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

Overweight and Obesity Clinical Trial

— PREPARE CHILD  

Official title:

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

Clinical Trial Details — Status: Recruiting

Administrative data

• NCT number: NCT05798494
• Other study ID #: Prepare Child AUH
• Status: Recruiting
• Phase: N/A
• Start date: June 1, 2023
• Est. completion date: April 11, 2028

Study information

• Verified date: January 2024
• Source: University of Aarhus
• Contact: Ulla K Opstrup, MD, PhD
• Phone: +4522370857
• Email: ullaopst[@]rm.dk
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

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 27-44.9 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.

Description:

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 18-36), overweight or obese (BMI 27-44.9 kg/m^2), between 18-38 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.

Recruitment information / eligibility

• Status: Recruiting
• Enrollment: 140
• Est. completion date: April 11, 2028
• Est. primary completion date: April 11, 2025
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years to 38 Years

Eligibility

Maternal inclusion Criteria:

• Maternal pre-pregnancy BMI 27.0-44.9 kg/m^2

• Maternal age range 18-38 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

Related Conditions & MeSH terms

• Body Composition
• Body Weight
• Child Nutrition Sciences
• Overweight
• Overweight and Obesity
• Pediatric Obesity
• Pregnancy Related
• Weight Loss

Intervention

Behavioral:
• Weight loss intervention
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.

Locations

Country	Name	City	State
Denmark	Aarhus University Hospital	Aarhus

Sponsors (4)

Lead Sponsor	Collaborator
Ulla Kampmann Opstrup	Jens Meldgaard Bruun, Professor Steno Diabetes Center Aarhus, Aarhus University Hospital, Per Ovesen, Professor Dept. of Obsteterics, Aarhus University Hospital, Sine Knorr, PhD Steno Diabetes Center Aarhus, Aarhus University Hospital

Country where clinical trial is conducted

Denmark, 

References & Publications (1)

Rasmussen L, Knorr S, Antoniussen CS, Bruun JM, Ovesen PG, Fuglsang J, Kampmann U. The Impact of Lifestyle, Diet and Physical Activity on Epigenetic Changes in the Offspring-A Systematic Review. Nutrients. 2021 Aug 17;13(8):2821. doi: 10.3390/nu13082821. — View Citation

