due to 4 perinatal deaths; a total of 72 adolescent mother- baby pairs were included in this analysis. Anthropometric measurements were undertaken immediately after delivery following stabilization as well maternal body mass index (BMI) was calculated using standard formula.2). It was noted that about 64% of undernourished mothers delivered LBW baby. Linear regression analyses of neonatal weight and length as dependent variables revealed that in both cases, maternal height had the most significant impact. It showed 12.9% (birth weight) and 16.1% (birth length) of variation. Moreover, the proportion of LBW was 75%, 52.3% and 25% among short (height ≤145 cm), average (146–155cm) and tall (>155cm) mothers , respectively. Short mothers had 2.74 and 9.0 fold greater risk of delivering LBW baby than average and tall mothers. In contrast, mean birth weight and length of baby was lower in short mother than their counterparts.The prevalence of low birth weight (LBW) in the present study was 52.8%. The results revealed that 30.6% of mothers were undernourished (BMI<19.8 kg/mThis study revealed that maternal height had the strongest significant impact on neonate size. This strong association could have serious health implications for Bengalee adolescent mothers. However, since this is a preliminary finding, it needs validation using a larger sample of adolescent mothers. Low Birth Weight   is a majIt was well documented that malnourished mothers are more prone to have LBW babies  and pregThe causes and consequences of LBW in different ethnic groups are of much interest to epidemiologists, health care workers, human biologists and biological anthropologists. However, studies that have compared these risk factor profiles of teenage mothers are lacking from West Bengal. Moreover, to the best of the author's knowledge there exists no published report, which deals with the association of maternal anthropometric characteristics and neonatal size among adolescent Bengalee mothers.In view of this, this study was undertaken to identify maternal anthropometric characteristics, which most strongly influence neonate weight and length among Bengalee mothers.A hospital based cross-sectional study was undertaken during 2004 in a Government general hospital in south Kolkata, India. This hospital serves the needs of patients belonging to the lower - middle class socio-economic group. The estimated minimum sample size (n=72) was calculated using a formula to determine sample size for single proportion population, taking the prevalence (p) of LBW 23% in the same hospital  and desiMonthly family income was recorded to the nearest year and Rupee (Rs.), respectively. All anthropometric measurements were undertaken immediately (within 24 hours) after delivery following stabilization using standard protocol . Mother Ethical Committee of the Society for Applied Studies, Kolkata, approved the study protocol. A written consent was obtained from each mother. Information on age, ethnicity, years of formal schooling, maternal obstetric, age, family size and monthly income were obtained using a questionnaire.The distributions of continuous variables were not significantly skewed thus, not necessitating their transformation. Linear regression analyses were used to test for the impact of independent variables. Neonate birth length and weight were used as dependent variables. Odds ratio (OR) and 95% confidence interval (CI) was calculated using standard formula. All statistical analyses were performed using SPSS for windows version 7.5. Statistical significance was considered at p < 0.05.The average monthly family income was Rupees 3665.30 (SD ±2166.7). It must be noted here that the exchange rate during the study period was 46 rupees to approximately 1 USD. The mean age and gestational age were 18.3 (0.8) years and 38.1 (2.4) weeks, respectively. The mean family size and formal years of schooling were 4.7 (2.4) person and 5 (3.3) years, respectively.2. Moreover, Mean birth weight and birth length baby was 2474 (409) gram and 47.3 (2.1) cm, respectively.Anthropometric characteristics of maternal and neonates expressed as means, standard deviation, minimum and maximum values are presented in 2) and about 64% of undernourished mothers delivered LBW baby. Moreover, the proportion of LBW was 75 %, 52.3% and 25% among short (height ≤145 cm), average (146–155cm) and tall mothers (>155cm). Short mothers had 2.74 (95%CI: 0.67–12.04) and 9 (95% CI: 1.24–78.65) fold greater risk of being LBW baby than average and tall mothers. There was a significantly decreasing trend with advancement of maternal height . In contrast, mean birth weight and length of baby was lower for short mothers, mean birth weight  was 52.8%. The results revealed that 30.6% of mothers were undernourished  and 16.1% (birth length) of variation. Maternal weight and head circumference also had significant impact on neonate weight and length. It appeared that none of the other maternal anthropometric characteristics had significant effect on the two dependent variables The mean maternal height and weight in this study were similar to those reported by the