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STUDY QUESTION Can we detect DNA methylation differences between ART children that underwent embryo culture in different media? SUMMARY ANSWER We identified no significant differences in site-specific or regional DNA methylation between the different culture medium groups. WHAT IS KNOWN ALREADY Embryo culture in G3 or K-SICM medium leads to differences in embryonic, neonatal and childhood outcomes, including growth and weight. The methylome may mediate this association as the period of in vitro culture of ART treatments coincides with epigenetic reprogramming. STUDY DESIGN, SIZE, DURATION This study was conducted as a follow-up to a previous culture medium comparison study in which couples were pseudo-randomized to embryo culture in G3 or K-SICM medium. Of the resultant singletons, 120 (n = 65 G3, n = 55 K-SICM), were recruited at age 9. PARTICIPANTS/MATERIALS, SETTING, METHODS The ART children provided a saliva sample from which the methylome was analysed using the Infinium MethylationEPIC array. After quality and context filtering, 106 (n = 57 G3, n = 49 K-SICM) samples and 659 708 sites were retained for the analyses. Differential methylation analyses were conducted using mixed effects linear models corrected for age, sex, sample plate and cell composition. These were applied to all cytosine-guanine dinucleotide (CpG) sites, various genomic regions (genes, promoters, CpG Islands (CGIs)) and as a targeted analysis of imprinted genes and birth weight-associated CpG sites. Differential variance was assessed using the improved epigenetic variable outliers for risk prediction analysis (iEVORA) algorithm and methylation outliers were identified using a previously defined threshold (upper or lower quartile plus or minus three times the interquartile range, respectively). MAIN RESULTS AND THE ROLE OF CHANCE After correcting for multiple testing, we did not identify any significantly differentially methylated CpG sites, genes, promoters or CGIs between G3 and K-SICM children despite a lenient corrected P-value threshold of 0.1. Targeted analyses of (sites within) imprinted genes and birth weight-associated sites also did not identify any significant differences. The number of DNA methylation outliers per sample was comparable between the culture medium groups. iEVORA identified 101 differentially variable CpG sites of which 94 were more variable in the G3 group. LARGE SCALE DATA Gene Expression Omnibus (GEO) GSE196432 LIMITATIONS, REASONS FOR CAUTION To detect significant methylation differences with a magnitude of WIDER IMPLICATIONS OF THE FINDINGS The results of this study are reassuring, suggesting that if there is an effect of the culture medium on DNA methylation (and methylation-mediated diseases risk), it does not differ between the two media investigated here. The findings concur with other methylome studies of ART neonates and children that underwent embryo culture in different media, which also found no significant methylome differences. STUDY FUNDING/COMPETING INTEREST(S) Study funded by March of Dimes (6-FY13-153), EVA (Erfelijkheid Voortplanting & Aanleg) specialty programme (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. The authors do not report any conflicts of interest relevant to this study. TRIAL REGISTRATION NUMBER Dutch Trial register—NL4083

BackgroundThe prenatal exposure to maternal n-6 and n-3 polyunsaturated fatty acids (PUFAs) might influence the development of social competence and internalizing and externalizing behaviours of the child, because of the numerous functions of PUFAs within the nervous system.MethodsTo analyse the association of selected maternal PUFAs (i.e., AA, EPA, DHA, total n-6, total n-3, and the n-6:n-3 ratio) measured during gestation with childhood social competence and problem behaviours, we examined 311 mother-child pairs from the Maastricht Essential Fatty Acid Birth (MEFAB) cohort. For each woman, PUFA-specific changes in relative concentrations were calculated by identifying the best-fitting curve of PUFA concentration by linear splines of gestational age. The associations of changes in maternal PUFAs in early and late pregnancy with childhood social competence, total problems, internalizing and externalizing behaviours, measured with the Child Behaviour Checklist 4/18 at age 7, were investigated with linear regression analyses adjusted for maternal and children's socio-demographic characteristics.ResultsIn late gestation (i.e., from gestational week 30), an increase in AA was associated with higher social competence, while a decrease in total n-6 was associated with lower externalizing behaviours. No other significant associations were found.DiscussionIn this prospective study, increasing maternal AA and decreasing total n-6 were associated with improved social competence and externalizing behaviours, respectively, in 7-year old children. Nonetheless, the clinical significance of the identified associations is modest and further investigations are warranted to clarify the relationship between maternal AA and total n-6 during pregnancy and childhood social and behavioural development.

Background/objectives Polyunsaturated fatty acid (PUFA) status during pregnancy has been suggested to influence offspring obesity and cardiometabolic health. We assessed whether prenatal PUFA exposure is associated with rapid infant growth, childhood BMI, and cardiometabolic profile.Subjects/methods In the Dutch MEFAB (n = 266) and Greek RHEA (n = 263) cohorts, we measured n-3 and n-6 PUFA concentrations in cord blood phospholipids, which reflect fetal exposure in late pregnancy. We defined rapid infant growth from birth to 6 months of age as an increase in weight z-score >0.67. We analyzed body mass index (BMI) as continuous and in categories of overweight/obesity at 4 and 6 years. We computed a cardiometabolic risk score at 6-7 years as the sum of waist circumference, non-high-density lipoprotein cholesterol and blood pressure z-scores. Associations of PUFAs with child health outcomes were assessed using generalized linear models for binary outcomes and linear regression models for continuous ones after adjusting for important covariates, and for the pooled estimates, a cohort indicator.Results In pooled analyses, we found no association of PUFA levels with rapid infant growth, childhood BMI (beta per SD increase in the total n-3:n-6 PUFA ratio = -0.04 SD; 99% CI: -0.15, 0.06; P = 0.65 at 4 years, and -0.05 SD; 99% CI: -0.18, 0.08; P = 0.78 at 6 years), and overweight/obesity. We also found no associations for clustered cardiometabolic risk and its individual components. The results were similar across cohorts.Conclusions Our findings suggest that PUFA concentrations at birth are not associated with later obesity development and cardiometabolic risk in childhood.

Background: It has been suggested that prenatal exposure to n-3 long-chain fatty acids protects against asthma and other allergy-related diseases later in childhood. The extent to which fish intake in pregnancy protects against child asthma and rhinitis symptoms remains unclear. We aimed to assess whether fish and seafood consumption in pregnancy is associated with childhood wheeze, asthma and allergic rhinitis. Methods: We pooled individual data from 60 774 mother-child pairs participating in 18 European and US birth cohort studies. Information on wheeze, asthma and allergic rhinitis prevalence was collected using validated questionnaires. The time periods of interest were: infancy (0-2 years), preschool age (3-4 years), and school age (5-8 years). We used multivariable generalized models to assess associations of fish and seafood (other than fish) consumption during pregnancy with child respiratory outcomes in cohort-specific analyses, with subsequent random-effects meta-analyses. Results: The median fish consumption during pregnancy ranged from 0.44 times/week in The Netherlands to 4.46 times/week in Spain. Maternal fish intake during pregnancy was not associated with offspring wheeze symptoms in any age group nor with the risk of child asthma [adjusted meta-analysis relative risk (RR) per 1-time/week = 1.01, 95% confidence interval 0.97-1.05)] and allergic rhinitis at school age (RR = 1.01, 0.99-1.03). These results were consistently found in further analyses by type of fish and seafood consumption and in sensitivity analyses. Conclusion: We found no evidence supporting a protective association of fish and seafood consumption during pregnancy with offspring symptoms of wheeze, asthma and allergic rhinitis from infancy to mid childhood. This work was supported by the European Community’s Seventh Framework Program [EU- FP7- HEALTH-2009-single-stage-241604]. Details of funding per cohort are available at IJE online.

