Healthy Lifestyle Before and During Pregnancy to Prevent Childhood Obesity - the PRE-STORK-trial

Total fat mass (in gram) assessed by air displacement plethysmography (PEA POD).

During pregnancy foetal body weight is determined by ultrasonographic method and infant body weight is measured on a scale.

During pregnancy abdominal circumference is determined by ultrasonographic method and infants are measured to the nearest 1.0 cm.

During pregnancy femur length is determined by ultrasonographic method and infants are measured to the nearest 1.0 cm.

During pregnancy head circumference is calculated by biparietal diameter and occiput-frontal diameter by ultrasonographic method and infants are measured to the nearest 1.0 cm.

Measured by the represented amount of amniotic fluid, cerebral media and umbilical artery doppler flow of the foetus.

Body mass index (BMI) is body weight relative to height (kg/m^2) and BMI z-score is called BMI standard deviation (s.d.) scores and is a measure of relative weight adjusted for child age and sex.

Measured by air displacement plethysmography (PEA POD).

Measured by air displacement plethysmography (PEA POD) and lean mass is also based on this assessment.

The gestational age is registered when giving birth.

Measured as percent of normal weight related to gestational age.

Assessed as the proportions of infants being born LGA

Assessed as the proportions of infants being born SGA.

Assessed as the proportions of infants born with Apgar score <7.

Assessed by the weight of the placenta (in gram).

Changes in faecal microbiota composition.

Body composition (fat mass (g), and fat free mass (g)) is assessed by air displacement plethysmography (PEA POD).

Body composition (fat percent (%) ) is assessed by air displacement plethysmography (PEA POD).

Body composition (fat mass (g) and fat free mass (g)) is assessed by dual energy x-ray absorptiometry scans.

Body composition (fat percent (%)) is assessed by dual energy x-ray absorptiometry scans.

Body mass index (BMI) is body weight relative to height (kg/m^2) and BMI z-score is called BMI standard deviation (s.d.) scores and is a measure of relative weight adjusted for child age and sex.

Assessed as the proportion of women who achieve a weight loss of ≥10% at time of pregnancy (or two years after randomisation without achieving pregnancy)

Assessed as the proportion of women without obtaining pregnancy or live-born offspring within a period of two years.

Maximum observed glucose concentration, time to maximum observed glucose concentration and baseline-subtracted area under the plasma glucose concentration time curve (0-120 min), and two-hour plasma glucose concentration are assessed during a 75 gram oral glucose tolerance test.

Fasting insulin concentration is assessed prior to a 75 gram oral glucose tolerance test.

Area under the insulin concentration time curve (0-60 min), area under the insulin concentration time curve (0-120 min), maximum observed insulin concentration, time to maximum observed insulin concentration, baseline-subtracted area under the insulin concentration time curve (0-60 min) and baseline-subtracted area under the insulin concentration time curve (0-120 min), Matsuda index and homeostasis model assessment-insulin resistance (HOMA-IR) are assessed during a 75 gram oral glucose tolerance test.

Glucagon assessed during a 75 gram oral glucose tolerance test.

Glucagon-like peptide-1 (GLP-1) assessed during a 75 gram oral glucose tolerance test.

Peptide tyrosine tyrosine (PYY) assessed during a 75 gram oral glucose tolerance test.

Gastric inhibitory peptide (GIP) assessed during a 75 gram oral glucose tolerance test.

Ghrelin assessed during a 75 gram oral glucose tolerance test.

Measured by 10-day coefficient of variance (CV) of glucose levels measured by continous glucose monitoring (CGM) and during a 75 gram oral glucose tolerance test.

Calculated as weight (kg) divided by height(m)^2.

Measured by scale to nearest 0.1 kg

Measured to the nearest 0.5 cm.

Body composition measured by Dual-energy X-ray absorptiometry scans prior to and after pregnancy. During pregnancy measured by bioelectrical impedance analysis.

Bone density is measured by Dual-energy X-ray absorptiometry scans prior to and after pregnancy. During pregnancy measured by bioelectrical impedance analysis.