WHO  collaborMost importantly, this study provided strong evidence that maternal height had the strongest significant impact on newborn size (birth weight and birth length). It is well established that socio-economic status and ethnicity influences height. Stunting is a consequence of long-term poor nutritional intake and is the best indicator of decreased growth in children over an extended period. Stunting has been associated with poorer cognition and school achievement in later childhood . StuntinThus, this study clearly indicated the strong influence of maternal height on neonate size among adolescent Bengalee mothers. Maternal height alone accounted for 12.9% and 16.1% variation in neonate weight and length, respectively. This strong association could have serious health implications among adolescent mothers in the developing country where most of them suffer form prolonged nutritional deficiency. However, since this is a preliminary finding, it needs validation using a larger sample of adolescent Bengalee mothers. If validated, an appropriate maternal height cut-off point for neonate size could be derived. Based on this cut-off point, high-risk adolescent pregnancies can be identified for appropriate nutritional intervention.


Background. Although twin gestation is well recognized to be associated with impaired fetal and postnatal growth, specific data about body composition of twins in the first month of life are scarce. Objective. The aim of this study was to compare the body composition of twins, evaluated with air-displacement plethysmography, to that of singletons of similar gestational age and adequacy of growth, during the first month of life. We tested the hypothesis that the quality of growth would be similar. Methods. Anthropometric and air-displacement plethysmography measurements were performed in 18 pairs of twins and in 36 singleton neonates, longitudinally, from birth to the 30th day of life. Each twin was matched to a singleton infant of similar gestational age and birth weight z-score. Results. With regard to anthropometric measures, the only difference was a lower weight in twins versus singletons on the 15th day of life. With regard to body composition, we did not find any difference between groups at any time point. Fat mass increased significantly from day 1 to day 30 in both twins and singletons. Conclusion. In terms of body composition, twins do not differ from singletons of similar gestational age and weight, either at birth or in early postnatal life. Early nutrition, growth, and subsequent health are closely interrelated . Twins gEvaluation of body composition (BC) allows assessment of quality of growth in terms of fat mass (FM) and fat-free mass (FFM) and may potentially lead to a better nutritional management in infants with impaired growth . SpecifiThe aim of this study was to compare, by ADP, the BC of twins to that of singletons. We tested the hypothesis that quality of growth would be similar.Parents provided written consent; the study was approved by the ethics committee of our institution.Twins and singletons with a gestational age (GA) between 35 and 40 weeks inborn admitted from 01.2011 to 09.2012 were recruited at birth. Exclusion criteria were intensive care support, oxygen supplementation, intravenous hydration, congenital malformations or chromosomal abnormalities, and having no consent.z-score (±0.3).We conducted a longitudinal, observational preliminary study. Each twin was matched to a singleton infant of similar GA  and birth weight (BW) z-score was calculated from the BW of infants obtained in the delivery room  and BW mean and standard deviation of a Caucasian reference population [BW pulation . Sex wasAnthropometric measures were taken at birth and on days of life 15 and 30. Recumbent length was measured to the nearest 0.2 mm . Head circumference (HC) was measured as occipitofrontal circumference to the nearest 1 mm with a graduated, nonstretchable, flexible tape measure. FM and FFM were measured by an ADP system  in all subjects at three time points: during the first 24 hours of life and on days of life (DOL) 15 and 30. A detailed description of the PeaPod operating principles, validation, and measurement procedures is provided elsewhere [Infants >2500 g at birth were managed within a rooming-in program. They were exclusively breastfed, provided their weight loss did not exceed 10% of BW. All babies <2500 g at birth received 80 mL/Kg/day of breast or formula milk. Feeding was recorded at every measurement as exclusive breastfeeding, partial or predominant breastfeeding, and formula feeding (WHO definitions).t-test for paired samples was used to compare data of twins versus singletons; a chi-square test or Fisher's test was used to compare proportions. Repeated measurements were compared with a one-way analysis of variance (ANOVA). The association between two continuous variables and the independent effect of multiple variables on a dependent continuous variable were tested with linear and multiple regression analysis, respectively. A cluster correction included in the multiple regression analysis accounted for the matching