Background: Previous research shows that, besides risk factors in adult life, the early-life environment can influence blood pressure and hypertension in adults. However, the effects of residential traffic exposure and residential greenness in the early-life on blood pressure in young adulthood are currently unknown. Methods: Ambulatory (24-h) blood pressures of 278 twins (132 pairs) of the East Flanders Prospective Twins Study were obtained at the age of 18 to 25 years. Prenatal and adulthood residential addresses were geocoded and used to assign prenatal and postnatal traffic and greenness indicators. Mixed modelling was performed to investigate blood pressure in association with greenness while adjusting for potential confounding factors. Results: Night-time systolic blood pressure was inversely associated with greenness at the residential address in twins living at the same address their entire life (non-movers, n = 97, 34.9%). An interquartile increase in residential greenness exposure (1000 m radius) was associated with a 3.59 mmHg (95% CI: -6.0 to -1.23; p = 0.005) lower adult night systolic blood pressure. Among twins who were living at a different address than their birth address at time of the measurement (n = 181, 65.1%), night-time blood pressure was inversely associated with residential surrounding greenness at adult age as well as with residential greenness in early-life. However after additional adjustment for residential greenness exposure in adulthood, only residential greenness exposure in early-life was significantly associated with night systolic blood pressure. While no significant effect of adult residential greenness with adult blood pressure was observed, while accounting for the early-life greenness exposure. Conclusions: Lower residential greenness in the early-life environment was independently associated with a higher adult blood pressure. This indicates that residential greenness has persistent effects on blood pressure. This investigation is supported by the EU research council "project ENVIRONAGE" (ERC-2012-StG 310,890) and Flemish Scientific Fund (G073315 N). Since its start, the East Flanders Prospective Twin Study has been partly supported by grants from the Fund of Scientific Research Flanders and Twins, a non-profit Association for Scientific Research in Multiple Births (Belgium).

Objectives Maternal pre-pregnancy weight is known to affect foetal development. However, it has not yet been clarified if gestational weight gain is associated with childhood behavioural development. Methods We performed a pooled analysis of two prospective birth cohorts to investigate the association between gestational weight gain and childhood problem behaviours, and the effect modification of maternal pre-pregnancy BMI. In total, 378 mother–child pairs from the Maastricht Essential Fatty Acids Birth cohort (MEFAB) and 414 pairs from the Rhea Mother–Child cohort were followed up from early pregnancy to 6–7 years post-partum. At follow up, parents assessed their children’s behaviour, measured as total problems, internalizing and externalizing behaviours, with the Child Behaviour Checklist. We computed cohort- and subject-specific gestational weight gain trajectories using mixed-effect linear regression models. Fractional polynomial regressions, stratified by maternal pre-pregnancy BMI status, were then used to examine the association between gestational weight gain and childhood problem behaviours. Results In the pre-pregnancy overweight/obese group, greater gestational weight gain was associated with higher problem behaviours. On average, children of women with overweight/obesity who gained 0.5 kg/week scored 25 points higher (on a 0–100 scale) in total problems and internalizing behaviours, and about 18 points higher in externalizing behaviours than children whose mothers gained 0.2 kg/week. Inconsistent results were found in the pre-pregnancy normal weight group. Conclusions for Practice Excessive gestational weight gain in women with pre-pregnancy overweight/obesity might increase problem behaviours in school-age children. Particular attention should be granted to avoid excessive weight gain in women with a pre-pregnancy overweight or obesity.

The East Flanders Prospective Twin Survey (EFPTS) is a registry of multiple births in the province of East Flanders, Belgium. Since its start in 1964, over 10,000 twin-pairs have been registered. EFPTS has several unique features: it is population-based and prospective, with the possibility of long-term follow-up; the twins (and higher order multiple births) are recruited at birth; basic perinatal data are recorded; chorion type and zygosity are established; since 1969, placental biopsies have been taken and frozen at -20°C for future research. Since its origin, the EFPTS has included placental data and allows differentiation of three subtypes of monozygotic twins based on the time of the initial zygotic division: the dichorionic-diamniotic pairs (early, with splitting before the fourth day after fertilization), the monochorionic-diamniotic pairs (intermediate, splitting between the fourth- and the seventh-day postfertilization) and the monochorionic-monoamniotic pairs (late, splitting after the eighth day postfertilization). Studies can be initiated taking into account primary biases, those originating 'in utero'. Such studies could throw new light on the consequences of early embryological events and the gene-environment interactions as far as periconceptional and intrauterine environment are concerned. ispartof: TWIN RESEARCH AND HUMAN GENETICS vol:22 issue:6 pages:454-459 ispartof: location:England status: published

Several studies in singletons have shown that maternal exposure to ambient air pollutants is associated with restricted fetal growth. About half of twins have low birth weight compared with six percent in singletons. So far, no studies have investigated maternal air pollution exposure in association with birth weight and small for gestational age in twins. We examined 4760 twins of the East Flanders Prospective Twins Survey (2002-2013), to study the association between in utero exposure to air pollution with birth weight and small for gestational age. Maternal particulate air pollution (PM10) and nitric dioxide (NO2) exposure was estimated using a spatial temporal interpolation method over various time windows during pregnancy. In the total group of twins, we observed that higher PM10 and NO2 exposure during the third trimester was significantly associated with a lower birth weight and higher risk of small for gestational age. However, the association was driven by moderate to late preterm twins (32-36 weeks of gestation). In these twins born between 32 and 36 weeks of gestation, birth weight decreased by 40.2 g (95% CI: -69.0 to -11.3; p=0.006) and by 27.3 g (95% CI: -52.9 to -1.7; p=0.04) in association for each 10 mu g/m(3) increment in PM10 and NO2 concentration during the third trimester. The corresponding odds ratio for small for gestational age were 1.68 (95% CI: 1.27-2.33; p=0.0003) and 1.51 (95% CI: 1.18-1.95; p=0.001) for PM10 or NO2, respectively. No associations between air pollution and birth weight or small for gestational age were observed among term born twins. Finally, in all twins, we found that for each 10 mu g/m(3) increase in PM10 during the last month of pregnancy the within-pair birth weight difference increased by 19.6 g (95% CI: 3.7-35.4; p=0.02). Assuming causality, an achievement of a 10 mu g/m(3) decrease of particulate air pollution may account for a reduction by 40% in small for gestational age, in twins born moderate to late preterm.

Background: Even before the onset of age-related diseases, obesity might be a contributing factor to the cumulative burden of oxidative stress and chronic inflammation throughout the life course. Obesity may therefore contribute to accelerated shortening of telomeres. Consequently, obese persons are more likely to have shorter telomeres, but the association between body mass index (BMI) and leukocyte telomere length (TL) might differ across the life span and between ethnicities and sexes. Objective: A collaborative cross-sectionalmeta-analysis of observational studies was conducted to investigate the associations between BMI and TL across the life span. Design: Eighty-seven distinct study samples were included in the meta-analysis capturing data from 146,114 individuals. Studyspecific age- and sex-adjusted regression coefficients were combined by using a random-effects model in which absolute [base pairs (bp)] and relative telomere to single-copy gene ratio (T/S ratio) TLs were regressed against BMI. Stratified analysis was performed by 3 age categories ("young": 18-60 y; "middle": 61-75 y; and "old": >75 y), sex, and ethnicity. Results: Each unit increase in BMI corresponded to a-3.99 bp (95% CI: -5.17, -2.81 bp) difference in TL in the total pooled sample; among young adults, each unit increase in BMI corresponded to a -7.67 bp (95% CI:-10.03,-5.31 bp) difference. Each unit increase in BMI corresponded to a -1.58 × 10-3 unit T/S ratio (0.16% decrease; 95% CI: -2.14 × 10-3, -1.01 × 10-3) difference in ageand sex-adjusted relative TL in the total pooled sample; among young adults, each unit increase in BMI corresponded to a -2.58 × 10-3 unit T/S ratio (0.26% decrease; 95% CI: -3.92 × 10-3, -1.25 × 10-3). The associations were predominantly for the white pooled population. No sex differences were observed. Conclusions: A higher BMI is associated with shorter telomeres, especially in younger individuals. The presently observed difference is not negligible. Meta-analyses of longitudinal studies evaluating change in body weight alongside change in TL arewarranted.

