We propose to recruit 300 Aboriginal pregnant mothers and their newborns from the Six Nations Reserve and follow them prospectively to the age of 3 years. Approval was received from the McMaster/Hamilton Health Sciences Research Ethics Board (REB) on April 19, 2012 as well as the Six Nations Band Council REB on May 22, 2012.
Data collection
Stage I: Antenatal data
Pregnant mothers are recruited through self referral, or referral to the study from local health care providers (midwives, nurses, primary care physicians, obstetricians). A log of all interested subjects is kept, and the main reasons for exclusion or refusal to participate is recorded. At the baseline visit, between 24–28 weeks of pregnancy, information on age, parity, medical and pregnancy history, cigarette smoking exposure of mother, father and family members, drugs and alcohol exposure, family structure (i.e. marital status, and number of children in the house), community of birth, mother tongue, cultural practices, psychosocial characteristics, as well as socioeconomic factors (i.e. household income, education, and employment) is collected (Table
2). The pregnant mother also has a number of anthropometric measurements taken during this visit. A digital scale is used to record body weight to the nearest 100 g. Height is measured using a stadiometer to the nearest 1 cm and mid upper arm circumference to the nearest 0.1 cm using a plastic measuring tape. Maternal BMI is calculated using weight and height at baseline (kg/m
2). The mother’s pre-pregnancy weight is recorded. Skinfold thickness (triceps and subscapular) is measured to the nearest 0.2 mm, using skinfold calipers (Holtain, UK), for the prediction of body fat using prediction equations [
25]. Systolic and diastolic blood pressure is measured using an automated BP monitor (OMRON Intelli Sense, Model HEM-757). All ultrasound reports are obtained and used to establish gestational age and to assess fetal growth characteristics.
Table 2
Proposed measures and timing of measures in ABC
Demographics -
| X | | | | | |
Age | X | | | X1
| X1
| X1
|
HCN | X | | | X1,3
| X1,3
| X1,3
|
Address/Postal Code | X | | | X1
| X1
| X1
|
Family Doctor - Info | X | | | X1
| X1
| X1
|
Midwife/ObGyn Info | X | | | | | |
Expected Delivery Date | X | | | | | |
Medical History
| X | | | | | |
Diabetes | X | | | | | X1
|
Increased blood pressure | X | | | | | X1
|
Increased cholesterol | X | | | | | X1
|
Other major medical history | X | | | | | X1
|
Family History | X | | | | | |
Medications Used | X | | | | | X1
|
Past Pregnancy Info
| X | | | | | |
GTPAL | X | | | | | |
Still Births | X | | | | | |
Past Gest. DM | X | | | | | |
Pre-Eclampsia | X | | | | | |
Low Birth Weight | X | | | | | |
Premature Birth | X | | | | | |
Social Determinants
| X | | | | | |
Years Living on the Reserve | X | | | | | |
Place of Birth | X | | | | | |
Religious Practices | X | | | | | |
Annual Household Income | X | | | X1
| X1
| X1
|
Occupation | X | | | X1
| X1
| X1
|
Marital Status | X | | | X1
| X1
| X1
|
Education | X | | | | | X |
Social Support | X | | | X1
| X1
| X1
|
Domestic Violence | X | | | X1
| X1
| X1
|
Depression | X | | X1
| X1
| X1
| X1
|
Health Behaviours
| | | | | | |
Cigarette Exposure | X | | | X | X | X |
Diet/Infant feeding | X | | X3
| X3
| X3
| X3
|
Activity/Sedentary Behaviours | X | | X3
| X3
| X3
| X3
|
Physical Exam
| X | | | X | X | X |
Blood Pressure | X | X2
| | X3
| X3
| X1,3
|
Height/Length | X | X2
| X3
| X3
| X3
| X1,3
|
Weight | X | X2
| X3
| X1,3
| X1,3
| X1,3
|
Waist and Hip Circumference | X | X2
| | X3
| X3
| X1,3
|
Skin Folds | X | X2
| | X3
| X3
| X1,3
|
Head Circumference (baby only) | | X2
| X | X | X | X |
Fetal Ultrasound | X | | | | | |
Blood Analysis
| X | | | | | |
Hemoglobin | X | | | | | |
Glucose | | X2
| | X3
| X3
| X3
|
75 g OGTT (0, 60, 120 min) | X | | | | | |
Insulin | X | X2
| | X3
| X3
| X3
|
Adiponectin | X | X | | X | X | X |
Leptin | X | X2
| | X | X | X |