of data. Statistical significance was assumed at P < 0.05. Statistical analyses were performed using MedCalc software rel. 9.3.9.0  and STATA .Variables were expressed as mean (standard deviation) as normally distributed. A Student's A total sample size of 68 infants was required to detect a 2% difference in the percent FM, with a power of the study of 80% and an alpha error of 0.05 (equation for paired studies).P = 0.05). Dizygotic twin couples were 16/18.P < 0.001). Day 1 BC measurements were performed at 21.6 (9.5) hours of life in twins and at 22.0 (9) hours of life in controls (P = 0.9).Weight measured on day 1 by ADP was lower than BW obtained in the delivery room in both groups (P < 0.001).No difference in growth or in % FM between twins and singletons was detected at any study point, apart from a lower weight of twins versus singletons at DOL 15, which did not result in a different BC. FM significantly increased from day 1 to day 30 in both groups , the % FM on DOL 1 was not significantly associated with twin rather than singleton status. Female sex was weakly associated with a higher %FM on day 1 (P = 0.05).In a multiple regression analysis model  The follow-up is short and might fail to show long-term effects of postnatal factors  on BC. (3) We could not evaluate the effect of zygosity, umbilical cord insertion, or fusion of placentas on BC [This is a preliminary report with several limitations. (1) The sample size is relatively small. However, it can detect a 2% difference in % FM and matching by In conclusion, BC of twins does not differ from the one of singletons of similar GA and adequacy of growth, both at birth and in early postnatal life. A rapid FM gain occurs in the first month of life in this population. Our findings need to be confirmed in larger cohorts of twins.


The protein product of sFLT1 is a scavenger of vascular endothelial growth factor (VEGF) in the extracellular environment and plays a key role in the inhibition of placental angiogenesis. In terms of fetal development, inhibition of placental angiogenesis leads to impaired nutrition and hence to growth retardation. Various genes related to DNA methylation and oxidative stress showed also changed expression in relation to arsenic exposure but were not related to birth outcome parameters. In conclusion, this study suggests that increased expression of sFLT1 is an intermediate marker that points to placental angiogenesis as a pathway linking prenatal arsenic exposure to reduced birth weight.There is increasing epidemiologic evidence that arsenic exposure in utero is associated with adverse pregnancy outcomes and may contribute to long-term health effects. These effects may occur at low environmental exposures but the underlying molecular mechanism is not clear. We collected cord blood samples of 183 newborns to identify associations between arsenic levels and birth anthropometric parameters in an area with very low arsenic exposure. Our core research aim was to screen for transcriptional marks that mechanistically explain these associations. Multiple regression analyses showed that birth weight decreased with 47 g (95% CI: 16–78 g) for an interquartile range increase of 0.99 μg/L arsenic. The model was adjusted for child’s sex, maternal smoking during pregnancy, gestational age, and parity. Higher arsenic concentrations and reduced birth weight were positively associated with changes in expression of the  III+ and MMA, are reactive, have longer half-times in the tissues and considered more toxic.Arsenic (As) is a naturally occurring semi-metallic element that can contaminate groundwater used for drinking and irrigation in areas where it’s abundant. In Asia, millions of people are chronically exposed to a high level of As in their drinking water from wells drilled into As-rich geologic strata iAs and its methylated metabolites pass the placenta. Concentrations measured in maternal blood correlate with concentrations in cord blood Levels of As in breast milk are very low, even in areas with highly contaminated drinking water. In a study conducted in Bangladesh in utero or early-life exposure to As at high levels in relation to elevated risk of adverse birth outcome in areas where As concentrations in drinking water that rise far above 40 μg/L (Table S1). However, epidemiological data reporting the effects at the very low exposure end, i.e. below the current WHO drinking water guidelines of 10 μg/L, are currently scarce. The most recent studies investigating low ranges of exposure reported that blood As levels were associated to reduced birth weight in utero As exposure in the development of later life disease.There is a fairly consistent picture of in vitro studies and animal studies.Currently, the molecular mechanisms of action at higher As levels are partly understood. Toxicity pathways that have been suggested include DNA methylation AQP9 was identified as a potential placental biomarker for As exposure, while phospholipase ENPP2 was positively associated with infant birth weight.To the best of our knowledge, only one study exists in which gene expression had been investigated in relation