Background: Increasing evidence suggests that long-chain polyunsaturated fatty acid (LCPUFA) availability in utero could program later health.Objective: The objective of the study was to explore whether prenatal LCPUFA availability could be involved in programming cardiometabolic disease risk at childhood. Methods: Data of 242 mother child pairs from the Maastricht Essential Fatty Acid Birth (MEFAB) cohort were used. Multi-variable linear regression analysis was applied to identify associations between maternal LCPUFA concentrations around weeks 11, 22 and 32 of pregnancy and at time of delivery and cardiometabolic risk factors of their children (glucose metabolism, blood lipids, and blood pressure) at age 7.Results: Maternal eicosapentaenoic acid (20:5n-3) at week 11 of pregnancy was negatively associated with children's glucose (B=-0.34 mmol/L; 95% CI: -0.56, -0.12). Positive associations were found between maternal linoleic acid (18:2n-6) at time of delivery and children's proinsulin (B=0.25 pmol/L; 95% Cl: 0.08, 0.41); maternal 3-docosapentaenoic acid (22:5n-3) at week 11 and children's total cholesterol (B=1.23 mmol/L; 95% Cl: 0.45, 2.01) and low-density-lipoprotein cholesterol (B=1.12 mmol/L; 95% CI: 0.42, 1.82); and maternal osbond acid (22:5n-6) at week 22 and tetracosadienoic acid (24:2n-6) at week 32 and children's diastolic blood pressure (B=16.86 mmHg; 95% Cl: 7.63, 26.08 and B=17.75 mmHg; 95% Cl: 6.37, 29.94, respectively).Conclusion: Our findings suggest that maternal omega-6 (n-6) fatty acids may be of particular importance in relation to children's glucose metabolism and blood pressure, whereas omega-3 (n-3) fatty acids seem particularly related to blood lipids at childhood. In general, the strength of the associations appeared stronger with fatty acid concentrations in early pregnancy compared to late pregnancy. (C) 2015 Elsevier Ltd. All rights reserved.

OBJECTIVESNighttime blood pressure (BP) has been shown to be superior to daytime BP in predicting hypertension related target organ damage and cardiac mortality. In our Georgia Cardiovascular Twin Study, we showed that apart from the genes that also influence daytime BP, specific genetic determinants explained 44% and 67% of the nighttime systolic BP (SBP) and diastolic BP (DBP) heritabilities, respectively. Here, we determined whether these results could be confirmed in a much larger twin cohort of young adults with 24-hour ambulatory BP measurements.METHODSAmbulatory BP was available in 703 white twins (308 pairs and 87 singletons, aged 18-34 years, 50% males) from the Prenatal Programming Twin Study. A bivariate quantitative genetic twin model was used to analyze daytime and nighttime BP. We conducted a meta-analysis to compare and integrate results from the 2 twin cohorts.RESULTSModel fitting showed no sex differences for any of the measures. Heritabilities were 0.60 and 0.51 for SBP and 0.54 and 0.46 for DBP at daytime and nighttime. The specific heritability due to novel genetic effects emerging during the nighttime was 0.21 for SBP and 0.26 for DBP, which comprised 41% and 57% of the total nighttime heritability for SBP and DBP, respectively. Meta-analysis confirmed absence of cohort differences with very similar combined results.CONCLUSIONSIn addition to genes that influence both daytime and nighttime BP, a large part of the heritability is explained by genes that specifically influence BP at night.

Lower prenatal exposure to n-3 PUFA relative to n-6 PUFA has been hypothesised to influence allergy development, but evidence remains largely inconsistent. In the Dutch Maastricht Essential Fatty Acid Birth (MEFAB) (n 293) and Greek RHEA Mother–Child (n 213) cohorts, we investigated whether cord blood phospholipid PUFA concentrations are associated with symptoms of wheeze, asthma, rhinitis and eczema at the age of 6–7 years. Information on allergy-related phenotypes was collected using validated questionnaires. We estimated relative risks (RR) and 95 % CI for associations of PUFA with child outcomes using multivariable generalised linear regression models. In pooled analyses, higher concentration of the n-3 long-chain EPA and DHA and a higher total n-3:n-6 PUFA ratio were associated with lower risk of current wheeze (RR 0·61; 95 % CI 0·45, 0·82 per sd increase in EPA+DHA and 0·54; 95 % CI 0·39, 0·75 per unit increase in the n-3:n-6 ratio) and reduced asthma risk (RR 0·50; 95 % CI 0·31, 0·79 for EPA+DHA and 0·43; 95 % CI 0·26, 0·70 for the n-3:n-6 ratio). No associations were observed for other allergy-related phenotypes. The results were similar across cohorts. In conclusion, higher EPA and DHA concentrations and a higher n-3:n-6 fatty acid ratio at birth were associated with lower risk of child wheeze and asthma. Our findings suggest that dietary interventions resulting in a marked increase in the n-3:n-6 PUFA ratio, and mainly in n-3 long-chain PUFA intake in late gestation, may reduce the risk of asthma symptoms in mid-childhood.

Background: Telomere attrition is extremely rapid during the first years of life, while lifestyle during adulthood exerts a minor impact. This suggests that early life is an important period in the determination of telomere length. We investigated the importance of the early-life environment on both telomere tracking and adult telomere length. Methods: Among 184 twins of the East Flanders Prospective Twin Survey, telomere length in placental tissue and in buccal cells in young adulthood was measured. Residential addresses at birth and in young adulthood were geocoded and residential traffic and greenness exposure was determined. Results: We investigated individual telomere tracking from birth over a 20 year period (mean age (SD), 22.6 (3.1) years) in association with residential exposure to traffic and greenness. Telomere length in placental tissue and in buccal cells in young adulthood correlated positively (r = 0.31, P < 0.0001). Persons with higher placental telomere length at birth were more likely to have a stronger downward shift in telomere ranking over life (P < 0.0001). Maternal residential traffic exposure correlated inversely with telomere length at birth. Independent of birth placental telomere length, telomere ranking between birth and young adulthood was negatively and significantly associated with residential traffic exposure at the birth address, while traffic exposure at the residential address at adult age was not associated with telomere length. Conclusions: Longitudinal evidence of telomere length tracking from birth to adulthood shows inverse associations of residential traffic exposure in association with telomere length at birth as well as accelerated telomere shortening in the first two decades of life. This investigation is supported by the EU research council “project ENVIRONAGE” (ERC-2012-StG 310890), and the Flemish Scientific Fund (G073315N). Dr. Bijnens holds a fellow-ship from the Marguerite-Marie Delacroix foundation. Since its start, the East Flanders Prospective Twin Survey has been partly supported by grants from the Fund of Scientific Research Flanders and Twins, a non-profit Association for Scientific Research in Multiple Births (Belgium).

BACKGROUND: Prenatal exposure to polyunsaturated fatty acids (PUFAs) may influence childhood growth. However, available evidence mostly derived from short-term studies is inconsistent.OBJECTIVE: To assess whether fetal PUFA exposure is associated with height and body mass index (BMI), a common measure of adiposity, from 6 months to 23 years of age.METHODS: In the MEFAB cohort, we assessed cord blood phospholipid n-3 and n-6 PUFA levels, reflecting fetal exposure in late pregnancy. For 250 (45.2% females) participants, we collected a total of 1770 (n= 802 for females) repeated growth measurements from infancy to young adulthood. We examined sex-specific associations of PUFAs with height and BMI at different developmental ages (infant: 6 months; toddler: 2 years; pre-schooler: 4 years; school-aged child: 7 years; adolescent: 12 years; and young adult: 23 years) using fractional polynomial mixed models adjusted for important covariates.RESULTS: Higher n-3 PUFA levels were associated with higher infant length in males (β= 0.44cm [95% CI: 0.07, 0.82] per SD increase), whereas, for females, higher n-6 PUFA concentrations were associated with lower length in infancy (β= -0.69cm [95% CI: -1.08, -0.30] per SD increase). A higher ratio of n-3 to n-6 PUFAs was associated with higher infant length in both sexes (β= 0.40cm [95% CI: 0.01, 0.78] and 0.42cm [95% CI: 0.05, 0.79] per unit increase for males and females, respectively). These associations were not detectable later in childhood and young adulthood. No associations with BMI were found at any time point examined.CONCLUSIONS: Our findings suggest a small sex-specific influence of PUFA status at birth on length in infancy, but this does not persist in later life up to young adulthood. PUFA status at birth does not seem to affect BMI from infancy till young adulthood.

