PRE-Pregnancy Weight Loss And the Reducing Effect on CHILDhood Overweight - Aarhus

Study Overview
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Brief Summary
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The study is a single site parallel randomized controlled study. The study will be assessing the effect of a 10% pre-conceptional weight loss intervention vs a control group among healthy couples where the prospective mother is pregnant and overweight or obese (BMI 28-45 kg/m^2). The couples in intervention group will receive dietitian counseling and participate in physical activity sessions to attain the 10% weight loss. The overall objective is to test whether a comprehensive pre-conceptional parental weight loss intervention effectively reduces the risk of offspring overweight, and adiposity and its complications compared to a control group.

Detailed Description
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The study is a single site parallel randomized controlled study. The study will be assessing the effect of a pre-conceptional weight loss intervention vs a control group among healthy couples where the prospective mother is pregnant (recruited at gestational age 32-34), overweight or obese (BMI 28-45 kg/m^2), between 18-35 years and planning an additional pregnancy within the following 3 years. The investigators will recruit a total of 140 healthy pregnant couples who will be randomized 1:1 to either intervention or control, stratified according to maternal pre-pregnancy BMI and previous or present diagnosis of gestational diabetes. The couples in intervention group will receive dietary counseling by a dietitian from 3 months post-partum of the 1st child until birth of the 2nd child. The goal is to achieve a healthy lifestyle and a moderate weight loss of 10% according to pre-pregnancy weight during the first year. Hereafter, the couples will be guided in weight maintenance until pregnancy of the second child. During pregnancy the goal is to achieve a healthy weight gain. Couples in intervention group will also participate in physical activity sessions every 2nd week and advised to walk with moderate intensity at least 2-4 times a week for at least half an hour. The overall objective is to test whether a comprehensive pre-conceptional parental weight loss intervention effectively reduces the risk of offspring overweight, and adiposity and its complications compared to a control group. The 1st baby born before intervention is started will be the index child and the metabolic and anthropometric parameters of the 1st child will be compared to parameters of the second child to assess the effect of the parent's healthy lifestyle between the two pregnancies. The investigators hypothesize that parental weight loss intervention, initiated before conception, will facilitate lower parental insulin resistance, inflammation, body weight and adiposity, incretin responses and energy expenditure compared to usual care. For the offspring the investigators hypothesize that the intervention will reduce adverse pregnancy outcomes with a short-term reduction in the risk of being born large for gestational age with excessive fat mass and a long-term reduction in the risk of developing overweight or obesity as a consequence of both social (family approach) and biological (epigenetic modifications) pathways.

Official Title
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PRE-Pregnancy Weight Loss And the Reducing Effect on CHILDhood Overweight - Aarhus

Conditions
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Overweight and Obesity, Weight Loss, Pregnancy Related, Child Nutrition Sciences, Body Composition

Intervention / Treatment
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* Behavioral: Weight loss intervention


Participation Criteria
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Eligibility Criteria
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Maternal inclusion Criteria: Maternal pre-pregnancy BMI 27.0-44.9 kg/m^2 Maternal age range 18-35 years Connected to Aarhus University Hospital as place of birth Pregnant Paternal Inclusion Criteria: Paternal pre-pregnancy BMI 18.5-44.9 kg/m^2 Paternal age range 18-55 years Parental Inclusion Criteria: Couples (male and female) Planning pregnancy within 3 years Provided voluntary informed consent Danish or English speaking Intention to permit the planned offspring to participate in the follow-up study Able to store biological samples from the offspring at home in the freezer in a box given by the study personnel Parental Exclusion Criteria: Diabetes mellitus (Type 1 or 2) Previous or present eating disorder Allergy towards ingredients in the very low calorie diet products Severe heart, liver or kidney disease Conception by in vitro fertilization Any medical condition or concomitant medication as judged by the medical responsible Adherence to vegan diets or other diets interfering with the dietary guidelines in the study Participation in other clinical trial that can affect the results of the current study Engagement in elite sports or similar strenuous exercise ≥5 h/week at inclusion Blood donation or transfusion within the past month before screening Blood donation during the study Inability or unwillingness to follow the study protocol and instructions given by the study personnel A detailed description of the exclusion criteria is given below: Medical conditions as known by the participant: Diabetes mellitus (type 1 and 2) History or diagnosis