to As exposure at the low-dose end, i.e. in New Hampshire (United States) In the present paper we present the results of a cross sectional study of the association between birth outcomes and biomarkers of fetal exposure to As in a sample of mother–child pairs of the general population of Flanders. The As levels in the cohort are all very low. In addition, we measured gene expression of cord blood cells as an intermediate biomarker to screen biological pathways that may relate As exposure to the health outcome.Written informed consent was provided by all mothers that participated in this study. The mothers also signed the informed consent on behalf of the children enrolled in the study. The study protocol was approved by the ethical committee of the University of Antwerp (Reference UA A08 09).th percentile of birth weight for each week of pregnancy defined by externally obtained sex-specific reference weight curves 2001–2010 . Covariate/confounder data of individual health and lifestyle were obtained via questionnaires. The characteristics of the study population (N = 183) are presented in Table S2).The study was a part of a biomonitoring program for environmental health surveillance in Flanders, Belgium Total As exposure was measured in cord and maternal blood samples. Cord blood was collected immediately after birth, while maternal blood was collected during the mothers’ stay at the maternity unit of the hospital. Arsenic concentration in whole blood was measured by high resolution inductively coupled plasma-mass spectrometry (ICP-MS) The gene expression pattern of blood cells were analysed with an Agilent microarray platform. Aliquots of cord blood (2–3 mL) were collected in Tempus Blood Tubes (Applied Biosystems) as quickly as possible after delivery. After collection, the content of the tubes was mixed by inverting the tubes. Samples were kept at 4°C at the hospital (max 48 hours) and frozen at −20°C upon arrival in our lab.Total RNA was extracted from the blood samples using the Tempus Spin RNA isolation kit (Applied Biosystems) according to manufacturer’s instructions. RNA yields (13–160 μg/sample) were measured using the NanoDrop Spectrophotometer (NanoDrop Technologies). The integrity of the RNA was tested with capillary gel-electrophoresis using RNA 6000 Chips (Agilent Technologies) analyzed on the Agilent 2100 Bioanalyzer. RIN values ranged from 7 to 9.6. Samples were stored at −80°C.Total RNA was amplified and labelled to generate complementary RNA (cRNA) labelled with cyanine (Cy)3-CTP using the Low Input Quick Amp Labelling (one color) kit (Agilent Technologies) according to the manufacturer’s instructions. The single-stranded, labelled cRNA was purified with Qiagen’s RNeasy mini spin columns (Qiagen). Yield and specific activity (dye-incorporation rate) were determined using the NanoDrop spectrophotometer.2-scale). For sequences that are replicated on the array, mean) was calculated. Feature Extraction files include quality control parameters for each of the sequences to assess reliability of the data. A reliable sequence is defined as a sequence 1) of which the intensity is higher than the feature noise level, 2) of which the intensity is below the feature saturation level, 3) that is not a population outlier, 4) of which the spot is uniform, and 5) of which the background is not an outlier. This information was taken into account in the analysis as described in section ‘Statistical analysis’.For each sample, 1.65 μg cRNA was hybridized per microarray  during 17 h using the automated HS4800TM pro hybridization station (Tecan), according to the manufacturer’s instructions. The arrays were scanned with an Agilent scanner (G2565BA). The arrays were subjected to a primary quality control using the Agilent Feature Extraction Software (Version 10.7). For each feature on the array, the software accurately determines the intensity , which were used as input for further processing and analysis . Normalization between arrays was performed by means of quantile normalization upon logarithmic transformation of the data . cDNA synthesis and qPCR reactions were performed as described previously a priori fixed known confounders, gender of the newborn and smoking of the mother during pregnancy, that were included irrespective of their significance level. Other influencing factors (covariates) were included when significant at the 0.05 level in the multiple regression model. The following covariates were considered: gestational age (only for birth weight and head circumference), maternal age, parity, stress/pressure during pregnancy, maternal education level, (passive) smoking before pregnancy, alcohol use before/during pregnancy, maternal height, maternal body mass index (BMI), equivalent income, infections/complication during pregnancy, use of folic acid during pregnancy and caesarean section . Effect modification (interaction) was analyzed in models including main effects and cross-product terms, and by doing stratified analyses. A p-value <0.20 for the effect of the cross-product was taken as an indication of interaction. Assumptions of normality, constancy of