Lipid Profile | X | X2
| | X3
| X3
| X3
|
CBC | | X2
| | X3
| X3
| X3
|
Aliquots for Future Analysis | X | X2
| | X3
| X3
| X3
|
DNA Long-term Storage | X | X2
| | | | |
Birth Visit
| | | | | | |
Type of Delivery | | X | | | | |
Duration of Labour | | X | | | | |
Premature Labour | | X | | | | |
Blood Loss | | X | | | | |
Birth Weight | | X | | | | |
APGAR scores (1 and 5 min) | | X | | | | |
Adverse outcomes | | X | | | | |
Placenta & Cord Blood | | X | | | | |
Stool | | | | X3
| | |
Breastmilk | | X4
| | | | |
Dietary and Physical Activity assessment: We have previously developed and validated a FFQ for Aboriginal people in Canada [
8], which is administered during the second trimester visit, as well as at 6 months and 1 year postpartum visits. Information on maternal activity during pregnancy is collected for activities in 5 domains – occupational, discretionary exercise, household chores, sedentary activities, hobbies and sleep. Maternal sedentary behaviours will include daily screen time (computer, television, video games). Activity and sedentary behaviour are collected at baseline and at each annual visit.
Psychosocial Assessment: SES is assessed by recording annual household income, employment, education and marital status. Information is gathered about chronic stressors in the home, workplace and community and stressful life events. Adequacy of social support to the mother is measured using a questionnaire to evaluate the emotional, instrumental, informational, and appraisal components of social support. Depression in the mother is assessed by the Kessler-10 scale (K-10) which is a 10-item scale with five response categories ranked on a 5-point scale [
26]. Intimate partner violence is assessed using the 2-item Woman Abuse Screening Tool short version [
27]. All psychosocial questionnaires are administered at the baseline visit, at 6 months postpartum and annually thereafter.
Laboratory Assessments: The classification of maternal glycemic status is critical to determine glucose-metabolic status during the second trimester of pregnancy. All non-diabetic mothers will undergo the 75-gram oral glucose tolerance test (OGTT) between 24–28 weeks of gestation. This test is chosen to avoid the high false negative rate using the 50 gram glucose challenge test among some non-white populations [
28]. Three blood samples are collected: fasting, 60, and 120 minutes [
29]. Some local analysis are performed immediately (i.e. glucose, complete blood count) using standardized assays, and the remainder are processed, shipped and stored at the Clinical Trials Clinical Research Laboratory (Hamilton Health Sciences) for future analysis (i.e. lipids, adiponectin, leptin, insulin, the buffy coat for DNA extraction).
Stage 2: Birth
At the time of birth, details including birth outcomes for the mother and baby (e.g. type of delivery, APGAR scores, problems during delivery, length of stay) are collected. A cord blood sample for biochemistry (i.e. glucose, insulin, lipids, adiponectin, leptin), DNA and additional serum and plasma aliquots for future analysis is collected from each baby. Newborn’s physical characteristics including birth weight, skin fold thickness, length, abdominal, head, and arm circumference and blood pressure are measured within 72 hours after birth.
Assessment of Body Composition in Newborn and Infants: In infants, percent body fat can be estimated by a prediction equation derived from four skinfold measures [
30]. This method has been validated against DXA in newborns [
31] and among children aged 4–10 years [
32]. The correlation coefficient of equation-estimated percent body fat in newborns compared to DXA is 0.92 and among children aged 4–10 years, 0.88 [
31]. The reliability of these estimates range from 99.5 to 99.8% [
30‐
32]. In the ABC all newborns and infants will have skinfold thickness measured (biceps, triceps, subscapular, and suprailiac) at birth and at each annual visit.