of eating disorder (e.g. restrained eating, disinhibition, emotional eating) Any significant medical condition as assessed by the investigator (e.g. dysregulated thyroid disease or reproductive diseases) Any significant psychiatric disorder (i.e. schizophrenia, bipolar disease or depression) as assessed by the investigator Severe chronic heart, liver and kidney disease Polycystic ovary syndrome with irregular cycle Medication: Current use of medication or use within the previous three months with a potential to affect body weight or pregnancy as judged by the investigator (e.g. systemic corticoids) Personal/other: Severe food allergies, food intolerances or dislike expected to interfere with the study, including allergy towards ingredients in the VLCD products Engagement in elite sports or similar strenuous exercise ≥5 h/week Blood donation or transfusion within the past month before screening Planned blood donation for other purpose than this study during participation Alcohol abuse, as judged by the investigator, within the previous 12 months Drug abuse, as judged by the investigator, within the previous 12 months Psychological or behavioral problems which, in the judgement of the investigator, would lead to difficulty in complying with the study protocol Participation in other clinical trials within the past three months or intention to do so during the study, which are judged by the investigator to affect the present study Unable to consume the interventional product for religious reasons, swallowing disorders, other physiological reasons or any other reasons for not being able to follow the recommended diet Inability or unwillingness to give written informed consent or communicate with study personnel Inability or unwillingness to follow the study protocol and instructions given by the study personnel Illiteracy or inadequate understanding of Danish or English language. Any other condition that judged by the investigator may interfere with the adherence to the study protocol

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 35 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: 140 couples will be recruited and randomized to either intervention og control. Participants includes the mother, father and 1st and 2nd child, therefore the total number of participants will be 560.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Intervention<br>Intervention group will receive dietary counseling by a trained dietitian from 3 months post-partum of the 1st child until birth of the 2nd child. The mother will receive advice to provide a moderate caloric restriction (-500 to 1000 kcal/d) and attend physical activity sessions to attain a 10 % weight loss according to pre-pregnancy weight. If the father is also overweight or obese, he will receive the same advice. | Behavioral: Weight loss intervention<br>* Subjects will receive dietary counseling by a dietitian from 3 months post-partum of the 1st child until birth of the 2nd child. The mother (and father if overweight, BMI ≥25 kg/m^2) will receive advice to provide a moderate caloric restriction (-500 to 1000 kcal/d) and power walk 2-4 times a week for a half an hour and attend physical activity sessions every 2nd week to attain a minimum weight loss of approximately 0,5-1,0 kg/week. The weight loss will last until a 10% weight loss (according to pre-pregnancy weight) has been achieved or until 1-year post-partum. If the subject has not achieved a weight loss of 10%, she or he will receive Very low-calorie diet (VLCD) until target weight loss is reached, though no longer than 8 weeks. After weight loss subjects will receive counseling to maintain weight loss and healthy weight gain during pregnancy until birth of the 2nd child.<br>|
| No Intervention: Control<br>The control group will receive standard care and will not receive any dietary advice prior to pregnancy. During pregnancy control subjects will receive dietary advice according to standard care which means no counselling from a dietitian unless there are special needs (e.g., gestational diabetes), then they will receive counseling from a dietitian in accordance with standard care. | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Neonatal fat mass | Offspring's neonatal fat mass (grams) assessed by air displacement plethysmography (Pea POD) | Collected within the first week of birth (strived <48 hours) | 
| Difference in epigenetic changes in child 1 and 2 | Cord blood from offspring will be collected at birth and stored at -80 °C until use. At the Department of Molecular Medicine AUH genomic DNA will be extracted from peripheral blood using standard methods e.g., QIAmp® Mini Kit (Qiagen, Germany). 1 μg of genomic DNA will be bisulfide converted using Illumina iScan (platform). The methylation level will be measured using Infinium MethylationEPIC DNA Analysis BeadChip (Array type) at Eurofins Genomics AS (Denmark) or similar methods. The stated required sample size (140 couples) is calculated based on the neonatal fat mass. As no other studies have performed methylation analysis using WGBS in similar cohorts, precise power calculations on epigenetic changes are impractical. However, since the EPICOM study found differentially methylated regions in only 20 cases and 20 controls using Illumina's 450K methylation assay, the power to elucidate epigenetic changes is considered sufficient in the planned study. | Assessed at birth | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Obstetrics outcome - length of gestational age | Length of gestational age | Assessed at birth of both offsprings | 