variance, independence (randomness), and linearity were checked with informal diagnostic plots and formal tests . Linear and logistic regression analyses were used to quantify the effect on different growth parameters associated with an interquartile range (IQR) increase of the exposure to As. The exposure-response relations were adjusted for Table S4). Genes related to the first 3 processes were retrieved from the Ingenuity Knowledge Base , the list of As- related genes was obtained from a recent publication boys = 14, ngirls = 17), and ‘low’ smaller than geometric mean minus standard deviation . P-value below 0.05 was used as a cut-off for statistical significance. We aimed to follow a similar approach to select ‘high’ and ‘low’ birth weight babies. For this, adjusting birth weight a priori for gestational age was necessary to categorize the samples. A linear regression model - including a quadratic term - was fitted separately for male en female babies with gestational age as the independent variable and birth weight as the outcome variable. For each individual, the ratio between observed birth weight and the estimated birth weight at the same gestational age was calculated. Following Ln-transformation of this ratio, ‘high’ and ‘low’ birth weight samples were identified. ‘High birth weight’ means Ln-transformed ratio above mean plus standard deviation , and ‘low birth weight’ smaller than mean minus standard deviation . Analogue to As exposure, we tested whether ‘low birth weight’ samples show differences in expression as compared to ‘high birth weight’ samples for boys and girls separately. Following a targeted approach (focused on a subset of genes among the whole array), we decided to not correct for multiple testing. Transcripts for which the microarray data were not reliable for all samples being considered (according to the criteria mentioned previously) were excluded from statistical analysis.For the statistical analysis of the transcriptome data Matlab was used, version 2012b. For our analysis we focused on a subset of genes that were associated with intrauterine growth, DNA methylation, oxidative stress, and As-related genes (Model 1). Secondly, As exposure should be a significant predictor of the expression of gene ‘X’ (the mediator) in cord blood cells. In addition, the expression of gene ‘X’ should be a significant predictor of birth weight (model 3). In the final step (model 4), it must be confirmed that the expression of gene ‘X’ is a significant predictor of birth weight, while controlling for As exposure. The estimated change in birth weight related to As exposure (βAs) in model 4 should be less compared to model 1 to demonstrate mediation. Maximum evidence for mediation (full mediation) occurs if by inclusion of the expression of gene ‘X’, βAs drops to zero. In partial mediation βAs in model 4 is weaker, yet still significant, compared to model 1. In that case, gene expression of gene ‘X’ accounts for some – but not all – of the observed association between As exposure and birth weight. The following covariates were considered: gender, gestational age, smoking during pregnancy, and parity. Effect modification (interaction) was analyzed in models including main effects and cross-product terms.To demonstrate mediating effects of gene expression in the association between As exposure and birth weight, a mediation analysis was performed by the procedure described by Baron and Kenny (1986) sFLT1 as measured with microarray by use of qPCR, the correlation between both measures was calculated for transcript NM_001159920. Input values obtained from 30 samples were log2-transformed fold changes relative to one of the samples (which was randomly selected). Analogue, correlation between qPCR data obtained from NM_001159920 and NM_001160030 . For NM_001159920, the difference in expression among ‘high’ and ‘low’ As exposure was analyzed by independent two-sample t-test  and compared with differential expression measured by microarray.For confirmation of the expression of The geometric mean value of As in cord blood was 0.56 μg/L (95% CI: 0.47–0.66 μg/L). The As levels ranged up to 14.4 μg/L, with one value below the limit of detection (LOD) of 0.028 μg/L (replaced by LOD/2), and an interquartile range of 0.99 μg/L. As levels in maternal and cord blood were highly correlated ). The correlation between both measures is visualized by Table S2).Fourteen of the newborns (7.7%) were born as SGA-babies, the gestational age ranged from 34 to 42 weeks with a median of 40 weeks (The multiple regression models showed a significant association (p<0.05) between increasing As concentrations in cord blood and an increased risk of SGA and lower birth weight. For an increase of As cord blood levels with the interquartile range ( = 0.99 μg/L), the risk for SGA babies increased with 38% (95% CI: 11–71%) and birth weight decreased with 47 g (95% CI: 16–78 g) Table 1.The associations were not modified by sex when looking at the cross-product term of sex with As levels . When doing stratified analyses, the associations between As and birth outcomes were still statistically significant in