Introduction: Concentrations of the fish fatty acids EPA and DHA are low among Dutch women of reproductive age. As the human brain incorporates high concentrations of these fatty acids in utero, particularly during third trimester of gestation, these low EPA and DHA concentrations may have adverse consequences for fetal brain development and functioning.Methods: Analyses were conducted using longitudinal observational data of 292 mother-child pairs participating in the MEFAB cohort. Maternal AA, DHA, and EPA were determined in plasma phospholipids - obtained in three trimesters-by gas-liquid chromatography. Cognitive function was assessed at 7 years of age, using the Kaufman Assessment Battery for Children, resulting in three main outcome parameters: sequential processing (short-term memory), simultaneous processing (problem-solving skills), and the mental processing composite score. Spline regression and linear regression analyses were used to analyse the data, while adjusting for potential relevant covariates.Results: Only 2% of the children performed more than one SD below the mental processing composite norm score. Children with lower test scores ( = 25%). Fully-adjusted linear regression models did not show associations of maternal AA, DHA, or EPA status during any of the pregnancy trimesters with childhood sequential and simultaneous processing.Conclusion: Maternal fatty acid status during pregnancy was not associated with cognitive performance in Dutch children at age 7.

It is hypothesized that prenatal and early postnatal exposure to n-3 long-chain polyunsaturated fatty acids (LCPUFAs) is negatively associated with adiposity later in life. We conducted a systematic review and meta-analysis to evaluate whether maternal n-3 LCPUFA supplementation in pregnancy and/or lactation exerts a beneficial effect on adiposity status in childhood. We searched six electronic databases till 20 May 2014 for randomized controlled trials (RCTs) of n-3 LCPUFA supplementation to pregnant and/or lactating women that reported data on body mass index (BMI), waist circumference, sum of skinfold thicknesses or body fat mass in children. Adiposity measures were grouped into three age categories: preschool children (13 years). Trial quality was assessed. We conducted fixed-effect and random-effects meta-analyses to combine study-specific estimates of differences between the supplemented and control groups. A total of 6 RCTs (9 publications) involving 2847 participants were included. Summary estimates showed no effect of maternal supplementation on BMI in preschool (standardized mean difference (SMD)=0.07, 95% confidence interval (CI)=-0.22, 0.36, P=0.65) and school-aged children (SMD=0.12, 95% CI=-0.06, 0.30, P=0.20). Because of sparse data, it was not possible to pool study results relating to other adiposity measures. There is currently no evidence to support that n-3 LCPUFA supplementation during pregnancy and/or lactation favourably affects child adiposity. Further high-quality trials are needed.European Journal of Clinical Nutrition advance online publication, 13 August 2014; doi:10.1038/ejcn.2014.158.

Background In contrast to the postnatal period, little is known about telomere length (TL) during prenatal life. The decrease in placental TL remains unknown, although intra uterine growth retardation and preeclampsia are associated with shorter placental TL. The aim of this study is to assess the decrease of placental TL during the third trimester of gestation and to explore the role of potential “growth influencing factors”. Methods The study sample consisted of 329 live-born twins from the East Flanders Prospective Twin Survey. TL was determined using a multiplex quantitative PCR method. Gestational age, sex, birth order, placental characteristics, parity, maternal and paternal age, diabetes, hypertension, smoking, alcohol use, and socio economic status (SES) were considered “growth influencing factors”. Bivariable multilevel regression analysis with “growth influencing factors” was performed. Results Placental TL ranged from 4.3 kbp to 84.4 kbp with a median of 10.8 kbp. Ln(TL) decreased in a linear fashion with an estimated TL decreasing from 13.98 kbp at 28 weeks to 10.56 kbp at 42 weeks. The regression coefficient of gestational age became smaller if considered together with SES ( b = −0.017; p = 0.08) or diabetes ( b = −0.018; p = 0.07) and bigger if considered together with parity ( b = −0.022; p = 0.02), indicating that part of the association between gestational age and telomere length is explained by these three confounding factors. Conclusion Placental TL decreases during the third trimester of gestation of live-born twins with approximately 25% indicating that telomere shortening may play a role in aging of the placenta.

Prenatal polyunsaturated fatty acid (PUFA) concentrations may be involved in the prenatal programming of adiposity. In this study we therefore explored the association between maternal PUFA concentrations, measured up to four times during pregnancy, and offspring adiposity at age 7 in 234 mother-child pairs of the Maastricht Essential Fatty Acid Birth cohort. Only dihomo-gamma-linolenic acid (DGLA, an n-6 fatty acid) concentration was associated with adiposity: per standard deviation increase in relative DGLA concentration, BMI increased by 0.44 kg/m(2) (CI95: 0.16, 0.72), sum of skinfolds increased by 3.41 mm (CI95: 1.88, 4.95), waist circumference increased by 1.09 cm (CI95: 0.40, 1.78), and plasma leptin concentration increased by 0.66 mu g/l (CI95: 0.20, 1.11). In conclusion, maternal DGLA throughout gestation was associated with increased BMI and some additional measures of adiposity at age 7. This suggests that maternal DGLA might play a role in or reflect the prenatal programming of adiposity. (C) 2014 Elsevier Ltd. All rights reserved.

Objectives: Blood pressure variability (BPV) and its reduction in response to antihypertensive treatment are predictors of clinical outcomes; however, little is known about its heritability. In this study, we examined the relative influence of genetic and environmental sources of variance of BPV and the extent to which it may depend on race or sex in young twins.Methods: Twins were enrolled from two studies. One study included 703 white twins (308 pairs and 87 singletons) aged 18-34 years, whereas another study included 242 white twins (108 pairs and 26 singletons) and 188 black twins (79 pairs and 30 singletons) aged 12-30 years. BPV was calculated from 24-h ambulatory blood pressure recording.Results: Twin modeling showed similar results in the separate analysis in both twin studies and in the meta-analysis. Familial aggregation was identified for SBP variability (SBPV) and DBP variability (DBPV) with genetic factors and common environmental factors together accounting for 18-40% and 23-31% of the total variance of SBPV and DBPV, respectively. Unique environmental factors were the largest contributor explaining up to 82-77% of the total variance of SBPV and DBPV. No sex or race difference in BPV variance components was observed. The results remained the same after adjustment for 24-h blood pressure levels.Conclusions: The variance in BPV is predominantly determined by unique environment in youth and young adults, although familial aggregation due to additive genetic and/or common environment influences was also identified explaining about 25% of the variance in BPV.

