The Generation R Study: design and cohort update 2010

The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. Mothers with a delivery date between April 2002 and January 2006 were eligible. Extensive assessments have been carried out in mothers and fathers and are currently performed in their children (Tables 3, 4). Assessments in pregnancy were planned in early pregnancy (gestational age < 18 weeks), mid-pregnancy (gestational age 18–25 weeks) and late pregnancy (gestational age > 25 weeks). These measurements are considered as first, second and third trimester measurements. The fathers were assessed once in pregnancy. The children form a prenatally recruited birth-cohort that will be followed until young adulthood. In the preschool period, which in the Netherlands refers to the period from birth to the age of 4 years, data collection was performed by a home-visit at the age of 3 months, questionnaires at the ages of 2, 6, 12, 18, 24, 30, 36 and 48 months and visits to the routine child health centres at the ages 2, 3, 4, 6, 11, 14, 18, 24, 30, 36 and 45 months (Table 4). Additional, more detailed assessments of fetal and postnatal growth and development have been conducted in a randomly selected subgroup of Dutch children and their parents at a gestational age of 32 weeks and postnatally at the ages of 1.5, 6, 14, 24, 36 and 48 months (Tables 3, 4).

Dutch is defined as two parents and four grandparents born in the Netherlands. From the age of 5 years onwards, regular detailed hands on assessment are performed in all children and their parents in a well-equipped, dedicated research center.

Eligible mothers were those who were resident in the study area at their delivery date and had a delivery date from April 2002 until January 2006. We aimed to enroll mothers in early pregnancy (gestational age < 18 weeks) but enrolment was allowed until birth of their child. Midwives and obstetricians informed eligible mothers about the study at their first prenatal visit in routine care, handed out the information package and asked these mothers to make an appointment for the first ultrasound examination. The study staff contacted these mothers by phone for additional information about the study and in person at the ultrasound examination to obtain informed consent. Mothers who were not approached in pregnancy, were approached and enrolled in the first months after birth of their child when newborns visit the routine child health centers [2]. The fathers were not approached directly by the study staff but the mothers were informed about the importance of involvement of the fathers in the study.

In total, 9,778 mothers were enrolled in the study (Fig. 1). Of these mothers, 91% (n = 8,880) was enrolled in pregnancy. Only partners from mothers enrolled in pregnancy were invited to participate. In total, 71% (n = 6,347) of all fathers was enrolled. The general characteristics of the mothers and fathers are presented in Table 5. Of all participating mothers, enrolment was in early pregnancy in 69% (n = 6,691), in mid-pregnancy in 19% (n = 1,918), in late pregnancy in 3% (n = 271) and at birth of their child in 9% (n = 898). Of all pregnant women enrolled, 94% (n = 8,356), 6% (n = 516) and 0.1% (n = 8) were first, second and third pregnancies in the study, respectively. A total of 1,232 pregnant women and their children were enrolled in the subgroup for additional detailed studies. Ethnicity of participating mothers and partners was defined according the classification of Statistics Netherlands [71, 72]. The largest ethnic groups were the Dutch, Surinamese, Turkish and Moroccan groups. The ethnic distribution differed only moderately from that of the population in the study area [71]. Mean household income in Rotterdam is about €1600 per month and the percentage subjects with a secondary or higher education level in Rotterdam is 56% [71]. The educational level of participating mothers and their partners was classified in groups according to the classification of Statistics Netherlands [73]. Ethnic background, educational level and occupational status are of major interest and are studied as determinants of health and behavioural outcomes [74‐91]. Both household income and highest followed educational level in mothers and fathers in the study cohort suggest a selection towards a higher socioeconomic status than in the whole study. This pattern is similar as in other large scale cohort studies [92]. However, differences between the population and cohort characteristics may also be due to selective missing values of ethnicity and socio-economic status in the questionnaires. Socio-economic status is related to various perinatal and postnatal health outcomes and of major interest in the study [93‐109].

Among the live births, 51% were male and 49% female. These percentages are similar to the population figures in the Netherlands and in Rotterdam [71]. The percentages of children born preterm or with low birth weight are smaller than expected on the basis of the population figures. This seems to reflect a selection toward a relative more healthy study population. Estimation of the precise number of eligible pregnant women in the study area is difficult since there is no satisfactory registry of pregnancies. Therefore, it was not attempted to identify overall response rates of pregnant women. Since the children form a prenatally recruited birth-cohort, the overall response of the study has been established at birth and is 61%.

As described above, 9,778 mothers were enrolled in the study and gave birth to 9,745 known live born children. The logistics of the postnatal follow up studies were embedded in the municipal routine child care system and restricted to the study area. In total 1,163 children lived outside this definite study area at birth and were therefore not approached for the postnatal follow up studies during the preschool period. Of the remaining 8,582 children, 689 (8%) mothers did not give consent for the postnatal phase of the study leaving 7,893 children for the postnatal follow-up studies. Reasons for non-participation in the postnatal follow up studies were primarily time restrictions or plans for moving from the study area. Of the total of 7,893 mothers, 598 (7.5%) gave a restricted consent and did not want to participate in the questionnaire studies. In total 7588 (78%) children or their parents participated in at least one measurement during the preschool period, of whom 6845 (90%) completed at least one questionnaire.

