NFFD is a randomized, blinded, controlled trial with two parallel groups performed in southern Norway, encompassing the cities of Kristiansand and Mandal and the more rural surrounding areas. The protocol for the trial is previously published [17]. Midwives at eight healthcare clinics enrolled participants between September 2009 and February 2013. Women were eligible if they were nulliparous, with a singleton pregnancy of ≤20 gestational weeks, had a pre-pregnancy body mass index (BMI) ≥19 kg/m2, were literate in Norwegian or English, and provided signed, informed consent. Exclusion criteria were pre-existing diabetes, disabilities precluding participation in a physical fitness program (based on national and international recommendations) [18], on-going substance abuse, or planned relocation outside the study area before delivery. The first 20 participants comprised a feasibility study. The protocol was modified to include a lower age limit of 18 years and to allow randomisation after initial questionnaires and blood tests were completed, in order to assure that participants were sufficiently motivated and avoid missing data. Participating clinics documented attendance of 4245 women during the inclusion period, of whom we estimate that 1610 were nulliparous (Fig. 1) [16].
Ethics, consent and permissions
The trial was performed in accordance with the Declaration of Helsinki. The present study is a planned secondary analysis of the NFFD trial, and was included in the consent and ethical approval of the trial. The Norwegian Regional Committee for Medical Research Ethics South-East-C approved the trial and modifications (REK reference 2009/429), including specific approval for the storage and analysis of frozen serum in Research Biobank Notification no. 2594. Signed, informed consent was obtained from all participants.
Randomisation and blinding
After receiving signed consent forms and confirming that blood tests and questionnaires were completed, a research nurse assigned participants consecutively to the intervention or control arm of the study utilizing a computer-generated list with 1:1 allocation ratio and blocks of 20. All examinations, blood test evaluations and scoring of questionnaires were performed by assessors blinded to group allocation.
Intervention
Details of NFFD’s dietary and physical activity components and the rationale behind them are previously published [17, 19]. The dietary component was based on ten recommendations designed to increase awareness of food choices, with advice to increase intake of water, vegetables and fruit and reduce snack food consumption. There was no calorie restriction or specific limitation of fats or carbohydrates. Counselling was performed twice, by phone, with a four to six week interval. Counsellors were either experienced clinical dieticians or graduate students in public health, trained and supervised by the NFFD team. The physical activity component consisted of access to twice-weekly exercise classes at a local gym facility, led by physical therapists or students in sports science, trained and quality-controlled by the NFFD team. Attendance was recorded. Participants were encouraged to engage in 30 min of moderate-intensity physical activity on three additional days per week. Lifestyle recommendations were reinforced with booklets, access to a NFFD internet site, and invitation to one cooking class and one evening meeting with information on the NFFD trial and the value of regular exercise and healthy diet in pregnancy.
Participants in the control group received routine prenatal care following Norwegian standard: eight prenatal appointments, including one second-trimester ultrasound examination, with additional care as needed, provided free of charge. Routine care includes a booklet with advice on prenatal nutrition, physical activity and recommendations for weight gain based on current Institute of Medicine (IOM) guidelines (normal-weight: 11.5–16 kg, overweight: 7–11.5 kg, obese: 5–9 kg) [20].
Measurements
The primary aims of the NFFD trial were to examine if intervention resulted in differences in GWG, birth weight of term infants, the proportion of term infants >4000 g, maternal fat percent at 36 gestational-weeks, and the incidence of operative deliveries. Maternal glucose levels at 30 gestational-weeks was a primary endpoint, while the proportion of women with elevated 2-h glucose tolerance tests and measurement of hormones related to glucose metabolism were secondary endpoints of the trial. Assessment of the subgroup of overweight/obese women was specified in the trial protocol.
