Introduction 1 2 1 2 To describe normal and abnormal growth, development and health from fetal life until young adulthood; To identify biological, environmental and social determinants of normal and abnormal growth, development and health from fetal life until young adulthood; To examine the effectiveness of current strategies for prevention and early identification of groups at risk. Table 1 Main outcomes per research area Growth and physical development Fetal growth patterns and organ development Pregnancy complications Postnatal growth patterns Obesity Risk factors for development of cardiovascular disease Risk factors for type 2 diabetes Behavioural and cognitive development Maternal and paternal psychopathology Fetal and postnatal brain development Behaviour, psychopathology and cognition Neuromotor development Chronic pain Attachment Stress reactivity Diseases in childhood Infectious diseases in childhood Development of the immune system Asthma and asthma related symptoms Paroxysmal neurological disorders Health and healthcare Quality of life Health care utilization Effectiveness of screening programmes Table 2 Main determinants Biological determinants Parental anthropometrics and blood pressure Fetal and postnatal growth characteristics Endocrine and immunological factors Genetic variants Environmental determinants Maternal and childhood diet Parental life style habits (including smoking, alcohol consumption) Housing conditions Social determinants Parental education, employment status and household income Parental marital status Ethnicity 2 Design and biological samples collection 2 2 Additionally, more detailed assessments of fetal and postnatal growth and development are conducted in a randomly selected subgroup of Dutch children and their parents, referred to as the Generation R Focus Cohort. Studies conducted in this subgroup examine etiological associations using more time-consuming methods that cannot be applied in the whole cohort. 1 Fig. 1 Design data collection biological specimens  The Generation R Study is approved by the Medical Ethical Committee of the Erasmus Medical Center, Rotterdam. Mothers and their partners received written and oral information about the study. Participants were asked for their written informed. Study cohort 3 Table 3 Characteristics of mothers and their partners   Mothers Partners (n = 9,778) (n = 6,347) Gestational age at enrolment (%)     Early pregnancy 69 –     Mid-pregnancy 19 –     Late pregnancy 3 –     Birth 9 – Pregnancy number in study (%)     1st pregnancy  94 –     2nd pregnancy  6 –     3rd pregnancy  0.1 – a 29.7 (5.3) 32.7 (5.8) Parity (%)     0 55 –     1 31 –     ≥2 14 – Ethnicity (%)     Dutch, other-European  58 70     Surinamese 9 6     Moroccan  7 4     Turkish  9 6     Dutch Antilles  4 3     Cape Verdian  4 2     Others  9 9 Education (vocational training) (%)     Lower  13 8     Intermediate 45 41     Higher  42 51 Values are percentages  a Logistics All biological samples were bar coded with a unique laboratory number. Blood or urine samples collected at one visit have the same bar code with an additional specific tube number. This combination forms a unique registration number. All following steps in processing, storing and data management of the samples are linked to this unique number. All blood samples from the mother and partner were taken by research nurses and temporally stored at our research centre or one of the obstetric departments at room temperature for a maximum of 3 h. At least every 3 h, these blood samples were transported to a dedicated laboratory facility of the regional laboratory in Rotterdam, the Netherlands (STAR-MDC) for further processing and storage. Participants delivered their child either at home or at one of the hospitals in Rotterdam. The midwife or obstetrician collected cord blood samples. Subsequently, courier services with a 7-days and 24-h availability were responsible for transportation of these cord blood samples to our laboratory within 2 h. After collection and transportation, all blood and urine samples have been centrally processed and stored at the STAR-MDC laboratory. Samples for DNA extraction have initially been stored as EDTA whole blood samples and subsequently transported to the Erasmus Medical Center for further processing. 