After resting for a minimum of 10 minutes heart rate measured 3 times with 2 min in between each measurement.

After resting for a minimum of 10 minutes systolic and diastolic blood pressure is measured 3 times with 2 min in between each measurement.

Fasting plasma glucose is measured by blood sampling after an overnight of fasting.

HbA1c is measured by blood sampling after an overnight of fasting.

Cholesterol is measured by blood sampling after an overnight of fasting.

High-density lipoprotein (HDL) is measured by blood sampling after an overnight of fasting.

Low-density lipoprotein (LDL) is measured by blood sampling after an overnight of fasting.

Very-low-density lipoprotein (VLDL) is measured by blood sampling after an overnight of fasting.

Triglycerides is measured by blood sampling after an overnight of fasting.

Apolipoprotein B/apolipoprotein A1 ratio assessed by blood sampling after an overnight of fasting.

Liver function by Fibro-Scan (a proxy for liver fibrosis) measured by Fibro-Scan (UL-based elastography) and liver fat is measured by the controlled attenuation parameter score in decibel per meter.

Liver function by Fibro-Scan (a proxy for liver fibrosis) is measured by Fibro-Scan (UL-based elastography) and liver stiffness is measured by shear wave velocity in kilopascal.

Food preference is measured using the Steno Biometric Food Preference Task after an overnight of fasting.

Pharmacodynamic endpoints for bone homeostasis derived from bone turnover markers carboxy-terminal collagen crosslinks (CTX) as a marker for bone resorption after an overnight of fasting.

Pharmacodynamic endpoints for bone homeostasis derived from bone turnover markers type 1 amino-terminal pro-peptide (P1NP) as a marker for bone formation after an overnight of fasting.

Pharmacodynamic endpoints for bone homeostasis derived from bone turnover markers osteocalcin (OC) as a marker of bone formation after an overnight of fasting.

High-sensitivity C-reactive protein (hs-CRP) is measured by blood sampling after an overnight of fasting.

Tumour necrosis factor-α (TNF-α) is measured by blood sampling after an overnight of fasting.

Interleukin-6 (IL-6) is measured by blood sampling after an overnight of fasting.

Lipopolysaccharide-binding protein (LBP) is measured by blood sampling after an overnight of fasting.

Waist and hip circumference is measured to the nearest 0.1 cm.

Resting energy expenditure is meaured using indirect calorimetry.

Exercise capacity is measured by maximal exercise test with direct measurement of maximal oxygen consumption.

Assessed as the proportion of women with prediabetes.

Assessed as the proportion of women diagnosed with gestational diabetes according to the Danish guidelines

Exercise capacity is measured indirectly with the decive Seismofit® which estimates the maximal oxygen consumption by a 3-minute test during rest using seismocardiography.

Daily physical activity, sedentary time and sleep are measured with an 3-axis accelerometer (AX3, Axivity) for seven consecutive days. Two devices are placed on lower back and the thigh, respectively.

Physical exercise duration and intensity, daily physical and sleep are measured in the intervention group by a activity watch.

Defined as systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 90 mmHG

If hypertension occurs during pregnancy, information on the findings below will be obtained:

i. proteinuria > 0,3 gram/liter ii. Thrombocytopenia (< 100x109/l) iii. Renal insufficiency (serum creatinine > 100 mmol/l) iv. Impaired liver function (elevated transaminases to twice the normal concentration) v. Pulmonary edema vi. Cerebral or visual symptoms vii. Utero-placental dysfunction (SGA corresponding to an ultrasound-estimated weight below -15 %)

Defined as diagnosed hypertension before gestional week 20

Destinction between

• Non-instrumental vaginal delivery
• Instrumental vaginal delivery
• Elective caesarean section
• Emergency caesarean section (grade 1,2 or 3)

Successful breastfeeding 12 weeks after delivery, defined as exclusive breastfeeding (using formula supplements less than once a week)

Analyses will consist of analysing macronutrient content (protein, fat, carbohydrates, and calories), vitamins and minerals

Assessed as the proportion of women who initiate fertility treatment during the trial period

Body weight is measured using a scale to the nearest 0.1 kg.

