Study population and data collection

This study included 768 mother-newborn pairs (singletons) selected from the ongoing ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort in the province of Limburg in Belgium. The study protocol was approved by the Ethical Committee of Hasselt University and East-Limburg Hospital in Genk (Belgium) and has been carried out according to the declaration of Helsinki. Written informed consent was obtained from all participating mothers. In total, 768 mothers with a singleton pregnancy and all with a pre-pregnancy BMI below 40 kg/m2 were recruited from February 1, 2010, to February 1, 2015, between Friday 1200 hours and Monday 0700 hours. The mother’s ability to fill out questionnaires in Dutch was a criterion for selection. The overall participation rate was 61 %. Because DNA was missing or of bad quality for cord blood DNA (n = 14) or for placental DNA (n = 57) or because telomere length measurements were too variable between triplicate measurements for cord blood telomere (n = 10) or placental telomere (n = 8), and because of missing data on maternal weight gain during pregnancy (n = 1), the final sample consisted of 743 mother-newborn pairs to study associations with cord blood telomere lengths and 702 mother-newborn pairs to study associations with placental telomeres (Additional file 1: Figure S1).

Data on maternal pre-pregnancy weight, weight before delivery, and height were collected from the medical records at the hospital. Maternal height and weight were measured without shoes, wearing light clothes to the nearest centimeter and weight to the nearest 0.1 kg at the first antenatal visit of each pregnancy (weeks 7–9 of gestation). BMI was defined as weight in kilograms divided by the square of height in meters. Gestational age was estimated based on ultrasound data. Pre-pregnancy BMI was categorized into three categories; normal was defined as BMI below 25 kg/m2, overweight when BMI was greater than or equal to 25 kg/m2 and below 30 kg/m2, and obese was defined when BMI was greater than or equal to 30 kg/m2. Furthermore, the women were weighed on admission to the delivery ward. Maternal pregnancy weight gain (weight before delivery minus pre-pregnancy weight) was categorized according to the Institute of Medicine guidelines – we defined insufficient and excessive gestational weight gain in relation to maternal pre-pregnancy BMI (for underweight: total weight gain < 13 and > 18 kg; normal weight: total weight gain < 11.5 and > 16.0 kg; for overweight: total weight gain < 7.0 and > 11.5 kg; for obesity: total weight gain < 5.0 and > 9.0 kg, respectively) [48].

Study questionnaires were completed in the post-delivery ward to provide detailed information on maternal age, paternal age, maternal education (as a measure for socioeconomic status), smoking status, parity, ethnicity, and pregnancy complications. Maternal smoking status was assessed as former smokers who had quit smoking before pregnancy and smokers who continued smoking during pregnancy. Maternal education was coded low when mothers only went to primary school and did not obtain a diploma, middle as they obtained a high school diploma and high when they obtained a college or university degree.

Newborn’s ethnicity was classified as European-Caucasian when two or more grandparents were European or non-European when at least three grandparents were of non-European origin. Information on pregnancy complications were collected from the medical records for each mother. Pregnancy complications was coded as absent if mothers did not experience any pregnancy complications or as present if mothers experienced one or more pregnancy complications. Included pregnancy complications were gestational diabetes, hypertension, infection diseases, pre-eclampsia, vaginal bleeding, and hyper- or hypothyroidism. Perinatal parameters were obtained after birth such as birth date, newborn gender, birth weight, and Apgar score. The ENVIRONAGE birth cohort is generalizable to the gestational segment of the population at large as it did not differ from all births in Flanders as to maternal age, education, parity, newborn gender, ethnicity, and birth weight (Additional file 1: Table S1) [49].

Cord blood and placental tissue collection

Umbilical cord blood was drawn immediately after delivery in BD Vacutainer® plastic whole blood tubes with spray-coated K2EDTA (BD, Franklin Lakes, NJ, USA). Samples were centrifuged at 3200 rpm for 15 min and blood tubes were stored at −80 °C. Buffy coat was removed upon DNA extraction. Placentas were collected and stored at −20 °C within 10 minutes after delivery. Placental biopsies were taken at the fetal site and stored at −80 °C upon DNA extraction as described previously [50]. For each placenta, four different biopsies were taken at four standardized sites across the middle region of the placenta, at 4 cm approximately from the umbilical cord. First, we determined within-placental average relative telomere length variation in 14 different placentas based on the four different biopsies. This average within placental variation was 11.7 %. Because of the low variation in telomere length within the placenta for different biopsies we used only one biopsy (1–2 cm3) taken to the right of the main artery for placental telomere length assessment.

Average relative telomere length measurement

DNA was extracted from cord blood buffy coat and placental tissue using the QIAamp DNA Mini Kit (Qiagen, Inc., Venlo, The Netherlands). DNA quantity and purity was assessed by a Nanodrop 1000 spectrophotometer (Isogen, Life Science, Belgium). DNA integrity was assessed by agarose gel-electrophoresis. Average relative telomere length was measured by a modified quantitative real-time PCR (qPCR) protocol as described previously [51]. To ensure a uniform DNA input of 5 ng for each qPCR reaction, samples were diluted and checked using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Europe). Telomere and single copy-gene reaction mixture and PCR cycles used are given in Additional file 1: Text S1. All measurements were performed in triplicate on a 7900HT Fast Real-Time PCR System (Applied Biosystems) in a 384-well format. On each run, a 6-point serial dilution of pooled buffy coat or pooled placental DNA was run to assess PCR efficiency as well as eight inter-run calibrators to account for inter-run variability. Relative average telomere lengths were calculated using qBase software (Biogazelle, Zwijnaarde, Belgium) and were expressed as the ratio of telomere copy number to single-copy gene number (T/S) relative to the average T/S ratio of the entire sample set. Before our study, we performed an interlaboratory comparison of our telomere assay with a US reference lab to standardize the protocol. We achieved coefficients of variation (CV) within triplicates of the telomere runs, single-copy gene runs, and T/S ratios of 0.68 %, 0.41 %, and 6.4 %, respectively, for cord blood telomeres. For placental telomeres, we achieved CVs of 0.70 %, 0.45 %, and 6.9 %, for telomere runs, single-copy gene runs, and T/S ratios, respectively.

Statistical analysis

All statistical analyses were performed using the SAS 9.3 statistical software (SAS Institute Inc., Cary, NC, USA). Continuous variables were tested for normality. Average relative cord blood and placental telomere lengths showed skewed distribution and were log10 transformed to improve normal distribution. To study potential confounding structure in our dataset, we assessed the distributions of continuous variables (ANOVA) and proportions of categorical variables (χ2 test) across different classes of maternal pre-pregnancy BMI (normal, overweight, and obese). Pearson correlation (unadjusted analysis) and multiple linear regressions were applied to address the association between maternal pre-pregnancy BMI and cord blood or placental telomere lengths. In a first model, we adjusted for maternal and paternal age, maternal education, newborn gender, gestational age and birth weight. In a second model, we additionally adjusted for parity, maternal smoking status, newborn ethnicity, cesarean section, and pregnancy complications. Finally, we introduced maternal net weight gain during pregnancy to our models as a continuous variable as well as a categorical variable coded as sufficient and insufficient weight gain using the definition of Institute of Medicine guidelines (see Study population and data collection). In order to test whether the association of gestational weight gain on newborn telomere length is influenced by maternal pre-pregnancy BMI, we tested the interaction between weight gain and pre-pregnancy BMI. We ran different sensitivity analyses in which we separately excluded mothers from non-European origin, those with pre-pregnancy BMI less than 18.5, those who underwent cesarean section, or those who experienced complications during pregnancy.