| Obstetric - medication use | Medication use during labor | Collected from initiation of labor until completed birth of both offsprings | 
| Obstetric - delivery mode | Delivery mode (rupture of membranes or premature contractions, vaginal, cesarean section, instrumental delivery, miscarriage and stillbirth). | Collected at birth of both offsprings | 
| Obstetric - pregnancy complications | Pregnancy complications e.g. miscarriage, abortion (induced and indication for abortion), hydramnios, gestational hypertension, preeclampsia, need for maternal corticosteroid treatment for fetal lung maturation, urinary tract infection) will be collected from medical record. | Collected at gestational age (GA) 34-36 and after birth of both offsprings | 
| Obstetric - Neonatal unit admissions | Neonatal unit admissions | Collected at birth and until 12 months after birth of both offsprings | 
| Obstetric - obstetrics complications | Obstetrics complications (e.g. preterm prelabour rupture of the membranes, shoulder dystocia, birth trauma (perinatal rupture), postpartum hemorrhage, maternal infections, maternal hospitalization). | Collected at birth of both offsprings | 
| Obstetrics - birth defects/abnormalities | Birth defects/abnormalities | Collected at birth of both offsprings | 
| Neonatal morbidities | Neonatal morbidity (e.g. major congenital malformations (ICD10 Q00-Q99 or requiring medical or surgical treatment), birth injury, respiratory distress, transient tachypnoea, neonatal hypoglycemia, systemic infections, admission to neonatal intensive care unit, number of admission days, hyperbilirubinemia), deaths. | Collected at birth of both offsprings | 
| Fetal and offspring growth - body weight | Fetal body weight will be measured with ultrasound during pregnancy and offspring body weight will be measured using an electronic scale. | Collected from medical record during pregnancy, measured in gestational age (GA) GA34-GA36 via ultrasound in both pregnancies, at birth and 3, 12 and 18 months after birth of both offsprings | 
| Fetal and offspring growth - length | Fetal length will be measured with ultrasound during pregnancy and offspring length will be measured using an infantometer as appropriate for measuring infant, baby and toddler length. | Collected from medical record during pregnancy, measured in GA34-GA36 via ultrasound in both pregnancies, and measured at birth and 3, 12 and 18 months after birth of both offsprings | 
| Fetal and offspring growth - abdominal circumference | Abdominal circumference will be measured with ultrasound during pregnancy and offspring abdominal circumference will be measured three times to the nearest 0.1 cm using a stretch-resistant measuring tape | Collected from medical record during pregnancy, measured in GA34-GA36 via ultrasound in both pregnancies, and measured at birth and 3, 12 and 18 months after birth of both offsprings | 
| Fetal and offspring growth - crown circumference | Crown circumference will be measured with ultrasound during pregnancy | Collected from medical record during pregnancy, measured in GA34-GA36 via ultrasound in both pregnancies | 
| Fetal and offspring growth - femur length | Femur length will be measured with ultrasound during pregnancy | Collected from medical record during pregnancy, measured in GA34-GA36 via ultrasound in both pregnancies, | 
| Fetal growth - fractional limb volume | Fractional limb volume will be measured with ultrasound during pregnancy | Collected from medical record during pregnancy, measured in GA34-GA36 via ultrasound in both pregnancies, | 
| Offspring growth - BMI z-score | Offspring body mass index (BMI) z-score (BMI adjusted for age and sex). | Collected at birth and 3, 12 and 18 months after birth of both offsprings | 
| Offspring growth - weight for gestational age | Weight for gestational age. Measured as percent of normal weight related to gestational age. | Collected at birth and 3, 12 and 18 months after birth of both offsprings | 
| Offspring growth - large for gestational age | Large for gestational age. Prevalence of infants being large for gestational age (LGA). | Collected at birth of both offsprings | 
| Offspring growth - small for gestational age | Small for gestational age. Prevalence of offspring being small for gestational age (SGA). | Collected at birth of both offsprings | 
| Offspring growth - apgar score | Apgar score (0-10). The higher the score, the better health condition of the offspring) | Collected at birth of offspring of both offsprings | 
| Offspring growth | Offspring circumference (thigh and upper arm) and skinfold (triceps, subscapularis and suprailiac). Circumferences will be measured to the nearest 0.1 cm using a stretch-resistant measuring tape and skinfolds are measured to the nearest 0.02 cm using a caliper to lightly pinch the skin and the underlying fat. | Collected at birth and 3, 12 and 18 months after birth of offspring. | 
| Offspring growth - body composition by the POD system | Offspring fat free mass (g) and fat mass (g) will be assessed by air displacement plethysmography using the POD system (Pea POD for 1-8kg) | Collected within the first week of birth and 3 months post partum in both offsprings | 
| Offspring blood pressure | Offspring blood pressure (diastolic and systolic blood pressure) will be measured three times after 10 minutes rest using an automated device placed on the arm and in direct contact with the skin. | Collected 12 and 18 months after birth of both offsprings | 