Height is measured to the nearest 0.5 cm.

BMI is calculated as weight (kg) divided by height(m)^2.

Fat mass (g) and fat free mass (g) are measured by bioelectrical impedance analysis after standardized conditions.

Fat percent (%) is measured by bioelectrical impedance analysis after standardized conditions.

After resting for a minimum of 10 minutes systolic and diastolic blood pressure is measured 3 times with 2 min in between each measurement.

Appetite sensation, daily physical activity level and wellbeing based on questionnaires.

Sleep patterns based on questionnaires.

Perception of weight-related stigma and self-esteem, social/partner support on eating habits and exercise based on questionnaires.

Quality of semen from the father (meassured as number of sperm cells).

Epigenetic profile of sperm cells

Epigenetic profile from umbilical cord blood

Epigenetic profile from placenta biopsies

Epigenetic profile in peripheral blood mononuclear cells

Changes in faecal microbiota composition,: (what species are present and how many different types of species are present with a sample) using Shannon index.

Associations between parent and child genetics and obesity-related phenotypes.

Proportion of women who experience complications during delivery (premature delivery, labour induction, vacuum extraction, shoulder dystocia, planned and emergency caesarean section, postpartum haemorrhage and stillbirth).

Proportion of children with neonatal complications including (simple and complex) asphyxia (represented by potential of Hydrogen (pH)-values from umbilical cord), hypoglycaemia, jaundice, and admittance to neonatal intensive care

TEG is measured by blood sampling after an overnight of fasting

Proportion of women who experience complications during pregnancy (gestational week 24 - 40) and post-partum (3 months post partum) (deep vein thrombosis, pulmonary embolism, gestational hypertension, preeclampsia, abortion)

International Normalized Ratio (combined measure for coagulation factor II, VII, and X). Blood sampled from umbillical cord at birth.

Concentration of haemoglobin is measured by blood sampling from umbillical cord at birth

Concentration of high-sensitivity CRP (hsCRP). Blood sampled from umbillical cord at birth

Concentration of alanine transaminase. Blood sampled from umbillical cord at birth

TEG is measured by blood sampled from umbillical cord at birth and from vein puncture three months after birth

Concentration of the endothelial marker thrombomodulin, is measured by blood sampling after an overnight of fasting.

Activated partial thromboplastin time (seconds)(APTT). Blood sampled from umbillical cord at birth.

International normalised ratio (INR) of time (seconds). Blood sampled from umbillical cord at birth.

Concentration of bilirubin. Blood sampled from umbillical cord at birth

Concentration of albumin. Blood sampled from umbillical cord at birth

Concentration of syndecan-1 is measured by blood sampling after an overnight of fasting.

Concentration of platelet endothelial cell adhesion molecule 1 (PECAM-1) is measured by blood sampling after an overnight of fasting.

The severity of pregnancy-related nausea and vomiting is meas-ured based on the questionnaire Pregnancy-Unique Quantification of Emesis and Nausea (PUQE)

Number of planned obstetric consultations after gestational week 20

Number of obstetric emergency consultations after gestational week 20

Number of diagnosed urinary tract infections during pregnancy

Number of admissions with vaginal bleeding episodes after gestational week 20

Admissions with premature contractions before gestational week 34

Admissions with shortened cervix less than 20 mm before gestational week 34

Defined as delivery before gestational week 34

Induction (mechanical or medical) of labour before spontaneous onset of labour

Use of epidural analgesia during birth

Use of scalp pH sampling during birth

Use of Oxytocin stimulation in active labour

Perineal lacerations, destinction between 1st, 2nd, 3rd, and 4th degree

Post-partum haemorrhage in mL

Length of admission and re-submission after delivery

Defined as haemoglobin less than 5.0 mmol/l

Diagnosed mastitis

Diagnosed Endometritis

Number of and total length of hospital admissions

Mood and depressions Edinburgh Postnatal Depression Scale 16 weeks after delivery

Quality of life (SF-36) 16 weeks after delivery