Background: Accelerated infant growth is associated with an al-tered, mostly adverse adult cardiometabolic risk profile. The impor-tance of genetic and environmental factors to these associations isunclear.Objective: The objective was to examine the importance of geneticand environmental factors in the associations between infant growthand adult cardiometabolic risk factors (anthropometrics, lipids, in-sulin sensitivity, leptin, blood pressure, and fibrinogen) in twins.Design: Cardiometabolic risk factors were assessed in 240 twinpairs (aged 18–34 y) from the East Flanders Prospective Twin Sur-vey. Infant growth was defined as change in weight z score. Weregressed intrapair differences in growth during 4 growth windows(0–1, 1–6, 6–12, and 12–24 mo) against intrapair differences in therisk factors in monozygotic and dizygotic twins separately.Results: Within monozygotic twin pairs only, associations betweeninfant growth and most adult lipids, glucose, leptin, and blood pres-sure (eg, systolic blood pressure: b = 5.95 mm Hg per change inz score, P = 0.01 in monozygotic twins; b = 21.64, P = 0.82 indizygotic twins from 12 to 24 mo) were found. Within dizygotictwin pairs only, associations between growth and triglycerides andfibrinogen (eg, fibrinogen: b = 0.07 ln mg/dL per change in z score,P=0.31 in monozygotic twins; b = 0.79, P = 0.01 in dizygotictwins from 0 to 1 mo) were identified. Most associations showeda detrimental effect of accelerated growth, but beneficial associa-tions were also identified (eg, total–to–high-density-lipoprotein cho-lesterol ratio: b = 20.22 per change in z score from 1 to 6 mo, P =0.008 in monozygotic twins).Conclusion: Our data showed that environmental factors play a rolein the associations between infant growth and most adult lipids,glucose, leptin, and blood pressure, whereas genetic factors are in-volved regarding triglycerides and fibrinogen. Am J Clin Nutrdoi: 10.3945/ajcn.112.039131.INTRODUCTIONCardiovascular disease is a major cause of mortality world-wide (1). The incidence of cardiovascular disease is increased inindividuals with an adverse cardiometabolic risk profile, whichincludes obesity, an unfavorable lipid profile, insulin resistance,and high blood pressure. It has become clear that besides theimportance of lifestyle, factors in early life are also involved inthe development of this risk profile. Low birth weight is relatedto an adverse cardiometabolic risk profile and cardiovasculardisease later in life (2–4). In addition, subsequent acceleratedpostnatal growth has been proposed as a mechanism that linkslow birth weight to an unfavorable cardiometabolic risk profile(5, 6). Growth during infancy (mostly defined as 0–2 y) has beenpositively associated with obesity (7), hypertension (8), an ad-verse cardiometabolic risk profile (9), and metabolic syndrome(10). On the contrary, there is also some evidence that slowinfant growth is associated with certain harmful cardiometabolicoutcomes in adulthood, eg, regarding lipids (11, 12), insulin re-sistance (13, 14), and stroke (15).Importantly, to regard “infancy” as one growth window from0 to 2 y may be too broad a definition. For example, it wasreported that the first 3 mo of life may be most important for thedevelopment of an adverse cardiometabolic risk profile (16). Inaddition, accelerated growth in the first 2 wk of life is relatedto adult endothelial dysfunction, a precursor of atherosclerosis

BACKGROUND The natural dizygotic (DZ) twinning rate has been proposed as a reliable and useful measure of human fecundity, if adjusted for maternal age at twin birth. The aim of this study was to analyze age-adjusted trends in natural DZ twinning rates over the past 40 years using data from the 'East Flanders Prospective Twin Survey (EFPTS)'. METHODS This study involved 4835 naturally conceived twin pregnancies between 1969 and 2009 from the population-based Belgian 'EFPTS'. Age-adjusted trends in the incidence of natural DZ twin pregnancies were calculated using a generalized linear model with Poisson distribution. RESULTS Both the natural DZ twinning rates and maternal age at twin birth increased in a linear fashion from 1969 to 2009. When age-adjusted, we found that the trend in the natural DZ twinning rate was stable during the whole time period. CONCLUSIONS According to our population-based data and after age-adjustment, a stable natural DZ twinning rate could be observed over the last four decades. Under the assumption that the spontaneous DZ twinning rate is a sensor of fecundity, this indicates a stable 'high' fecundity for this population.

BACKGROUND: PGD is nowadays a well-established alternative to prenatal diagnosis. However, information with respect to couples' motives and profiles for choosing PGD is scarce. METHODS: A prospective cohort of 264 couples referred for PGD was interviewed semi-structurally after intake, and follow-up data were collated after 6-8 years. Outcome measures were: the primary choice shortly after intake (PGD intention), and their definitive use, until maximum 8 years later (PGD use). Logistic regression analysis was performed with clinical impact of the genetic disorder, couples' experiences, obstetric history and psychosocial factors as putative predictors. RESULTS: About 53.4% of the couples showed PGD intention. The experience of one or more miscarriages, the loss of an affected child and the absence of (acceptable) alternatives for the female partner positively contributed to PGD intention. For PGD use (45.8% of couples), infertility, a history of pregnancy termination(s) and the absence of alternatives according to the female partner were positive determinants. A living affected child reduced PGD use. Mode of inheritance and clinical impact of the disorder did not contribute. CONCLUSIONS: Fewer than 50% of the referred couples actually started PGD treatment. Personal experiences and reproductive history [the presence of a living affected child, infertility or a history of termination of pregnancy (TOP)] were more important determinants of eventual PGD use than the mode of inheritance or the expected clinical impact of the disorder.

Background and Purpose— Results from case-control and case-case studies indicate that a positive family history of stroke (FHstroke) is an independent risk factor for lacunar stroke. Different lacunar stroke phenotypes can be distinguished on the basis of the presence of asymptomatic lacunar infarcts (aLACs), ischemic white-matter lesions, or brain microbleeds. The aim of the present study was to determine whether familial aggregation of stroke was different for lacunar stroke phenotypes. Methods— In 157 patients with a first-ever lacunar stroke, a complete first-degree FHstroke was obtained by a standardized questionnaire and additional interview. Lacunar stroke patients were categorized successively into groups, depending on the presence of aLACs, ischemic white-matter lesions, and brain microbleeds on magnetic resonance imaging. Results— Fifty-two percent of patients reported a positive FHstroke in at least one of their first-degree relatives. In younger (P Conclusions— A family history of stroke might be an independent risk factor for the lacunar stroke phenotype with aLACs at younger ages, suggesting a role for genetic factors in this phenotype caused by diffuse vasculopathy.

Both zygosity and chorionicity provide important information in twin research. The East Flanders Prospective Twin Survey (EFPTS) determines zygosity and chorionicity at birth and therefore provides a gold standard for the testing of diagnostic parameters that can be used to determine chorionicity. The aim of the present study was to investigate whether birthweight discordancy can be used as an indicator of chorionicity. The study sample consisted of 4,060 live-born twin pairs from the EFPTS. We studied MZ twins, using univariate and multivariate logistic regression analyses to calculate odds ratios (OR) and 95% confidence intervals (CI) of being MC in relation to discordancy level. Diagnostic parameters, including sensitivity and specificity, were calculated. A two-fold cross-validation was carried out and a bootstrap distribution with 10,000 samples was created to estimate the standard deviations. For discordancy levels of below 10%, 10–15%, 15–20%, 20–25% and above 25%, the ORs (95% CI) were 1.16 (0.91–1.47), 1.38 (1.05–1.80), 2.13 (1.51–3.01), 2.73 (1.73–4.29) and 2.81 (2.81–4.35) respectively. There were no gender differences. Sensitivity was 42.2% (SD 5.6%), specificity was 72.8% (SD 6.3%), positive predictive value was 72.8% (1.5%) and the negative predictive value was 39.2% (0.7%). In conclusion, although a higher discordancy level resulted in higher ORs of being an MC twin, birthweight discordancy level can only be used to some weak extent as a proxy for chorionicity, highlighting the need to assess and record chorionicity data in obstetrical units.

Children born small for gestational age are at increased risk of developing type 2 diabetes in adulthood. The satiety signal leptin that regulates food intake and energy expenditure might be a possible molecular link, as umbilical cord leptin levels are positively correlated with birth weight. In the present study, we examined whether common single nucleotide polymorphisms (SNPs) in the leptin (LEP; 19G>A) gene and its receptor (LEPR; Q223R and K109R) are associated with birth weight and adult metabolic risk factors for type 2 diabetes in twins.SNPs were genotyped in 396 monozygotic and 232 dizygotic twins (286 men and 342 women, mean age 25 years) recruited from the East Flanders Prospective Twin Survey. Data were analysed using linear mixed models.The LEPR K109R SNP was associated with birth weight (KK, KR and RR (95% confidence interval, CI): 2511 (2465-2557), 2575 (2516-2635) and 2726 (2606-2845) gram; P(additive)=0.001). Also the LEPR Q223R SNP showed a significant association with weight at birth (QQ, QR and RR (95% CI): 2492 (2431-2554), 2545 (2495-2595) and 2655 (2571-2740) gram; P(additive)=0.003). Furthermore, an interaction between the LEPR K109R and the Q223R SNP on birth weight was observed (P=0.014). G allele carriers of the LEP 19G>A SNP had higher high-density lipoprotein (HDL) cholesterol levels compared to 19A homozygotes (GX vs AA (95% CI): 1.62 (1.58-1.66) vs 1.49 (1.40-1.58) mmol l(-1); P(recessive)=0.013).This study indicates that leptin may act as a growth-promoting signal during fetal development, and suggests a possible role for the LEPR in explaining the inverse relationship between birth weight and the development of metabolic diseases in adulthood. Additionally, these results suggest that the LEP 19G>A SNP affect HDL cholesterol levels.