| Offspring heart rate | Offspring heart rate will be measured three times after 10 minutes rest using an automated device placed on the arm and in direct contact with the skin. | Collected 12 and 18 months after birth of both offsprings | 
| Offspring growth - self-reported body weight | Self-reported body weight | 18 months after birth the study staff will make an annual follow-up phone call to the parents to collect information about the offspring's self-reported body weight until the offspring is 18 of age | 
| Offspring growth - self-reported height | Self-reported height | 18 months after birth the study staff will make an annual follow-up phone call to the parents to collect information about the offspring's self-reported body weight until the offspring is 18 of age | 
| Adult weight | Adult body weight is measured to the nearest 0.1 kg using a calibrated scale | Intervention: each visit throughout study (GA34-36 of 1st child to 18mo. after birth of 2nd child). Control: In GA34-36 in both pregnancies, at study visits (3, 12 and 24mo. pp) and follow up visits (3, 12 and 18 months pp of 2nd child) | 
| Adult Height | Adult height will be measured to the nearest 0.5 cm using a wall-mounted stadiometer. | Collected in GA34-GA36 in index pregnancy | 
| Adult BMI | Calculated as weight (kg)/height(m)^2 | Collected in GA34-36 in 1st pregnancy, study visits 3, 12 and 24mo. pp after 1st child, GA34-36 in 2nd pregnancy and at follow up vist (3, 12 and 18mo. pp of 2nd child). | 
| Adult anthropometric | Waist- and hip circumference will be measured to the nearest 0.1 cm. During pregnancy and at birth the waist of the female will not be measured. | Intervention: each visit throughout study (GA34-36 of 1st child to 18mo. after birth of 2nd child). Control: In GA34-36 in both pregnancies, at study visits (3, 12 and 24mo. pp) and follow up visits (3, 12 and 18 months pp of 2nd child) | 
| Adult blood pressure | Blood pressure (diastolic and systolic blood pressure) will be measured three times with two minutes apart in right arm after 15 minutes rest using a validated automatic device. The reading is recorded to the nearest 1 mmHg. | Collected at GA34-36 in 1st pregnancy, study visits 3, 12 and 24 months pp. of 1st child, GA34-36 in 2nd pregnancy and 3 months pp after 2nd child | 
| Adult heart rate | Heart rate will be measured three times with two minutes apart in right arm after 5-10 minutes rest using a validated automatic device. The reading is recorded to the nearest 1 beats/minute for heart rate | Collected at GA34-36 in 1st pregnancy, study visits 3, 12 and 24 months pp. of 1st child, GA34-36 in 2nd pregnancy and 3 months pp after 2nd child | 
| Adult resting metabolic rate | Resting metabolic rate (calories/24-h) will be measured by indirect calorimetry using a ventilated hood system. Participants will lie in the supine position and rest for at least 30 min before measurements commence. The measurement will last for 20 min. | Collected at study visit 3, 12, and 24 months pp after 1st child. | 
| Adult resting respiratory quotient + substrate | Resting respiratory quotient (RQ), will be measured by indirect calorimetry using a ventilated hood system. Participants will lie in the supine position and rest for at least 30 min before measurements commence. The measurement will last for 20 min. Lipid, glucose and protein oxidation will be estimated. | Collected at study visit 3, 12, and 24 months pp after 1st child. | 
| Adult medication use | Use of medication will be collected throughout the study and for each medication the start date, stop date or continuing, form and indication will be noted in appropriate form. | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st. child | 
| Offspring medication use | Use of medication will be collected throughout the study and for each medication the start date, stop date or continuing, form and indication will be noted in appropriate form. | Birth, after 3, 12, and 18 months after birth of both offsprings | 
| Adult reproduction - Time to pregnancy | Duration of time to conception defined from selfreported time point they started to try get pregnant to registration of pregnancy. The time of conception will be calculated from the ultrasound scan in week 12 | Registration of 2nd. pregnancy (up to tree years from birth of 1.st. child) | 
| Adult glucose metabolism (OGTT) | 3-h oral glucose tolerance test (OGTT) will be performed at with 75g of glucose dissolved in 250 ml of water and added 1 gram of paracetamol. Blood will collected for measurements of blood glucose, hormones (insulin, C-Peptide, glucagon), paracetamol, glucagon-like-peptide-1 (GLP-1), Glucose insulinotropic polypeptide (GIP) and metabolites (FFA). Blood samples will be drawn at time 0, 15, 30, 45, 60, 90, 120 and 180 min | Performed at study visits 3, 12 and 24 months pp of 1st. child | 
| Adult Oral Glucose Tolerance Test (OGTT) during pregnancy (only female) | OGTT performed during pregnancy according to the screening guidelines for GDM will be obtained from the mother's medical journal in both pregnancies. | Collected in GA28 of 1st and 2nd pregnancy (performed after risk score at gestational age 20 and  28 or only  28) | 
| Placenta - weight (gram) | Placenta will be weighted after birth | Collected at birth of both offsprings | 
| VO2-max adult - cardiorespiratory fitness | The VO2-max test will be performed as an incremental exercise test on a bicycle ergometer. | Collected at study visit 3, 12 and 24 months pp after birth of 1st child | 