Outcome

Type	Measure	Description	Time frame	Safety issue
Other	Offspring feces - Microbiome	Will be analysed for microbiome. Collected at birth by researcher and home by parents preferable <24 hours before the planned visit.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - Short chain fatty acids	Will be analysed for short chain fatty acids. Collected at birth by researcher and home by parents preferable <24 hours before the planned visit.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - Metabolomics	Will be analysed for metabolomics. Collected at birth by researcher and home by parents preferable <24 hours before the planned visit.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - Proteomics	Will be analysed for proteomics. Collected at birth by researcher and home by parents preferable <24 hours before the planned visit.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - pH	Will be assessed for pH. Collected at birth by researcher and home by parents preferable <24 hours before the planned visit.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - Inflammation markers	Will be analysed for inflammation markers. Collected at birth by researcher and home by parents preferable <24 hours before the planned visit.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - consistency	Whenever a fecal sample is collected stool consistency of the collected sample will be collected with a questionnaire.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - frequency	Whenever a fecal sample is collected an estimate of stool frequency during the last week will be collected with a questionnaire.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring feces - gastrointestinal symptoms	Whenever a fecal sample is collected any gastrointestinal symptoms of the offspring during the last week will be collected with a questionnaire.	Collected within the first week after birth (but should avoid to collect the first fecal excretion after birth (meconium)), at study visit 3 and 12 months after birth of each offspring
Other	Offspring urine	Parents will be instructed to collect offspring's urine samples. A piece of bio-plastic will be placed in the diaper together with cotton balls to ensure that a sufficient amount of urine is absorbed into the cotton balls. Offspring urine will be analyzed for metabolomics.	Collected within the first week of birth and at 3 and 12 months follow up of each offspring
Other	Adult feces - Microbiome	Participants will collect a fecal spot sample at home maximum 24 hours before the planned visit.; Will be analysed for microbiome.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - Short chain fatty acids	Participants will collect a fecal spot sample at home maximum 24 hours before the planned visit. Will be analysed for short chain fatty acids.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - Metabolomics	Participants will collect a fecal spot sample at home maximum 24 hours before the planned visit.; Will be analysed for metabolomics	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - Proteomics	Participants will collect a fecal spot sample at home maximum 24 hours before the planned visit.; Will be analysed for proteomics.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - Inflammation markers	Participants will collect a fecal spot sample at home maximum 24 hours before the planned visit.; Will be analysed for inflammation markers.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - pH	Participants will collect a fecal spot sample at home maximum 24 hours before the planned visit.; Will be assessed for pH.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - consistency	Whenever a fecal sample is collected stool consistency of the collected sample will be collected with a questionnaire.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - frequency	Whenever a fecal sample is collected an estimate of stool frequency during the last week will be collected with a questionnaire.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult feces - gastrointestinal symptoms	Whenever a fecal sample is collected gastrointestinal symptoms during the last week will be collected with a questionnaire.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Adult urine	Participants will collect a urine spot sample at the study site. Adult urine will be analyzed for metabolomics.	Collected at GA34-GA36 in each pregnancy and at study visit 3, 12 and 24 months pp of 1st child
Other	Offspring nutritional milestones	Parents will be instructed to register the age of the offspring when it reaches the World Health Organization (WHO) nutritional milestones (age when the offspring was introduced to specific food items)	Collected at 12 and 18 months after offspring birth
Other	Offspring development, WHO milestones	Parents will be instructed to register the age of the offspring when it reaches the World Health Organization (WHO) developmental milestones (age when the offspring can sit, stands in crawl-position, stands with support, stand alone, walk with support, walk alone)	Collected at 12 and 18 months after offspring birth
Other	Adult physical activity	Physical activity and sleep will be assessed by an accelerometer and the females and males will wear the accelerometer for 7 days and 8 nights. The participants will be asked to keep a physical activity and sleep log for when they wake up and when they try to sleep	Collected at study visit 3, 12 and 24 months pp of 1st child
Other	Adult physical activity - Physical Activity Questionnaire (IPAQ)	Physical activity will be assessed by the International Physical Activity Questionnaire (IPAQ), in which time spent on strenuous and moderate activity and walks will be collected. The answers will be collected electronic via RedCap.	Collected at GA34-GA36 in each pregnancy, study visit at 3, 12 and 24 months pp of 1st child and 3 months pp visit after 2nd child
Other	Adult background quistionnaire, ethnicity,	All information will be collected electronic in RedCap.	Collected after screening in GA34-GA36 of 1st child
Other	Adult background quistionnaire, education	All information will be collected electronic in RedCap.	Collected after screening in GA34-GA36 of 1st child
Other	Adult background quistionnaire, parity	All information will be collected electronic in RedCap.	Collected after screening in GA34-GA36 of 1st child
Other	Adult background quistionnaire, pre-pregnancy weight	Pre-pregnancy weight, ethnicity, education and parity. All information will be collected electronic in RedCap.	Collected after screening in GA34-GA36 of 1st child
Other	Adult background quistionnaire, demographic information	All information will be collected electronic in RedCap.	Collected after screening in GA34-GA36 of 1st child
Other	Adult wellbeing - Depression, Major depression questionnaire (ICD-10)	Depression will be assessed by Major depression questionnaire (ICD-10). Mild depression: A score of 4 or 5 in two of the first three items. Plus a score of at least 3 on two or three of the last seven items.; Moderate depression: A score of 4 or 5 in two or three of the first three items. Plus a score of at least 3 on four of the last seven items.; Severe depression: A score of 4 or 5 in all of the first three items. Plus a score of at least 3 on five or more of the last seven items.; Major depression: The number of items is reduced to nine, as Item 4 is part of Item 5. Include whichever of the two items has the highest score (item 4 or 5).; A score on at least five items is required, to be scored as follows: the score on the first three items must be at least 4, and on the other items at least 3. Either Item 1 or 2 must have a score of 4 or 5.	Collected at GA34-GA36 in each pregnancy, study visit at 3, 12 and 24 months pp of 1st child and 3 months pp visit after 2nd child
Other	Adult wellbeing - Quality of life, Short form-36 (SF-36)	Quality of life will be assessed by the validated tool, Short form-36 (SF-36). The lower the score the more disability. The higher the score the less disability i.e., a score of zero is equivalent to maximum disability and a score of 100 is equivalent to no disability.	Collected at GA34-GA36 in each pregnancy, study visit at 3, 12 and 24 months pp of 1st child and 3 months pp visit after 2nd child