The assessment of fetal growth is an essential component of good antenatal care, especially for twins. The aims of this study are to develop twin-specific intrauterine 'growth' charts, based on cross-sectional birthweight data, for monochorionic and dichorionic twins according to sex and parity, and to detect twins at risk for neonatal death by comparing the use of twin-specific and singleton charts. The study sample consisted of 76,471 singletons and 8454 twins (4227 pairs) born in East Flanders (Belgium). Birthweights were analyzed using a nonlinear Gaussian regression. After 33 weeks of gestation, the birthweights of twins started to deviate from singletons (difference of 900 grams at 42 weeks). Birthweights of dichorionic twins continued to increase, whereas those of monochorionic twins decreased after week 40 (difference of more than 300 g at 42 weeks). After 31 weeks of gestation, neonatal mortality increased as centile decreased, and was especially high if birthweight was below the twin-specific third centile: .032 (below) versus .007 (above). Using singleton centiles, this was less obvious. In conclusion, twin-specific growth charts, taking chorionicity into account, are more accurate to detect twins at risk for neonatal death. Therefore the presented charts, based on cross-sectional birthweight data, enable an improved assessment of twin growth.

Insulin resistance and obesity are underlying causes of type 2 diabetes and therefore much interest is focused on the potential genes involved. A series of anthropometric and metabolic characteristic were measured in 240 MZ and 112 DZ twin pairs recruited from the East Flanders Prospective Twin Survey. Microsatellite markers located close to ABCC8, ADIPOQ, GCK, IGF1, IGFBP1, INSR, LEP, LEPR, PPARγ and the RETN gene were genotyped. Univariate single point variance components linkage analyses were performed using two methods: (1) the standard method, only comprising the phenotypic and genotypic data of the DZ twin pairs and (2) the extended method, also incorporating the phenotypic data of the MZ twin pairs. Suggestive linkages (LOD > 1) were observed between the ABCC8 marker and waist-to-hip ratio and HDL-cholesterol levels. Both markers flanking ADIPOQ showed suggestive linkage with triglycerides levels, the upstream marker also with body mass and HDL-cholesterol levels. The IGFBP1 marker showed suggestive linkage with fat mass, fasting insulin and leptin levels and the LEP marker showed suggestive linkage with birth weight. This study suggests that DNA variants in ABCC8, ADIPOQ, IGFBP1 and LEP gene region may predispose to type 2 diabetes. In addition, the two methods used to perform linkage analyses yielded similar results. This was however not the case for birth weight where chorionicity seems to be an important confounder.

Aims/hypothesis We determined the genetic contribution of 18 anthropometric and metabolic risk factors of type 2 diabetes using a young healthy twin population. Methods Traits were measured in 240 monozygotic (MZ) and 138 dizygotic (DZ) twin pairs aged 18 to 34 years. Twins were recruited from the Belgian population-based East Flanders Prospective Twin Survey, which is characterised by its accurate zygosity determination and extensive collection of perinatal and placental data, including information on chorionicity. Heritability was estimated using structural equation modelling implemented in the Mx software package. Results Intra-pair correlations of the anthropometric and metabolic characteristics did not differ between MZ monochorionic and MZ dichorionic pairs; consequently heritabilities were estimated using the classical twin approach. For body mass, BMI and fat mass, quantitative sex differences were observed; genetic variance explained 84, 85 and 81% of the total variation in men and 74, 75 and 70% in women, respectively. Heritability estimates of the waist-to-hip ratio, sum of four skinfold thicknesses and lean body mass were 70, 74 and 81%, respectively. The heritability estimates of fasting glucose, fasting insulin, homeostasis model assessment of insulin resistance and beta cell function, as well as insulin-like growth factor binding protein-1 levels were 67, 49, 48, 62 and 47%, in that order. Finally, for total cholesterol, LDL-cholesterol, HDL-cholesterol, total cholesterol:HDL-cholesterol ratio, triacylglycerol, NEFA and leptin levels, genetic factors explained 75, 78, 76, 79, 58, 37 and 53% of the total variation, respectively. Conclusions/interpretation Genetic factors explain the greater part of the variation in traits related to obesity, glucose intolerance/insulin resistance and dyslipidaemia. Electronic supplementary material The online version of this article (doi:10.1007/s00125-007-0784-z) contains supplementary material, which is available to authorised users.

The Maastricht Essential Fatty Acid Birth cohort (MEFAB) was established in 1989 to study the changes in fatty acid concentration during pregnancy and how this related to the fatty acid concentrations of the neonate. The original sample contains data of 1203 subjects. Some participants whom participated in the original MEFAB study also participated in follow-up studies at age 4, 7 or 12; 41%, 52% and 35% of eligible participants, respectively. Collected data include maternal fatty acid concentrations at multiple points during pregnancy and at birth and at age 7 for the child, but also anthropometric measurements during development, assessments of cognitive development, asthma, atopy and cardiovascular risk factors. Data of MEFAB have been used in 37 articles in peer-reviewed journals and 4 doctoral theses have been completed. Data of MEFAB is upon request available for new research questions.

The purpose of this study is to present curves of estimated placental growth in twins and to evaluate the relative contribution of gestational age, zygosity, chorionicity, fusion of the placentas, sex of the individual and of the twin pair, site of the umbilical cord insertion, birth order, maternal age, and parity. Perinatal data and placental data were obtained from 6315 live-born twin pairs from the East Flanders Prospective Twin Survey. Of 4318 twin pairs, with no missing values, the placental weights of different gestational ages were analyzed using a nonlinear multivariate Gaussian regression. Two groups were distinguished: (1) twins with two separate placentas, and (2) twins with only one placental mass (one placenta in case of monochorionic twins or two fused placentas in case of dichorionic placentas). Overall, placental weight was influenced by gestational age, fusion of the placentas, and parity. In the case of one placental mass, monozygotic dichorionic twins had the lowest weights. If two separate placentas were present, birth order played a role in favor of the first-born twin. For parity and zygosity, the differences were most pronounced between 27 and 29 weeks, whereas the difference for birth order was most pronounced between 33 and 37 weeks. In conclusion, basic physiological characteristics, routinely examined at birth, influence placental weight. Taking these covariates into account allows a better evaluation of the placental weight given a gestational age, as an indicator of growth.

Previous studies have shown that low birth weight (LBW) is a risk factor for renal impairment in adult life. The effects of LBW and renal function were studied by using twins, which allows distinguishing among fetoplacental, maternal, and genetic influences. Perinatal data were obtained at birth, and absolute creatinine clearance (not corrected for body surface area) was measured at a mean age of 25.6 yr in 653 individuals. Twins were considered both as individuals and as members of twin pairs. Statistical analyses were performed with and without adjusting for gestational age, zygosity, gender, age, body mass index, glucose level, BP, and smoking status. Creatinine clearance was 4 ml/min lower in twins with LBW (

The contribution of risk factors to blood pressure heritability estimates in young adults: the East flanders prospective twin study.Zeegers MP, Rijsdijk F, Sham P, Fagard R, Gielen M, De Leeuw PW, Vlietinck R.Maastricht University, Department of Epidemiology, Maastricht, the Netherlands. mpa.zeegers@epid.unimaas.nlThe heritability of blood pressure estimated in previous studies may be confounded by the influence of potential blood pressure risk factors. We applied the classical twin design to estimate the contribution of these covariates to blood pressure heritability. The study consisted of 173 dizygotic and 251 monozygotic twin pairs aged 18-34 years, randomly selected from the East Flanders Prospective Twin Survey. In a standardized examination, blood pressure and anthropometry was measured, a questionnaire was completed, and a fasting blood sample was taken. In univariate and bivariate modeling, diastolic and systolic heritability were estimated both unadjusted and adjusted for potential risk factors. Also, covariate interaction was modeled. Bivariate analysis gave heritability estimates of 0.63 (95%CI 0.55-0.59), 0.74 (95%CI: 0.68-0.79), and 0.78 (95%CI: 0.70-0.84) for diastolic, systolic, and cross-trait heritability, respectively. The remaining variances could be attributed to unique environmental influences. These heritability estimates did not change substantially in univariate analyses or after adjustment for risk factors. A sex-limitation model showed that the heritability estimates for women were significantly higher than for men, but the same genetic factors were operating across sexes. Sex and cigarette smoking appeared to be statistically significant interaction terms. The heritability of blood pressure is relatively high in young adults. Potential risk factors of blood pressure do not appear to confound the heritability estimates. However, gene by sex by smoking interaction is indicated

