Maternal antecedents of adiposity and studying the transgenerational role of hyperglycemia and insulin (MAASTHI): a prospective cohort study

The aim of this study is to prospectively assess the effects of glucose levels in pregnancy on the risk of adverse infant outcomes, especially in predicting the possible risk markers of later chronic diseases (Fig. 1). The primary objective of the proposed study is to investigate the effect of glucose levels in pregnancy on skinfold thickness (adiposity) in infancy as a marker of future obesity and diabetes in offspring. Because psychosocial environment could itself be an important independent predictor of adiposity and could confound its relationship with maternal hyperglycaemia, our secondary objective is to assess the association between psychosocial environment of mothers and adverse neonatal outcomes including adiposity. The specific exposure, outcome and potential confounders for these objectives are listed in Table 1.

For hypotheses 1, the exposure of interest is maternal blood glucose level. We are not examining gestational diabetes only, but are interested in exploring the effect of the continuous range of glucose as exposure from near normal ranges to hyperglycemia.

After the fasting blood sample is drawn, the pregnant woman will receive 75 g oral glucose load and a venous blood sample will be collected 2 hours later for estimating plasma glucose [40‐42]. Evaluation of the samples will be done through the central laboratory so as to minimize the confounding effects of analytic variation. The primary outcome of our study is skinfold thickness in infants as a measure of adiposity. Skinfold thickness is the most accurate method of measuring body fat rather than Body Mass Index (BMI) as this will provide a better indication of individual fat and risk of obesity. Skinfold thickness measurement is a traditional and reliable measure of fat percentage. From research it’s found that greater weight gain during pregnancy was associated with higher child’s body mass index, sum of triceps and subscapular skinfold thickness [43, 44]. The triceps, biceps and subscapular skinfolds will be measured as described by Tanner/Whitehouse using a Holtain Calipers (Holtain, U.K.). Triceps skin folds will be measured over the posterior belly of triceps muscle of the left arm, halfway between the acromion and the olecranon, on a line passing upwards from the olecranon in the axis of the limb, with the arm extended. Subscapular skinfold will be measured immediately below the angle of the left scapula, in the natural cleavage line of the skin, with the arm held by the side of the body. In addition, we will also assess weight for length and waist girth (in centimeters) as co-primary outcomes. Waist girth is measured by placing the tape around the abdomen immediately above the umbilicus ensuring that it is horizontal and marked at the end of expiration. The ultrasound scan records of the respondents are reviewed and the estimated foetal weight, biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL) is recorded. Mother’s weight and skinfold thickness are measured during antenatal period. Based on the literature, we have identified the following potential confounders based on a priori knowledge: age, BMI, family history of diabetes, gestational age, parity, past medical history, family history of hypertension and socio-economic status.

For hypotheses 2, the exposure of interest is social support, measured by means of a validated tool (Questionnaire) administered during second trimester of pregnancy. Social support to the mother will be measured using a questionnaire developed at St. John’s Research Institute to evaluate a broad range of social support (i.e., emotional, instrumental, informational, and appraisal) [45]. The Edinburgh Postnatal Depression Scale is administered to the respondents during pregnancy, after delivery and when the infant is 14 week old. Those who score greater than 13 in the EPDS scale are administered Global Mental Health Assessment Tool (GMHAT) for further diagnosis and are referred to the psychiatrist at the hospital for counselling and treatment. The outcome of interest is infant adiposity. The potential confounders include maternal age, parity, BMI, weight gain during pregnancy on fetal biometry measures, [46] diet, gestational age, lifestyle factors, and alcohol and tobacco use [46, 47]. The mother’s 24 hour diet recall is recorded during pregnancy. The infant’s feeding mode whether it is breastfed, time of first breast feed and exclusive breastfeeding at birth, duration of breastfeeding and complementary feeding practices for every child is recorded annually. In addition, we may include additional potential confounders in the models based on emerging literature.