boys, but not in girls both for SGA and birth weight. Both estimates were however in the same order, but due to power  not statistically significant in girls.Table S4). st quadrant is composed of transcripts that show higher expression among the group of high As and the group of SGA as compared to the group of low As and the group of LGA, respectively. Making the same comparisons, transcripts that show lower expression can be found in the 3rd quadrant. Aiming to identify molecules that might link the level of As exposure to elevated risk on SGA these quadrants are of special interest, since the other 2 contain genes that show opposite regulation of transcription in ‘high’ versus ‘low’ As exposure, as compared to ‘low’ versus ‘high’ birth weight. The corresponding gene symbol of transcripts with p-value smaller than 0.05 (dashed threshold lines) in either ‘high’ versus ‘low’ As exposure or ‘low’ versus ‘high’ birth weight is included on the plot. Among girls, two transcripts show stronger potential to link the level of As exposure to elevated risk on SGA. One of them is upregulated in ‘high’ As exposed and ‘low’ birth weight samples, i.e. a soluble transcript variant of FLT1 (NM_001159920). The corresponding p-value for differential expression between ‘high’ and ‘low’ As exposure equals 0.033, and 0.017 between ‘low’ and ‘high’ birth weight. The other transcript  is downregulated in expression in ‘high’ As exposed and ‘low’ birth weight samples. The corresponding p-value for differential expression between ‘high’ and ‘low’ As exposure equals 0.032, and 0.009 between ‘low’ and ‘high’ birth weight. For boys, no transcripts could be identified that show strong potential to link the level of As exposure to elevated risk on SGA.To identify molecules that might link the level of As exposure to elevated risk on SGA, cord blood gene expression was analyzed. After microarray quality control filtering, 158 different genes could be annotated to the function ‘embryonal growth’ according the Ingenuity Knowledge Base  or the transmembrane isoform  of the protein. The data obtained for the mFLT1 did not successfully pass quality control . For sFLT1, reported microarray data are restricted to NM_001159920 as the sequence spotted on the microarray is specific for that variant. Use of qPCR enabled us to measure two different variants of sFLT1, variant NM_001160031 was not detectable. Microarray and qPCR data of NM_001159920 correlated very well . The correlation between both measures is visualized by The expression of Figure S1). The list of genes retrieved from the Ingenuity Knowledge Base can be found in Table S4. The expression of MYC was positively associated to the As level in boys . In girls , respectively ARID4A and PLD6 were significantly up- and downregulated, with increasing As levels. However, none of these genes could be linked to decreased birth weight.Analogue to the approach described in the previous paragraph, genes involved in DNA methylation were analyzed . They all successfully passed quality control filtering (Table S4). None of them was found to be significantly different  in expression among ‘high’ versus ‘low’ exposure or among ‘low’ versus ‘high’ birth weight.To enable comparison with a recent publication Table S4). A volcano plot that includes p-value and fold change (in ‘high As’ versus ‘low As’) for all 152 genes for boys and girls separately can be found in Figure S2. Among boys , the expression of four genes differed between ‘high’ and ‘low’ As . In girls , BRCA1, MMP2, MMP9, PEMT, S100A12, SELK, TAT, and VNN1 were differentially expressed. The direction of regulation can be derived from the plot.Several studies indicate that oxidative stress may play a role in As toxicity. A list of genes that are known to be involved in oxidative stress responses was retrieved from the Ingenuity Knowledge Base . Including the interaction between gender and As exposure did not improve the result. Due to this, we limited the proceeding steps in mediation analysis and the statistical output in sFLT1. In the next step, we evaluated birth weight in function of sFLT1, while adjusting for gender, gestational age, smoking during pregnancy, and parity (sFLT1 expression and gender was significant (p = 0.0450) and therefore also included. A significant reduction in birth weight and sFLT1 expression was found for girls (p = 0.0061), but not for boys (p = 0.9775). Duplication in sFLT1 expression in girls was associated with a reduction in birth weight of 182 g . In the final model (sFLT1 was a significant predictor of birth weight among the population of girls (p = 0.0118), while controlling for As exposure. The reduction in birth weight associated with ‘high’ As exposure was less pronounced  as compared to the first model  in which the effect of gene expression was not included. This indicates that sFLT1 may be considered as a  mediator in the association between As exposure and reduced birth weight.Two genes showed changes in expression in response to As exposure that could also be linked to reduced birth weight, i.e. As a sensitivity analysis, it has been verified whether the associations