Background: High variation in telomere length between individuals is already present before birth and is as wide among newborns as in adults. Environmental exposures likely have an impact on this observation, but remain largely unidentified. We hypothesize that placental telomere length in twins is associated with residential traffic exposure, an important environmental source of free radicals that might accelerate aging. Next, we intend to unravel the nature-nurture contribution to placental telomere length by estimating the heritability of placental telomere length. Methods: We measured the telomere length in placental tissues of 211 twins in the East Flanders Prospective Twin Survey. Maternal traffic exposure was determined using a geographic information system. Additionally, we estimated the relative importance of genetic and environmental sources of variance. Results: In this twin study, a variation in telomere length in the placental tissue was mainly determined by the common environment. Maternal residential proximity to a major road was associated with placental telomere length: a doubling in the distance to the nearest major road was associated with a 5.32% (95% CI: 1.90 to 8.86%; p = 0.003) longer placental telomere length at birth. In addition, an interquartile increase (22%) in maternal residential surrounding greenness (5 km buffer) was associated with an increase of 3.62% (95% CI: 0.20 to 7.15%; p = 0.04) in placental telomere length. Conclusions: In conclusion, we showed that maternal residential proximity to traffic and lower residential surrounding greenness is associated with shorter placental telomere length at birth. This may explain a significant proportion of air pollution-related adverse health outcomes starting from early life, since shortened telomeres accelerate the progression of many diseases. Keywords: Telomeres, Traffic, Placenta, Twin

Twin studies with objective measurements suggest habitual physical activity (HPA) are modestly to highly heritable, depending on age. We aimed to confirm or refute this finding and identify relevant genetic variants using a candidate gene approach. HPA was measured for 14 days with a validated triaxial accelerometer (Tracmor) in two populations: (1) 28 monozygotic and 24 dizygotic same-sex twin pairs (aged 22 ± 5 years, BMI 21.8 ± 3.4 kg/m2, 21 male, 31 female pairs); (2) 52 and 65 unrelated men and women (aged 21 ± 2 years, BMI 22.0 ± 2.5 kg/m2). Single nucleotide polymorphisms (SNPs) in PPARD, PPARGC1A, NRF1 and MTOR were considered candidates. Association analyses were performed for both groups separately followed by meta-analysis. Structural equation modeling shows significant familiality for HPA, consistent with a role for additive genetic factors (heritability 57 %, 95 % CI 32–74 %, AE model) or common environmental factors (47 %, 95 % CI 23–65 %, CE model). A moderate heritability was observed for the time spent on low- and high-intensity physical activity (P ≤ 0.05), but could not be confirmed for the time spent on moderate-intensity physical activity. For PPARD, each additional effect allele was inversely associated with HPA (P ≤ 0.01; rs2076168 allele C) or tended to be associated with HPA (P ≤ 0.05; rs2267668 allele G). Linkage disequilibrium existed between those two SNPs (alleles A/G and A/C, respectively) and meta-analysis showed that carriers of the AA GC haplotype were less physically active than carriers of the AA AA and AA AC haplotypes combined (P = 0.017). For PPARGC1A, carriers of AA in rs8192678 spent more time on high-intensity physical activity than GG carriers (P = 0.001). No associations were observed with SNPs in NRF1 and MTOR. In conclusion, HPA may be modestly heritable, which is confirmed by an association with variants in PPARD.

Antibody titres in immunoglobulin preparations for intravenous use were tested against 24 different enterovirus serotypes and varied between 1:100 and 1:10,000 within a single batch. Differences up to 8-fold were found for homologous titres between two different batches that were prepared 6 years apart. The lowest titre obtained was 1 : 40. The observed differences within and between the two batches could not be explained by different incidence of serotypes of enteroviruses circulating at the time blood was collected. Differences in titres of up to 18-fold were observed when several strains of the same serotype were tested suggesting that intratypic variation influences antibody titres. It is concluded that immunoglobulin preparations contain antibodies against many enteroviruses, but that titres can be low and cannot be predicted from the incidence of any particular serotype circulating in the community. Because of intratypic variation, selection of a batch for specific treatment should be based on results obtained with the patient's own isolate, and not with a reference strain.

BACKGROUND: Lower educated people have a higher prevalence of metabolic risk factors (MRF), that is, high waist circumference (WC), high systolic blood pressure, low high-density lipoprotein cholesterol level, high triglycerides and high fasting glucose levels. Behavioural and psychosocial factors cannot fully explain this educational gradient. We aim to examine the possible role of genetic factors by estimating the extent to which education and MRF share a genetic basis and the extent to which the heritability of MRF varies across educational levels. METHODS: We examined 388 twin pairs, aged 18-34 years, from the Belgian East Flanders Prospective Twin Survey. Using structural equation modelling, a Cholesky bivariate model was applied to assess the shared genetic basis between education and MRF. The heritability of MRF across education levels was estimated using a non-linear multivariate Gaussian regression. RESULTS: Fifteen percent (P < 0.01) of the negative relation between education and WC was because of genes shared between these two traits. Furthermore, the heritability of WC was lower in the lowest educated group (65%) compared with the highest educated group (78%, P = 0.04). The lower heritabilities among the lower educated twins for the other MRF were not significant. The heritability of glucose was higher in the lowest education (80%) group compared with the high education group (67%, P = 0.01). CONCLUSION: Our findings suggest that genetic factors partly explain educational differences in WC. Furthermore, the lower heritability estimates in WC in the lower educated young adults suggest opportunities for environmental interventions to prevent the development of full-blown metabolic syndrome in middle and older age.

Low birth weight and accelerated infant growth are associated with cardiovascular disease in adulthood. Endothelial dysfunction is regarded as a precursor of atherosclerosis and is also related to infant growth. We aimed to examine whether an association between infant growth and endothelial function is already present during discrete periods of growth during the first 6 months of life in healthy term infants. A cohort of 104 newborns was studied in the first week after birth and reexamined at the age of 6 months. Maximum vasodilatation in response to acetylcholine (endothelium dependent) and nitroprusside (endothelium independent) was measured in the vasculature of the forearm skin, using laser Doppler flowmetry and iontophoresis. Growth was calculated as difference in Z scores for weight, length, weight-for-length, and head circumference. Multivariable multilevel linear regression was used for the analysis. Growth from 0 to 1 month (calculated as difference in weight) was the only window in the first 6 months of life that was significantly and inversely associated with endothelium-dependent vasodilatation at 6 months ( b =−11.72 perfusion units per Z score, P =0.01 in multivariable analysis). Birth size was not important when considered simultaneously with infant growth. Maximum endothelium-independent vasodilatation was not associated with birth size or growth parameters. We conclude that growth in the first month of life is inversely associated with endothelium-dependent vasodilatation at the age of 6 months in healthy term infants, regardless of birth size.