Stage 3: Follow-up after Delivery:
After delivery, mother and child dyad are further followed by e-mail or telephone at 6 weeks and 6 months to collect information on the infants weight and feeding practices, and in a face to face visit at 1, 2, and 3 years after birth. An annual blood sample will be collected from the infant to measure the complete blood count to screen for iron deficiency anemia, and for analysis of glucose, insulin, and lipids. We offer use of a secured study website for participants to enter the baby’s weight, length, and head circumference monthly recorded at their routine well baby visits.
Assessment of growth and body composition of the infant: Anthropometric measurements of the child are made annually. Infants are weighed to the nearest 10 g on an electronic scale; length is measured on an infantometer. Head, chest and mid upper arm circumference of the baby are measured to the nearest 0.1 cm using a plastic measuring tape. Skinfold measurements are measured to the nearest 0.2 mm, using skinfold calipers (Holtain, UK) for prediction of body composition. All measures are done by trained personnel, and inter-observer reliability testing is conducted. Crown-heel length which is measured using a length board until 18 months of age, and height will be measured using a Harpenden stadiometer after 18 months of age. Weight is measured with an electronic scale.
Breastfeeding, Infant diet, and Activity Assessment: Information on infant feeding practices is collected at 6 weeks and 6 months, and annually by interviewing the mother of the infant/child. Information on initiation of breastfeeding, exclusivity of breastfeeding, duration of breast feeding, and introduction of complementary foods is collected. A validated Infant Feeding Form is used and is a closed ended questionnaire with information about breastfeeding, other feeds and complementary feeds taken during last 7 days [
33]. Among mothers who are breastfeeding, breast milk is collected at 6 weeks postpartum. The samples are collected, frozen and stored for future analysis of macronutrient content and environmental toxins such as persistent organic pollutants [
34]. The goal is to evaluate the association of breast milk content with adiposity and related metabolic phenotypes. Furthermore, an intervention to promote breastfeeding in the community is being pilot tested as a sub-study. The primary objective of the intervention study is to determine if prenatal training in breastfeeding education of a family member or support person improves the rate of any breastfeeding at 6 weeks post-partum. At age one and 3 years, the mother will complete a 24-hour dietary recall for the child. An activity assessment in the growing child at each annual visit will be performed using a 24-hour activity recall developed and validated for use in young children.
Infant microbiome: Stool will be collected from infants at age 1 year. Prior to the annual visit, a stool collection kit will be mailed to families. The kit will contain diaper liners, collection bag and instructions. A diaper liner will be placed in the child’s diaper until stool has been deposited in the liner. The diaper liner with specimen will be placed in the collection bag and refrigerated. The samples will be collected at the annual visit and frozen for future analysis.
Genetic, Methylation, Gene Expression, Placenta Analysis: Mother’s and newborn’s DNA is extracted from the buffy coats and used in future genetic association and methylation studies. RNA from leukocytes are extracted from PaxGene tubes, which are collected from newborns at birth. A 1 cm × 1cm biopsy of the placenta is collected from all consenting mothers and stored in RNAlater to enable future placental gene expression and methylation analysis.
Grandmother’s Interviews: Grandmothers of Aboriginal ancestry are invited to participate in an individual semi-structured qualitative interview, ensuring that their beliefs are captured using a culturally-sensitive lens. The interview questions are designed to elicit grandmother’s beliefs regarding optimal health behaviours for a woman (1) before pregnancy, (2) during pregnancy, (3) the first 6 weeks postpartum, and (4) optimal behaviours for the family with the new baby in the first year of life. Questions probe their beliefs about diet and feeding practices, sleep, activity, smoking, alcohol, social support, mental health, and intimate partner relationships. The qualitative interviews are analyzed using a constant comparison technique to identify emergent themes, concepts and linkages and used to develop a theory. Grandmother’s beliefs will then be compared to existing evidence-based knowledge and the results of the ABC study and used to inform future education initiatives.