Physical examinations were planned at each visit in early pregnancy, mid-pregnancy and late pregnancy and included height, weight and blood pressure measurements of both parents. Overall response rates for these specific measurements in mothers and fathers are similar as the visit percentages presented in Fig. 1. Since there was a wide range of gestational age at each visit, these measurements are used in the analyses as gestational age adjusted measurements [110].

Mothers received four postal questionnaires and father received one postal questionnaire in the prenatal phase (Table 3). All questionnaires are available in three languages (Dutch, English and Turkish). If needed, further support for verbal translation of questionnaires is available in Arabic, Portuguese and French. Each questionnaire comprises about 25 pages and takes about 30–45 min to be completed [2]. Topics in these questionnaires were:

Overall response rates for these questionnaires varied from 77 to 91% (Fig. 1). However, the response rates of specific questions may be lower due to missing values within questionnaires.

Fetal ultrasound examinations were performed at each prenatal visit. Overall response rates for these ultrasound examinations were in general similar to the visit percentages given in Fig. 1. These ultrasound examinations were used for both establishing gestational age and assessing fetal growth patterns. These methods have previously been described in detail [111, 112]. Establishing gestational age by using the first day of the last menstrual period is not reliable for a variety of reasons including the large number of women who do not know their exact date, have irregular menstrual cycles or amenorrhea, use oral contraceptive pills or bleed in early pregnancy [113]. Using fetal ultrasound data such as crown-rump length or biparietal diameter for pregnancy dating seems to overcome these problems but does not allow growth studies of these measurements since no growth variability between subjects is assumed. Pregnancy dating-curves have been derived in a subsample of the cohort including subjects with complete data on both the first day of the last menstrual period and crown-rump length or biparietal diameter and used to date the gestational age [111]. Subsequently, longitudinal curves of all fetal growth measurements (head circumference, biparietal diameter, abdominal circumference and femur length) were created resulting in standard deviation scores for all of these specific growth measurements. Various sociodemographic and lifestyle related determinant seems to affect these fetal growth and birth outcomes [114‐119]. Also, specific fetal growth patterns seem to be associated with outcomes in childhood [120‐123]. We have demonstrated in a subgroup study among mothers with a known and reliable last menstrual period, that various life style related factors affect first trimester growth [124]. Placental haemodynamics including resistance indices of the uterine and umbilical arteries have been assessed in second and third trimester [125]. Detailed measurements of fetal brain, cardiac and kidney development have been performed in the subcohort [126‐129].

The obstetric records of mothers have been looked up in the hospitals and mid-wife practices. Specialists in the relevant field code items in these records, and used for validation studies for maternal reported data [130, 131]. The major pregnancy outcomes, including live births, induced abortion fetal or perinatal loss, pregnancy induced hypertension, preeclampsia, and gestational diabetes are known in 99% of all enrolled mothers. These outcomes are related to various exposures of interest [132‐148]. In all children known to be born alive, information about sex, birth weight and gestational age is available. Currently, efforts are made to link the Generation R dataset to existing national databases for pregnancy and the neonatal period [149, 150].

Blood samples were collected in early and mid-pregnancy and at birth. Procedures for collection, processing and storage of biological samples have been described previously in detail [151]. The planned amounts of venous blood taken were 35 and 20 ml in early pregnancy and mid-pregnancy, respectively, from the mother and 10 ml from father. At delivery, 30 ml cord blood was collected. Blood samples are currently available for 97, 83 and 67% of the participating mothers, partners and children, respectively. Due to several clinical complications in mother and child, it was not always possible to collect cord blood. Subsequently, availability rates of cord blood samples in children born preterm or with low birth weight are lower than expected. Plasma and serum samples has been distributed in small aliquots (each 250 μl) and stored at −80°C. Maternal and cord blood samples have been used for measuring dietary biomarkers (folate, homocystein, total vitamin B12, free vitamin B12) levels; angiogenesis biomarkers (soluble fms-like tyrosine kinase-1 (sFlft-1), Placental growth factor (PlGF)); thyroid hormone levels (thyroid-stimulating hormone (TSH), free thyroxine (FT4)) and thyroid antibody levels, inflammation markers (high-sensitivity C-reactive protein (hs CRP); and celiac disease and Helicobacter Pylori antibodies [152‐154]. Urine samples of mothers have been collected from February 2004 until November 2005 and are stored for future measurements. Response rates for these urine samples are 85, 97 and 96% in early, mid- and late pregnancy respectively. These urine samples are used for measurement of several environmental exposures, and metabolites. Urine samples have been used for measurement of Chlamydia, and pesticides, bisphenol A, and phthalates levels [155, 156].