reported were influenced by maternal BMI and pregnancy complications. Including BMI in each of the linear regression models shown in Table S1). Although maternal urinary As concentrations were not analysed in our cohort, we have data of urinary levels of total As and toxicologically relevant As , and dimethylarsinic acid (DMA)) in a cohort from 101 women aged between 20 and 40 sampled in the same campaign. In this population, which is representative for Flanders, the median levels of total As and toxicological relevant As were respectively 16.2 μg/L  and 4.3 μg/L . The observed effect on birth weight confirms findings at relatively low levels th percentile: 0.2 μg/L; 75th percentile: 1.2 μg/L) and is even about 10 times lower compared to both studies mentioned. No significant associations were found between As levels in cord blood and head circumference at birth and gestational age. The latter could be an indication that the associations between As and SGA and birth weight were not mediated by effects on gestational age.This study provides evidence that As exposure even at low environmental levels is associated with birth anthropometric parameters. Decrease in birth weight, independent of gestational age, and an increased risk for babies born SGA was related to increasing As concentrations in cord blood samples of newborn babies. The association was observed at an exposure range which is applicable to the exposure of ‘control subjects’ in other studies . However, the effect of differential expression of sFLT1 on birth weight was only significant in girls, while there was absence of evidence for an association between sFLT1 and birth weight for boys.We tested whether changes in gene expression in cord blood cells could reflect molecular links between As exposure and birth outcomes. Specific gene panels were selected that were previously already associated with intrauterine growth retardation, As exposure, or with potential mode of actions of As such as oxidative stress and DNA methylation. Of these, two genes showed changes in expression in response to As exposure that could also be linked to the risk of SGA in our birth cohort, i.e. sFlt1 is a natural soluble factor, it is the truncated version of VEGFR1 (or mFLT1) lacking transmembrane and intracellular signaling domains. sFLT1 is a key molecule in inhibition of placental angiogenesis, it inhibits angiogenesis by binding to free forms of VEGF and PlGF sFLT1 mRNA expression in circulating cells not exposed directly to ischemia sFLT1 expression was less than the rise in placental expression, it indicates that – at least for sFLT1 - blood cells mRNA reflect transcriptional changes in the placenta.In vitro studies using human umbilical vein endothelial cells confirmed that inorganic arsenite or arsenate (100 μM) significantly suppressed the activity of human eNOS Disturbed regulation of placental angiogenesis confirms in humans what has been demonstrated earlier in animal experiments. Sodium arsenite caused spontaneous abortion in mice via aberrant placental vasculogenesis and placental insufficiency sFLT1 expression was restricted to female neonates. However, it suggests that hormonal status may play a role in phenotypic modulation. In the context of our results, it can be hypothesized that co-regulatory gender dependent signals are involved in directing the sFLT1-pathway towards its specific outcome. Accumulating data suggest that As may act as an endocrine disruptor. It has been demonstrated that monomethylated trivalent As species inhibit binding of glucocorticoid (GR) and progesterone receptor (PR) with their DNA response elements that are crucial to hormone-driven gene transcription Although gender dichotomy in the mechanism of growth restriction driven by sFLT1 has not been reported before, a very recent study demonstrated sexual dimorphism in placental function in severe pre-eclampsia sFLT1 is a partial mediator – and not a full mediator - of the association between As exposure and birth weight. This means that expression of sFLT1, accounts for some – but not all – of the observed association. It implies that additional factors are involved that could not be identified in our study. Gene expression in cord blood was considered as a surrogate marker to reflect adverse reactions in the tissues targeted by As exposure. However, it cannot be assumed that gene expression measurements in cord blood always reflect gene expression changes within other tissues. This implies that potential mediators may be missed. In addition, levels of regulation that were not covered in our study – such as post transcriptional and epigenetic regulations – may provide additional mediators to explain the association between As exposure and reduced birth weight.Our results indicate that nd Flemish Environment and Health Study showed that the concentration of As in cord blood of newborns in the present study was linked with reduced birth weight and a higher risk of having a SGA baby, even though exposure in Flanders is low compared to other regions. Impaired fetal growth is a risk factor to develop metabolic problems later in life. Molecular data