OBJECTIVE: The fetal response to an adverse intrauterine environment - reflected in low birth weight - is thought to cause an increased risk for adult hypertension. A possible mechanism by which fetal adaptive responses contribute to hypertension is an adverse effect on endothelial function. Identifying individuals with endothelial dysfunction as early as possible may assist in understanding the inverse association between birth weight and hypertension. The present study aimed to identify determinants of endothelial vasodilatation in the first week of life. METHODS: One hundred and four term newborns were studied in the first week after birth with regard to maximum vasodilatation in response to acetylcholine (endothelium-dependent) and nitroprusside (endothelium-independent) in the vasculature of the forearm skin, by use of a laser-Doppler device and iontophoresis. Bivariable and multivariable linear regression with various familial, gestational and neonatal potential covariates were used for the analysis. RESULTS: In the bivariable analysis, maximum perfusion after administration of acetylcholine was positively associated with birth weight, length, head circumference and maternal education level, but negatively associated with maternal hypertension during pregnancy. In the multivariable analysis, head circumference [b = 11.9 perfusion units/z-score, P = 0.02] and hypertension during pregnancy (b = -25.3 perfusion units from nonhypertensive to hypertensive, P = 0.02) remained significantly associated. Maximum perfusion after administration of nitroprusside was not related to any of the anthropometric measures; it was, however, related to gestational age (b = -11.1 perfusion units/week, P = 0.009). CONCLUSION: This study showed that body size, head circumference in particular, is positively associated with endothelial vasodilatation in newborns, whereas hypertension during pregnancy is inversely associated with endothelial vasodilatation.

Background Positive family history of stroke is an independent risk factor for lacunar stroke. However, the magnitude of familial aggregation of a certain disease is better evaluated by the genetic relative risk. This is calculated by dividing the prevalence of specific disease in family members of patients by the prevalence of this disease in the general population. In a cohort of lacunar stroke patients, who were subtyped clinically and radiologically, we determined the genetic relative risk of stroke. Methods By questionnaire and additional interview, we obtained a complete first-degree family history of stroke. The prevalence of stroke in first-degree relatives of these lacunar stroke patients was compared to the self-reported prevalence of stroke in a Dutch community based cohort of elderly volunteers. Secondly, the influence of proband characteristics and family composition on parental and sibling history of stroke were evaluated. Principal Findings We collected data of 1066 first-degree relatives of 195 lacunar stroke patients. Strokes occurred in 13.5% of first-degree relatives. The genetic relative risk was 2.94 (95%CI 2.45–3.53) for overall first-degree relatives, 4.52 (95%CI 3.61–5.65) for patients' parents and 2.10 (95%CI 1.63–2.69) for patients' siblings. Age of proband and proband status for hypertension influenced the chance of having a parent with a history of stroke whereas the likelihood of having a concordant sibling increased with sibship size. Conclusions We found an increased genetic relative risk of stroke in first-degree relatives of patients with lacunar stroke. Our data warrant further genomic research in this well-defined high risk population for stroke.

Background: In recent decades, the overall rate of preterm births has increased. The aim of the present study was to examine whether this trend is also seen for multiple gestations. More specifically, we examined if there has been a decrease in gestational age for live born monozygotic (MZ) and dizygotic (DZ) twins and if there has been a simultaneous change in birthweight. The contributions of fertility treatments and Caesarean sections were taken into consideration. All analyses were carried out in two large European twin cohorts. Methods: Cross-sectional study of 6310 live born twin pairs, born between 1964-2007, from the Belgian East Flanders Prospective Twin Survey and 14 712 twin pairs, born between 1990-2006, from the Netherlands Twin Register. Multiple regression analyses were performed with gestational age as outcome variable, and multilevel analysis with birthweight as outcome variable. All analyses were performed with and without adjustment for zygosity, parity, maternal age, mode of conception and delivery and, for the analyses of birthweight, gestational age. Results:Gestational age decreased in a linear fashion from 1964 to 2007 with a decrease of 0.25 days per year in a similar way for MZ and DZ twins. Changes in birthweight depended on gestational age: up to 32 weeks, birthweight decreased and after 32 weeks birthweight increased. The frequency of infertility treatment and Caesarean sections, primiparity and advanced maternal age increased over the years, but none of these factors influenced the secular trends in gestational age and birthweight. Conclusions: The decrease in gestational age and change in birthweight in twins are sources of concern, especially for very preterm twins, for whom birthweight decreased. For twins born after 32 weeks, an increase in birthweight was observed and this is very likely the explanation for the decrease in gestational age. © 2010 The Author.

Objective:The maternally imprinted insulin-like growth factor 2 (IGF2) gene is an important fetal growth factor and is also suggested to have postnatal metabolic effects. In this study, we examined whether common polymorphisms in IGF2 (6815_6819delAGGGC, 1156T>C and 820G>A (ApaI)) and a microsatellite marker in the close vicinity of IGF2 were linked to or associated with birth weight and adult metabolic risk factors.Design and participants:Polymorphisms were genotyped in 199 monozygotic complete twin pairs, 109 dizygotic complete twin pairs, 15 single twins, 231 mothers and 228 fathers recruited from the East Flanders Prospective Twin Survey. Conventional and parent-of-origin specific linkage and association analyses were carried out with birth weight, adult body height and parameters quantifying obesity, insulin sensitivity and dyslipidaemia measured at adult age (mean age 25 years).Results:In the parent-of-origin specific association analysis, in which only the paternally inherited allele was incorporated, the 1156T>C SNP (single nucleotide polymorphism) showed significant association with IGF-binding protein 1 (IGFBP1) levels (T and C (mean (95% CI)): 13.2 (12.1-14.3) and 16.2 (14.6-18.0) ng ml(-1), P=0.002). No linkage was observed in either the conventional or in the parent-of-origin specific linkage analysis.Conclusion:This study suggests that paternally inherited alleles of a common polymorphism in the IGF2 gene affect IGFBP1 levels.International Journal of Obesity advance online publication, 23 June 2009; doi:10.1038/ijo.2009.126.

The aim of this study was to validate the overall preimplantation genetic diagnosis (PGD)-PCR procedure and to determine the diagnostic value. Genotyped embryos not selected for embryo transfer (ET) and unsuitable for cryopreservation after PGD were used for confirmatory analysis. The PGD genotyped blastomeres and corresponding embryos were compared, and morphology was scored on Day 4 post fertilization. To establish the validity of the PGD-PCR procedure and the diagnostic value, misdiagnosis rate, false-negative rate and negative predictive value were calculated. Moreover, comparison on the validity was made for the biopsy of one or two blastomeres. For the total embryo group (n = 422), a misdiagnosis rate of 7.1% and a false-negative rate of 3.1% were found. The negative predictive value was 96.1%. Poor morphology Day 4 embryos (Class 1) were over-represented in the embryo group in which the blastomere genotype was not confirmed by the whole embryo genotype. The misdiagnosis rate of Class 1 embryos was 12.5% and the false-negative rate 17.1%. Exclusion of these embryos resulted in a misdiagnosis rate of 6.1%, a false-negative rate of 0.5% and a negative predictive value of 99.3%. The two blastomere biopsies revealed a significant higher positive predictive value, lowering the misdiagnosis rate, whereas the negative predictive value remained the same. In conclusion, the PGD-PCR procedure is a valid diagnostic method to select unaffected embryos for ET. The misdiagnosis and false-negative rates decrease by rejecting Class 1 embryos for ET. The biopsy of a second blastomere improves the positive predictive value, lowering the misdiagnosis rate.

Chemical carcinogenesis induced by lifestyle factors like cigarette smoking is a major research area in molecular epidemiology. Gene expression analysis of large numbers of genes simultaneously using microarrays holds the opportunity to study the effects of such an exposure at the genome level yielding more mechanism-based information. Therefore, the aim of our study was to investigate multiple gene expressions in blood, indicative for the effects caused by cigarette smoke. Smoking-discordant monozygotic twin pairs (n=9) were studied to diminish influences of genetic background. Using a dedicated microarray containing 600 toxicologically relevant genes, we investigated which genes are differentially expressed in smokers compared to non-smokers. We also looked for genes of which the expression changes correlated with DNA adducts, a biomarker of effective dose for exposure to cigarette smoke carcinogens. The mean DNA adduct level in smokers differed significantly from that in non-smokers (mean +/- standard error 1.96 +/- 0.24 versus 1.17 +/- 0.16 adducts per 10(8) nucleotides, respectively; P=0.04). The genes of which the expression differed most significantly between smokers and non-smokers are ATF4, MAPK14, SOD2, CYP1B1 and SERPINB2. CYP1B1 and SOD2 can directly be linked to cigarette smoke exposure, whereas the other genes are associated with stress or environmentally induced response. Main functions of the genes influenced by cigarette smoking comprise carcinogen metabolism, oxidative stress response and anti-apoptosis.