Other	Adult wellbeing - Stress, Perceived Stress Scale	Stress will be assessed by validated tool, Perceived Stress Scale.Individual scores on the PSS can range from 0 to 40 with higher scores indicating higher perceived stress. Scores ranging from 0-13 would be considered low stress. Scores ranging from 14-26 would be considered moderate stress. Scores ranging from 27-40 would be considered high perceived stress	Collected at GA34-GA36 in each pregnancy, study visit at 3, 12 and 24 months pp of 1st child and 3 months pp visit after 2nd child
Other	Adult sleep	Sleep will be assessed by the validated tool, Pittsburgh sleep quality index questionnaire (PSQI). In scoring the PSQI, seven component scores are derived, each scored 0 (no difficulty) to 3 (severe difficulty). The component scores are summed to produce a global score (range 0 to 21). Higher scores indicate worse sleep quality.	Collected at GA34-GA36 in each pregnancy, study visit at 3, 12 and 24 months pp of 1st child and 3 months pp visit after 2nd child
Other	Adult dietary intake, 24-h recall	A 24 hour dietary recall is performed by a trained dietitian. Dietary recall will be registered and intake calculated using the food data program 'VITAKOST' (www.vitakost.dk).	Collected at GA34-GA36 in each pregnancy, at visit 3, 12 and 24 months pp of the 1st child and 3 months visit after 2nd child. Further collected at 6, 18, 30 and 36 months pp and in GA12 and GA20 of 2nd pregnancy in intervention group
Other	Adult dietary intake, FFQ	Couples will be asked to perform an electronic Food Frequency Questionnaire (FFQ). Information on frequence intake of various food groups during the last month will be collected.	Collected at GA34-GA36 in each pregnancy, at study visit 3, 12 and 24 months pp of the 1st child and 3 month follow up vist after 2nd child
Other	Offspring dietary intake	We will collect information on offspring dietary intake including breast feeding, use of formula and type as well as use of antibiotics. The intake frequency of different foods will be collected throughout a children questionnaire.	Collected within the first week of birth and 3, 12 and 18 months after birth off each offspring
Other	Breast milk - nutrients biomarkers	Participants will collect breast milk within the first week following birth, at 1 and 3 months pp after each birth. Participants will receive written instructions in milk collection at home. First drops will not be collected, hereafter manually expressed milk will be obtained in a sterile container. Obtained around midday and minimum two hours after last breastfeeding. Breast milk will be analyzed for nutrients biomarkers.	Collected within the first week of birth and 3 months pp after each birth
Other	Breast milk - Hormonal and inflammatory markers	Participants will collect breast milk within the first week following birth, at 1 and 3 months pp after each birth. Participants will receive written instructions in milk collection at home. First drops will not be collected, hereafter manually expressed milk will be obtained in a sterile container. Obtained around midday and minimum two hours after last breastfeeding. Breast milk will be analyzed for hormonal and inflammatory markers	Collected within the first week of birth and 3 months pp after each birth
Other	Breast milk - Metabolomics	Participants will collect breast milk within the first week following birth, at 1 and 3 months pp after each birth. Participants will receive written instructions in milk collection at home. First drops will not be collected, hereafter manually expressed milk will be obtained in a sterile container. Obtained around midday and minimum two hours after last breastfeeding. Breast milk will be analyzed for metabolomics	Collected within the first week of birth and 3 months pp after each birth
Other	Breast milk - Proteomics	Participants will collect breast milk within the first week following birth, at 1 and 3 months pp after each birth. Participants will receive written instructions in milk collection at home. First drops will not be collected, hereafter manually expressed milk will be obtained in a sterile container. Obtained around midday and minimum two hours after last breastfeeding. Breast milk will be analyzed for proteomics.	Collected within the first week of birth and 3 months pp after each birth
Other	Breast milk - Short chain fatty acids and related metabolites	Participants will collect breast milk within the first week following birth, at 1 and 3 months pp after each birth. Participants will receive written instructions in milk collection at home. First drops will not be collected, hereafter manually expressed milk will be obtained in a sterile container. Obtained around midday and minimum two hours after last breastfeeding. Breast milk will be analyzed for short chain fatty acids and related metabolites	Collected within the first week of birth and 3 months pp after each birth
Other	Adult participation - dietitian guidance	It will be recorded how many times of individual and group dietitian guidance the participant attends	Every 1-2 month from 3 months pp of 1st child to birth of 2nd child
Other	Adult participation - physical session and power walk	It will be recorded how many times the participant participates in physical activity sessions and how much the person have power walked	Every 2nd week from 3 months pp of 1st child to birth of 2nd child
Primary	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)
Primary	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	Obstetrics outcome - length of gestational age	Length of gestational age	Assessed at birth of both offsprings
Secondary	Obstetric - medication use	Medication use during labor	Collected from initiation of labor until completed birth of both offsprings
Secondary	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
Secondary	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
Secondary	Obstetric - Neonatal unit admissions	Neonatal unit admissions	Collected at birth and until 12 months after birth of both offsprings
Secondary	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
Secondary	Obstetrics - birth defects/abnormalities	Birth defects/abnormalities	Collected at birth of both offsprings
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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,
Secondary	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,
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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.
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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)
Secondary	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
Secondary	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).
Secondary	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)
Secondary	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
Secondary	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
Secondary	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.
Secondary	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.
Secondary	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
Secondary	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
Secondary	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)
Secondary	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
Secondary	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)
Secondary	Placenta - weight (gram)	Placenta will be weighted after birth	Collected at birth of both offsprings
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	Cord blood - pH	Cord blood will be drawn immediately after birth. pH will be assessed.	Collected at birth
Secondary	Cord blood - Epigenetics	Epigenetics analyses from whole blood. Will be analysed for methylation changes. metabolomics and exosome.	Collected at birth of both offsprings
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	Adult blood sample - Interferon gamma (IFN-?)	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-?	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
Secondary	Adult blood sample - Tumor Necrosis Factor alfa (TNF-a)	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-a	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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