Assuming an incidence of 5 % obesity in children born to mothers with euglycemia, [48, 49] and a relative risk of 1.5 in the hyperglycaemic group, our estimated sample size for 80 % power to detect a difference at a 95 % confidence level, is 2936. Further assuming a loss to follow-up of up to 60 %, we plan to recruit 5000 women. The assumed loss to follow up is comparable to another birth cohort followed up at a similar age in India (Mysore Parthenon birth cohort); however unlike cohorts recruited from the community, ours is a hospital based study with outcome assessment at routine immunisation visits during infancy which are generally well attended, we expect the eventual loss to follow up to be much lower in our study [50]. We have obtained approval to conduct the study at Jayanagar General Hospital, a 300 bedded hospital. In addition, the city municipal corporation (Brihat Bengaluru Mahanagara Palike : BBMP) governing the greater Bangalore metropolitan area has provided approval for public hospitals in the city to participate in the study. The study is initiated at Jayanagar General Hospital; the hospital registers around 200–400 new cases of pregnant women each month.

All pregnant women between the age of 18–45 years who are less than 32 weeks of gestation and a) plan to deliver in the study hospital and b) reside in the study area will be eligible for recruitment. The recruitment will be done after obtaining an informed consent and noting relevant socio-demographic details. The exclusion criteria includes history of diabetes, Hepatitis B infection, HIV positivity and inability to complete the oral glucose-tolerance test within 32 weeks of gestation.

The process of recruitment, follow-up and corresponding timelines in the cohort have been captured in the Fig. 3. Pregnant women with a gestational period of less than 32 weeks will be recruited. A baseline questionnaire will be administered that includes socio-economic status, Standard of Living Index, 24-hour dietary recall, dietary habits, physical activity, obstetrics history, psychosocial stressors and social support. Weight, height, sitting height, skinfold thickness of biceps, triceps and subscapular skinfold will be recorded (Table 2). Blood pressure will be measured using an automated BP apparatus. All women will undergo an OGTT at 24 to 32 weeks of gestation. They will be asked to fast for a minimum 8 h prior to the study visit, where fasting samples will be drawn. Subsequently, 75 g of glucose will be given and the postprandial sample will be drawn after two hours. In a sub-sample of pregnant women who have normal ranges of glucose in the second trimester, another OGTT at 33 to 38 weeks will be performed to explore the proportion of women who develop gestational diabetes in the later phase of the third trimester. In a sub-sample, HbA1c test will also be conducted based on age, BMI, family history of diabetes and parity. Maternal subcutaneous fat is a significant predictor of adverse pregnancy outcomes. The visceral and subcutaneous fat thickness around abdomen through ultrasound scan will be obtained. A trained sonographer will be performing the scan and strict measures will be taken to avoid gender determination. Visceral fat builds up between and around internal organs such as the stomach and intestines, and produces toxins that make the body resistant to insulin. Subcutaneous fat is found just beneath the skin, and total fat is the combination of visceral and subcutaneous fat. Depending on presence in hospital visits, the husband’s anthropometry and blood pressure will also be recorded.

The measurements on skinfold thickness and weight for length in infants will be taken at birth and at 14 weeks corresponding to the visit of the mother and child for the purpose of immunization. These measurements will be repeated once in every year for the remaining duration of the study. Follow-up at 1, 2, 3 and 4 years will be on the child’s birthday for all children. At the end of the 4 years of follow up, the glucose and insulin levels in children and mother will be tested (Fig. 3).

For questionnaires and anthropometric measurements, field team will be trained using strict protocols and the accuracy and interobserver and intraobserver reliability of their measurements assessed at the outset and every 6 months. Anthropometric equipment will be calibrated monthly and recordings logged. Biochemical assays carried out by a single laboratory will be quality assured by India’s National Accreditation Board for Testing and Calibration Laboratories (NABL). Quality control samples will be collected for the internal and external quality checks.

Structured questionnaires will be administered by trained interviewers for baseline assessment and follow-up. Data will be entered directly using tablet devices provided to the field staff with validation checks built in to minimise data entry errors. Trained phlebotomists will collect blood samples. Participants not attending follow-ups will be reminded through linkages with frontline health workers in the community.

We will collect 11 ml fasting venous blood sample and 2 ml postprandial sample 2- h after following a 75 g oral load of glucose for the laboratory investigations. The fasting sample will be collected in plain, EDTA and sodium fluoride vacutainers for storage, haemoglobin and glucose assays respectively. Blood samples will be centrifuged and transferred within an hour in cool boxes to a single central laboratory for assays with external quality assurance mechanisms in place. Relevant study assays will be carried out at each time point (Table 3) and remaining sample stored in aliquots for future analysis.