indicate that impaired placental vascularisation, by upregulation of sFLT1, might be a mechanistic basis that mediates the association between As exposure and reduced birth weight.This study provides new insight in low-dose prenatal As toxicity. Results of the 2Figure S1Genes associated to ‘DNA methylation’. This figure shows the potential of genes related to DNA methylation to link the level of As exposure to decreased birth weight for boys (A) and girls (B) separately. For each of the sequences, the significance ) of differential regulation between ‘high’ and ‘low’ As is plotted on the horizontal axis, and between ‘low’ and ‘high’ birth weight is plotted on the vertical axis. The direction of regulation (up or down) is included in the plot. The dashed threshold lines represent p-value equal to 0.05. For transcripts of which the p-value is smaller than 0.05 in either one of the comparisons, the corresponding gene symbol is included in the plot.(TIFF)Click here for additional data file.Figure S2versus ‘low Arsenic’) of genes related to oxidative stress (N = 152) for boys (A) and girls (B) separately. The dashed treshold lines correspond to p-value equal to 0.05. For transcripts of which the p-value is smaller than 0.05, the corresponding gene symbol is included in the plot.Genes associated to ‘Oxidative stress’. The volcano plot includes the p-value and fold change (in ‘high Arsenic’ (TIFF)Click here for additional data file.Table S1th percentile; P25 = 25th percentile; P75 = 75th percentile; P90 = 90th percentile; SGA: small for gestational age 1. Ahmad SA, Sayed MH, Barua S, Khan MH, Faruquee MH, et al. (2001) Arsenic in drinking water and pregnancy outcomes. Environ Health Perspect 109∶629-631. 2. Guan H, Piao F, Zhang X, Li X, Li Q, et al. (2012) Prenatal exposure to arsenic and its effects on fetal development in the general population of Dalian. Biol Trace Elem Res 149∶10-15. 3. Hopenhayn C, Ferreccio C, Browning SR, Huang B, Peralta C, Gibb H, Hertz-Picciotto I (2003) Arsenic exposure from drinking water and birth weight. Epidemiology 14∶593-602. 4. Hopenhayn-Rich C, Browning SR, Hertz-Picciotto I, Ferreccio C, Peralta C, et al. (2000) Chronic arsenic exposure and risk of infant mortality in two areas of Chile. Environ Health Perspect 108∶667-673. 5. Huyck KL, Kile ML, Mahiuddin G, Quamruzzaman Q, Rahman M, et al. (2007) Maternal arsenic exposure associated with low birth weight in Bangladesh. J Occup Environ Med 49∶1097-1104. 6. Milton AH, Smith W, Rahman B, Hasan Z, Kulsum U, et al. (2005) Chronic arsenic exposure and adverse pregnancy outcomes in bangladesh. Epidemiology 16∶82-86. 7. Rahman A, Vahter M, Ekstrom EC, Rahman M, Golam Mustafa AH, et al. (2007) Association of arsenic exposure during pregnancy with fetal loss and infant death: a cohort study in Bangladesh. Am J Epidemiol 165∶1389-1396. 8. Rahman A, Vahter M, Smith AH, Nermell B, Yunus M, et al. (2009) Arsenic exposure during pregnancy and size at birth: a prospective cohort study in Bangladesh. Am J Epidemiol 169∶304-312. 9. Rahman A, Persson LA, Nermell B, El AS, Ekstrom EC, et al. (2010) Arsenic exposure and risk of spontaneous abortion, stillbirth, and infant mortality. Epidemiology 21∶797-804. 10. von Ehrenstein OS, Guha Mazumder DN, Hira-Smith M, Ghosh N, Yuan Y et al. (2006) Pregnancy outcomes, infant mortality, and arsenic in drinking water in West Bengal, India. Am J Epidemiol 163∶662-669. 11. Xu L, Yokoyama K, Tian Y, Piao FY, Kitamura F, et al. (2011) Decrease in birth weight and gestational age by arsenic among the newborn in Shanghai, China. Nihon Koshu Eisei Zasshi 58∶89-95. 12. Yang CY, Chang CC, Tsai SS, Chuang HY, Ho CK, et al. (2003) Arsenic in drinking water and adverse pregnancy outcome in an arseniasis-endemic area in northeastern Taiwan. Environ Res 91∶29-34.Literature overview of adverse pregnancy outcomes in relation to As exposure. Abbreviations: CI: Confidence interval; OR = Odds ratio; P10 = 10(DOCX)Click here for additional data file.Table S2th percentile; P75 = 75th percentile; SGA = Small for Gestational Age.Descriptive statistics of the study population (183 mother-newborn pairs). Abbreviations: N = number of subjects; P25 = 25(DOCX)Click here for additional data file.Table S3Sequences of 5′ exonuclease PrimeTime™ assays that were used for qPCR analysis.(DOCX)Click here for additional data file.Table S4Subset of genes that were associated with intrauterine growth, DNA methylation, oxidative stress, and As-related genes. Quality control: +: Quality control passed for given sequence, −: Quality control not passed for given sequence. Embryonal growth: Number of sequences Agilent array = 294 (160 unique genes); Number of sequences Agilent array after quality control filtering = 289 (158 unique genes). DNA methylation: Number of sequences Agilent array = 102 (49 unique genes); Number of sequences Agilent array after quality control filtering = 101 (48 unique genes). Oxidative stress: Number of sequences Agilent array = 275 (154 unique genes); Number of sequences Agilent array after quality control filtering = 269 (152 unique genes).(DOCX)Click here for additional data file.