3 Response rates 4 4 5 6 Table 4 Response rates biological specimens  Eligible Available % (n) Mother     DNA 8,880 91 (8,055) Early pregnancy     Plasma (EDTA) 6,748 95 (6,398)     Serum 6,748 94 (6,337)     Urine 2,797 85 (2,375)  Mid-pregnancy     Plasma (EDTA) 8,241 93 (7,682)     Serum 8,241 93 (7,631)     Urine 3,380 97 (3,279) Late-pregnancy     Urine 3,918 96 (3,762) Partner     DNA 6,347 83 (5,289) Child     DNA 8,821 67 (5,908)     Plasma (EDTA) 8,821 66 (5,857)     Serum 8,821 66 (5,821) Number of eligible subjects reflects those participating in the study during pregnancy (DNA mother and partner) and visit our research centre in early, mid- or late pregnancy (plasma, serum and urine). The number of eligible subjects for urine sample collection is lower since this data-collection was added after starting the study. Number of eligible children reflects the number of live born children of mothers who were enrolled in the prenatal phase of the study with a known date of birth Table 5 Characteristics of partners with and without DNA available  Partners (n = 6,347) DNA available (n = 5,289) No DNA available (n = 1,058) Age (years) 32.8 (5.7) 32.2 (5.7) Ethnicity (%)     Dutch, other-European  68.2 66.1     Surinamese 6.8 6.6     Moroccan  3.8 6.2     Turkish  6.8 7.2     Dutch Antilles  2.9 3.4     Cape Verdian  2.7 1.9     Others  8.8 8.6 Values are percentages or means (standard deviation) Table 6 Characteristics of children with and without DNA available  Children (n = 8,821) DNA available (n = 5,908) No DNA available (n = 2,913) Male (%) 50.6 50.1 Birth weight (grams) 3,444 (520) 3,280 (671) Birth weight <2,500 g (%) 3.3 10.8 Gestational age (weeks) 40.1 (37.1–42.0) 39.9 (34.7–42.1) Gestational age <37 weeks (%) 3.9 11.2 Ethnicity (%)     Dutch, other-European  64.7 54.6     Surinamese 7.4 9.0     Moroccan  6.5 7.9     Turkish  7.5 9.5     Dutch Antilles  3.8 4.8     Cape Verdian  2.4 3.5     Others  7.7 10.7 Values are percentages, means (standard deviation) or median (95% range) DNA For DNA extraction, one 10 ml EDTA tube from mothers, partners and children at birth have been collected and stored directly as whole blood sample at −80°C. After completion of the blood sample collection, DNA extraction was started from white blood cells. First, DNA extraction from all children has been conducted manually using the Qiagen FlexiGene Kit (Qiagen Hilden, Germany). Currently, DNA extraction from 5 ml whole blood samples from the mothers and partners is performed by a Hamilton STAR multi-channel robot using AGOWA magnetic bead technology (at 72 samples per run) and is expected to be finished in spring 2008. Extracted DNA is automatically collected in stock tubes (2D Matrix, Micronic) that are organized in 96-wells format, but that can be individually addressed. These stock samples are split into two tubes that are stored at different locations. One of the stocks will then be used for normalising the DNA concentrations in 96 deep well (DW) plates. This protocol, as well as other manipulations of the DNA samples are performed on a Caliper ALH3000 pipetting robot (8/96/384 channels) with a Twister module and a 96/384 wells Tecan GENios plus UV reader. In total, 90 DNA samples are normalised per 96 DW plate with 6 water blancs per plate. Additionally, a random selection of 5% of the total number of samples are put into separate 96 DW plates, again with 6 blancs per plate, for control purposes. For normalisation, sample and diluent volumes are automatically calculated and produced to obtain equal DNA concentrations for all samples (25–50 ng/μl). From these stock 96-DW plates, 384 DW plates are created, which comprise 24 blancs each. Subsequently, replica PCR plates (384 wells) with a concentration of 1 ng/μl are created for genotype studies. Stock 96-well plates (DW and 2D Matrix, Micronic) are stored at −20°C at two different locations within the Erasmus Medical Center (Genetic Epidemiology Laboratory, Genetic Laboratory Internal Medicine). The 384 DW stock and PCR plates are stored at the Genetic Laboratory Internal Medicine, Erasmus Medical Center. All genotyping studies with Generation R DNA material are performed in-house at the high throughput genotyping facility in this Laboratory. Genome Wide Association database 4 5 7 8 Blood for phenotypes Results of analyses performed in blood samples (5 ml EDTA plasma) for routine care for pregnant women (Hb, Ht, HIV, HBsAg, Lues, Rhesus factor and irregular antibodies) are obtained from midwife and obstetric registries. ® 2 Fig. 2 Distribution of blood samples in the 96 trays (see text)  Urine 9 Other population-based birth cohort studies 10 14 Collaboration with these studies for funding, standardization, replication and quality management is necessary. Currently, we are exploring possibilities for formal collaboration. Requests for biological samples use The study has an open policy with regard to collaboration with other research groups. Request for collaboration and use of biological specimens should primarily be pointed to Albert Hofman (a.hofman@erasmusmc.nl). These requests are discussed in the Generation R Study Management Team regarding their scientific merits, study aims, overlap with ongoing studies, logistic consequences and financial contributions. General policy is that collaborating researchers and groups are responsible themselves for the finances and that all laboratory tests are conducted in the Eramus Medical Center, Rotterdam. After approval of the project by the Generation R Study Management Team and the Medical Ethical Committee of the Erasmus Medical Center, the collaborative research project is embedded in one of the four research areas supervised by the specific principal investigator.