Pre-pregnancy weight was self-reported. Participants were weighed at their healthcare clinic at inclusion, and at Sørlandet Hospital at 30 gestational-weeks (Tanita BC 418, Tokyo, Japan). Feasibility study participants reported their height; later participants were measured using a stadiometer (Seca Leicester, Hamburg, Germany). Pre-pregnancy BMI was calculated based on self-reported pre-pregnancy weight and measured height when available. Participants were weighed on admission to the delivery ward. If missing, last weight in the prenatal record was recorded with corresponding date. GWG at term was calculated for women delivering at ≥37 gestational-weeks with weight available within 2 weeks of admission.
Participants completed questionnaires at trial inclusion and at gestational-week 36, either electronically or in print. No questionnaires were completed at gestational-week 30. Diet was assessed by 43 food-frequency questions, analyzed using a pre-determined score (range 0–10, with higher score denoting healthier eating behavior). The score is previously described in detail, and has demonstrated acceptable test-retest reliability [19]. Physical activity was assessed with the International Physical Activity Questionnaire (IPAQ) short version, scored using IPAQ analysis algorithms. The IPAQ is validated in a Scandinavian population [21].
Prior to randomisation, fasting blood tests were assessed for evidence of pre-existing diabetes (defined as glucose ≥7.0 mmol/l) [22]. No participants were excluded on this basis. At gestational-week 30, plasma glucose was measured after overnight fast and again at 2-h after 75 g glucose load. All tests were performed at Sørlandet Hospital using a Cobas 6000 c501 chemistry analyzer (Roche Diagnostics). Glucose levels ≥7.0 mmol/l at fasting and/or ≥7.8 mmol/l at 2-h were classified as elevated, based on contemporary national [23] and WHO 2006 criteria [22], and participants and their primary care physicians were informed. Glucose at 2-h was missing for 12 participants (9 intervention, 3 control), primarily due to vomiting. Fasting serum samples were frozen and stored at −80 °C. Frozen samples were analyzed at Aker Hormone laboratory using a Modular E170 analyzer (Roche), batched to decrease interassay variation. Insulin was analyzed using non-competitive electrochemoluminescence immunoassay (Roche Diagnostics), with coefficient of variance of 4%. Leptin was analyzed using competitive radioimmunoassay (Millipore), with coefficient of variance of 7%. HOMA-IR was calculated as: (insulin(mU/l) x fasting glucose(mmol/l))/22.5. Leptin, insulin and HOMA-IR were missing for eight participants (3 intervention, 5 control), due to errors in freezing or transport. All missing values were considered missing completely at random. Three insulin and HOMA-IR values (1 intervention, 2 control) were excluded from analysis as outliers.
Sample size
We predicted a 20% prevalence of newborns with birth-weight > 4000 g in the control group based on 2005 statistics from the Norwegian birth registry [24], and determined empirically that a reduction to 10% in the intervention group would be clinically relevant. We calculated that 198 women were required in each study arm to demonstrate statistical significance with a power of 80%. We also expected a 10% incidence of GDM (based on 2-h glucose ≥7.8 mmol/l) [22, 23] in the control group, and determined that a reduction to 3% in the intervention group would be clinically significant. We calculated that we would have 80% power to detect a statistically significant difference between groups with 200 participants in each arm. To allow for participant drop-out and premature deliveries and to allow for analysis of subgroups, we planned to randomize 600 participants.
Statistics
Unadjusted comparison of intervention and control groups was performed using student t-test or chi-square test as appropriate. Difference between the randomized groups for continuous or binary variables was assessed using multiple linear or logistic regression models adjusted for age, education, income level and smoking at inclusion, pre-pregnancy BMI category and gestational age at measurement. Variables included in the adjusted analysis were chosen based on clinical relevance (pre-pregnancy BMI category and smoking) and/or measured differences between intervention and control group (gestational age at measurement) and/or measured differences between included and missing participants (age, education and income). Effect modification between randomized groups and patient characteristics on continuous outcomes was assessed by an interaction term in the multiple linear regression models. For binary outcomes, effect modification was assessed by the Breslow-Day test of homogeneity of odds ratios. No further adjustment for BMI category was performed when analysis was stratified according to pre-pregnancy BMI. P-values <0.05 were considered statistically significant. All tests were two-sided. We used SPSS for Windows version 21.0 for all statistical analyses.