| Cardiac function offspring - Left ventricular and right ventricular morphology | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of Interventricular septal thickness diastole and valve- and chamber dimensions will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - left centricular ejection fraction | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of left ventricular ejection fraction will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - fractional shortening | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of fractional shortening will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Systolic function, strain | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of strain will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Systolic function, strain rate | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of strain rate will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - TAPSE and RV fractional area change | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of TAPSE and RV fractional area change will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Diastolic function, left ventricular E/A-ratio | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of left ventricular E/A-ratio will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Diastolic function, tissue velocities (E', A', S') | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of tissue velocities (E', A', S') will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Diastolic function, strain | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of strain will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Overall cardiac function | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of Myocardial performance index (MPI) and cardiac torsion will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Pulmonary artery acceleration time | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of Pulmonary artery acceleration time will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Pulmonary artery pressure | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of Pulmonary artery pressure will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cardiac function offspring - Persistent ductus arteriosus (PDA) | ECG and echocardiography will be performed according to standard imaging protocol. Assessment of PDA open or closed and PDA shunt will be done. | Collected at birth and at 12 months follow up visit after birth of each child | 
| Cord blood - Telomore length | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for Telomore length. | Collected at birth of both offsprings | 
| Cord blood - pH | Cord blood will be drawn immediately after birth. pH will be assessed. | Collected at birth | 
| Cord blood - Epigenetics | Epigenetics analyses from whole blood. Will be analysed for methylation changes. metabolomics and exosome. | Collected at birth of both offsprings | 
| Adult body composition by DEXA scan | Lean mass (g), fat mass (g), abdominal fat (g), visceral fat (g) assessed by a Dual-energy X-ray absorptiometry (DEXA) -scan . | Collected at study visits 3, 12 and 24 months pp. of 1st child | 
| Adult bone markers by DEXA scan - bone mineral content | Bone mineral content (g) assessed by a Dual-energy X-ray absorptiometry-scan. | Collected at study visits 3, 12 and 24 months pp. of 1st child | 
| Adult bone markers by DEXA scan - bone area | Bone area (cm^2) assessed by a Dual-energy X-ray absorptiometry-scan. | Collected at study visits 3, 12 and 24 months pp. of 1st child | 
| Adult bone markers by DEXA scan - bone mineral density | Bone mineral density (g/cm^2) assessed by a Dual-energy X-ray absorptiometry-scan. | Collected at study visits 3, 12 and 24 months pp. of 1st child | 
| Adult blood sample - glucose | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for glucose | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - CD163 | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for CD163 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - HbA1c | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for markers of glucose metabolism HbA1c | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Kreatinin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for kreatinin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - eGFR | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for kreatinin and GFR will be estimated | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - C-reaktivt protein (CRP) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for CRP | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Alanintransaminase (ALAT) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for ALAT | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Gamma-GT (GGT) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for GGT | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Basisk fosfatase (BASP) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for BASP | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Bilirubin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Bilirubin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Ferritin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Ferritin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Hemoglobin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Hemoglobin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Leukocytter | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Leucocytter | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - platelets | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for platelets | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Triglyceride | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for triglyceride | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Cholesterol HDL | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Cholesterol HDL | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Cholesterol LDL | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Cholesterol LDL | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Total Cholesterol | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Total Cholesterol | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - vitamin B12 | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for B12 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - vitamin D3 | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for vitamin D3 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - c-peptide | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for c-peptide | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Insulin-like Growth Factor-1 (IGF-1) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IGF-1 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Insulin-like growth factor-binding protein-1 (IGFBP-1) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IGFBP-1 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Insulin-like growth factor-binding protein-3 (IGFBP-3) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IGFBP-3 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Fibroblast growth factor 21 (FGF-21) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for FGF-21 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Leptin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Leptin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Adiponectin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Adiponectin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Ghrelin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Ghrelin | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Growth Hormone (GH) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for GH | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Free fatty Acids (FFA) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for FFA | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Interleukin 6 (IL-6) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IL-6 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Interleukin 6 (IL-10) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IL-10 | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Interleukin-1 alpha (IL-1 alpha) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IL-1 alpha | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Interferon gamma (IFN-γ) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for IFN-γ | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult blood sample - Tumor Necrosis Factor alfa (TNF-α) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for TNF-α | Collected at GA34-GA36 in 1st pregnancy, study visit 3, 12 and 24 months pp of 1st child, at GA34 in 2nd pregnancy and at follow visit 3 months pp after 2nd child | 
| Adult female blood sample - Humant choriogonadotropin (hCG) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will only be collected from the female participant during pregnancy and be analysed for hCG | Collected at GA34-GA36 in both pregnancies | 
| Adult female blood sample - Progesterone | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will only be collected from the female participant during pregnancy and be analysed for Progesterone | Collected at GA34-GA36 in both pregnancies | 
| Adult female blood sample - Proinsulin C-peptide | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will only be collected from the female participant during pregnancy and be analysed for Proinsulin C-peptide | Collected at GA34-GA36 in both pregnancies | 
| Adult female blood sample - sex hormone binding globulin (SHBG) | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will only be collected from the female participant during pregnancy and be analysed for SHBG | Collected at GA34-GA36 in both pregnancies | 
| Adult female blood sample - Prolactin | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will only be collected from the female participant during pregnancy and be analysed for Prolactin | Collected at GA34-GA36 in both pregnancies | 
| Adult blood sample -Cortisol | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will be analysed for Cortisol | Collected at GA34-GA36 in both pregnancies | 
| Adult female blood sample -Estradiol | All blood samples will be collected by venous puncture in the antecubital vein after an overnight fast from at least 10 pm the night before. Samples will only be collected from the female participant during pregnancy and be analysed for Estradiol | Collected at GA34-GA36 in both pregnancies | 