DNA from both parents (whole blood) and children (cord blood) has been extracted, normalised and plated and is currently used for several genotype studies. A genome wide association scan (GWAS) using the Illumina 610 Quad platform is available for the participating children. For genotyping, we used the infrastructure of the Genetic Laboratory of the Department of Internal Medicine (www.​glimdna.​org) that was also used for creation of the GWAS datasets of the Rotterdam Study, a prospective cohort study among more than 10,000 adults [157, 158]. The GWAS dataset underwent a stringent QC process whereby Individuals with low call rates or sex mismatches were excluded, and SNPs with low call rates were excluded. In addition, ethnic composition of the sample was estimated by Identity-By-State Analysis, using Principal Component Analysis, and cryptic familial relationships were identified through Identity-By-Descent analysis]. To maximize genome coverage and allow inter-study comparisons, we used MACH (version 1.0.15) software to impute genotypes at each of the 2.5 million autosomal CEPH HapMap II (release 22) SNPs for Europeans and European-Americans [159]. Criteria such as high levels of missing data (SNP call rate < 98%), highly significant departures from Hardy-Weinberg equilibrium (P < 1.10–6), or low Minor Allele Frequencies (MAF < 1%) were used to determine which SNPs to include in the imputation step. Most GWAS analyses are strongly embedded in the recently started Early Growth Genetics (EGG) Consortium and Early Genetics and Longitudinal Epidemiology (EAGLE) Consortium, in which several birth cohort studies combine their GWAS efforts focused on multiple outcomes in fetal life, childhood and adolescence [160, 161]. The power of large scale consortia has been shown previously [162‐170]. Also, strategies for genetic scoring analyses are being developed to estimate predictive values of identified genetic variants [171, 172]. Although a GWAS is available in children, we use DNA from both children and parents for genotyping for candidate gene or replication studies [173‐182]. Missing data in DNA samples in children will be collected by blood sampling or other methods at follow-up measurements.

At the age of 3 months, home visits were performed to assess neuromotor development using an adapted version of Touwen’s Neurodevelopmental examination [183‐185]. Information about growth (length (height), weight, head circumference) is collected at each visit to the routine child health centres in the study area using standardized procedures. These visits are planned at the ages 2, 3, 4, 6, 11, 14, 18, 24, 30, 36 and 48 months. Response rates for these visits vary between 70 and 97% [186].

During the preschool period, mothers receive 8 questionnaires and fathers receive one questionnaire. Each questionnaire comprises about 25 pages. Items included in these questionnaires and their references are demonstrated in Table 6 [187‐223]. Response rates based on the number of send questionnaires are showed in Fig. 1. Due to logistical constraints and implementation of questionnaires after the first group of children reached a certain age, not all children received each questionnaire. Thus, although response rates may be similar, the absolute number of completed questionnaires could differ between different ages. Response rates presented in Fig. 1 are based on the number of send questionnaires.

During the preschool period, children participating in the subgroup studies have been invited six times to a dedicated research center. Measurements at these visits included physical examinations (height, weight, head circumference, skinfold thickness and waist- hip ratio, Touwen’s Neurodevelopmental Examination at the ages of 1.5, 6, 14, and 24 months) and ultrasound examinations (brain structures at 1.5 months and cardiac and kidney structures at the ages of 1.5, 6 and 24 months, abdominal fat at the age of 24 months) [224‐237]. Dual × Energy Absorptiometry (DXA) scanning was performed in a subgroup of children at the age of 6 months [238]. Similarly Fractional Exhaled Nitric Oxide (FeNO) has been measured in only 50% of all children at the ages of 6 and 24 months. Blood pressure was measured at the age of 24 months [239]. Observations of parent-child interaction and behaviour, such as executive function, heart rate variability, infant-parent attachment, moral development, and compliance with mother and child have been performed at the ages of 14, 36 and 48 months and with father and child at the age of 48 months [240, 241]. Biological materials have been collected, including bacterial colonization measured by nasal- and nasopharyngeal swabs at the ages of 1.5, 6, 14 and 24 months, cortisol day rhythm measured by repeated salivary samples at the age of 14 months and, if parents give consent, blood samples at the ages of 6, 14 and 24 months [242‐251]. Response rates for these blood samples are about 30%, mainly because of lack of parental consent.

From the age of 5 years onwards, we plan to invite all participating children to a well-equipped and dedicated research center in the Erasmus Medical Center—Sophia Children’s Hospital every 3 years. Currently, all children aged 5–6 years are invited to participate in hands on measurements, behavioural observations and biological sample collection. The total visit takes about 2.5 h and all measurements are grouped in thematic 20 min blocks. These measurements are focused on several health outcomes including asthma, bacterial carriage, infectious diseases, behaviour and cognition, body composition, eye and tooth development, heart and vascular development, kidney growth and function, and obesity. These measurements will be used as both outcomes and determinants for health outcomes in later life. Cardiovascular, metabolic and bone measurements are also conducted in mothers. Clinically relevant results are discussed with the parents and, if needed, children or mothers are referred to their general practitioner, paediatrician or other relevant health care provider. All children are expected to have visited our research center in summer 2011. Currently ideas for follow up studies including detailed imaging studies using Magnetic Resonance Imaging (MRI), are discussed and tested in subgroups.