| Offspring blood sample - glucose | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for glucose | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - c-peptide | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for c-peptide | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - insulin | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Insulin | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - IGF-1 | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for IGF-1 | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - IGFBP-3 | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for IGFBP-3 | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - IGFBP-1 | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for IGFBP-1 | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - CD163 | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for CD163 | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Ferritin | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Ferritin | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - HgbA1c | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for HgbA1c | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Hgb | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Hgb | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - CRP | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for CRP | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Triglyceride | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Triglyceride | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Cholesterol HDL | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Cholesterol HDL | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Cholesterol LDL | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Cholesterol LDL | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Total Cholesterol | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Total Cholesterol | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - FGF-21 | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for FGF-21 | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Leptin | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Leptin | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Adiponectin | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for Adiponectin | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - FFA | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for FFA | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - Growth Hormone (GH) | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for GH | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - cTroponin I (cTnI) | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for cTnI | Collected 12 months after birth in both offsprings | 
| Offspring blood sample - BNP | A non-fasting venous blood will be drawn. Parents will be instructed that the offspring should not have eaten two hours prior to the assessment. Samples will be analysed for BNP | Collected 12 months after birth in both offsprings | 
| Cord blood - c-peptide | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for C-peptide | Collected at birth of both offspring | 
| Cord blood -Insulin | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for Insulin | Collected at birth of both offspring | 
| Cord blood -IGFBP-3 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IGFBP-3 | Collected at birth of both offspring | 
| Cord blood -IGFBP-1 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IGFBP-1 | Collected at birth of both offspring | 
| Cord blood -IGF-1 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IGF-1 | Collected at birth of both offspring | 
| Cord blood -Ferritin | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for Ferritin | Collected at birth of both offspring | 
| Cord blood -cTnI | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for cTnI | Collected at birth of both offspring | 
| Cord blood -BNP | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for BNP | Collected at birth of both offspring | 
| Cord blood -CRP | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for CRP | Collected at birth of both offspring | 
| Cord blood -CD163 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for CD163 | Collected at birth of both offspring | 
| Cord blood -FGF21 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for FGF21 | Collected at birth of both offspring | 
| Cord blood -Leptin | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for Leptin | Collected at birth of both offspring | 
| Cord blood -Adiponectin | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for Adiponectin | Collected at birth of both offspring | 
| Cord blood -FFA | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for FFA | Collected at birth of both offspring | 
| Cord blood - Vitamin D3 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for Vitamin D3 | Collected at birth of both offspring | 
| Cord blood - HbA1c | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for HbA1c | Collected at birth of both offspring | 
| Cord blood - IL-6 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IL-6 | Collected at birth of both offspring | 
| Cord blood - IL-10 | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IL-10 | Collected at birth of both offspring | 
| Cord blood - IL-1 alpha | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IL-1 alpha | Collected at birth of both offspring | 
| Cord blood -TNF- alpha | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for-TNF- alpha | Collected at birth of both offspring | 
| Cord blood -IFN- gamma | Cord blood will be drawn immediately after birth and analysed. Blood will be analyzed for IFN- gamma | Collected at birth of both offspring | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Overweight, Pregnancy, Weight loss, Diet, Reducing, Epigenetics, Healthy Lifestyle, DNA Methylation, Exercise, Offspring's neonatal fat mass, Pea Pod, Infant, Large for Gestational Age, Body Fat Distribution