Women were assessed at baseline, at delivery, in the immediate in-patient postpartum period and then postpartum at 6 weeks, 3 months and 6 months. Assessments included demographic, biologic including anthropometric, behavioral, cognitive/psychological and sociocultural evaluations. See Table
1 for the assessment schedule. In recognition of participants’ time commitments, women receive gift card incentives following each assessment. Data collection time points for infants are birth, 3-months, 6 months and at 1 year of age. Additionally, infant weight data are obtained from their pediatricians until one year of life.
Table 1
Schedule of study assessments
Review study protocol & informed consent
| ✓ | | | | | | |
Confirmation inclusion/exclusion criteria
|
Demographics
| ✓ | | | | | | |
Biological
| Medical History | ✓ | | | | | | |
Blood Pressure | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Urine for protein and glucose | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Height | ✓ | | | | | | |
Prepregnancy Weight & BMI | ✓ | | | | | | |
Skin Fold Thickness (SFT) | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Waist, Hip and Arm Circumferences | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Genetics (Maternal buccal swab, umbilical cord and blood, placenta, adipose tissue†)‡ | | ✓ | | | | | |
Behavioral
| 24 Hour Dietary and Physical Activity Recalls | ✓ | | | | ✓ | | |
3-Factor Eating Questionnaire R18-modified | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Food Craving Inventory | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Pregnancy and Physical Activity Questionnaire (PPAQ) (modified postpartum) | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Breastfeeding-Infant Feeding Surveys | | | ✓ | ✓ | ✓ | ✓ | ✓ |
Pittsburgh Sleep Quality Index | ✓ | | | ✓ | ✓ | ✓ | |
Cognitive/psychological
| Spielberger Trait Anxiety Inventory | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Perceived Stress Scale | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Quality of Life SF-12 with RAND score | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Happiness Scale | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Edinburgh Postnatal Depression Scale | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Pregnancy & Weight Gain Attitude Scale (PWGAS), modified | ✓ | | | | | | |
Socio-cultural
| Weight gain advice and purposeful weight control attempts survey | ✓ | | | ✓ | ✓ | ✓ | |
Social Support | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Life Events | ✓ | | ✓ | ✓ | ✓ | ✓ | |
Biologic pathways (specimens & imaging)
‡
| Maternal Blood | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |
Umbilical Cord Blood | | ✓ | | | | | |
Placenta | | ✓ | | | | | |
Umbilical Cord | | ✓ | | | | | |
Adipose Tissue† (Subcutaneous & Omental) | | ✓ | | | | | |
Magnetic Resonance Imaging† | | | ✓ | | | | |
Mouthwash Buccal Epithelial Cells for DNA | ✓ | | | | | ✓ | |
Weight outcomes
| Maternal Weight | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | |
Neonatal Weight & Length | | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ |
Biological (Including Medical History, Clinical Assessments, Anthropometric Data and Genetics)
Baseline medical history is collected on the day of enrollment by study personnel through personal and medical record review. Characteristics surveyed include age at menarche, parity, interval from last pregnancy and obstetric history including prior complications like gestational diabetes, hypertensive disorders of pregnancy, prolonged bedrest and others. Medical chart review is performed to confirm or clarify aforementioned information and to retrieve information on medication use, pregnancy dating, and weight-related services such as nutrition consultations.
Estimated delivery date and thus gestational age is determined by chart review, based on 1st trimester ultrasound or clinical dating that agrees with 2nd trimester ultrasound [
49].
Blood pressure is measured three times in the right arm after 15 mins of sitting [
50] and with a 15 minute lapse between each measurement, using an automated Dinamap XL blood pressure monitor [
51].
Urine is evaluated for the presence of
protein or glucose using Siemens multistix 10SG reagent strips for urinalysis.
Height is measured using a 1 Seca 213 Portable Stadiometer.
Prepregnancy weight is self-reported at first prenatal visit and abstracted from prenatal record.
Pre-pregnancy body mass index (BMI) calculated as prepregnancy weight (kg)/height
2 (meters
2). BMI considered continuously and categorically as per World Health Organization criteria: underweight (<18.5 kg/m
2), normal (18.5-24.9 kg/m
2), overweight (25.0-29.9 kg/m
2) and obese (≥30 kg/m
2) [
3]. In accordance with prenatal care standard, weight and gestational age at each visit is recorded from digital clinical scales and was abstracted from records.
Skinfold thickness (SFT) measurements are performed at seven body sites (biceps, triceps, subscapular, iliac, costal, mid thigh, lower thigh), on the subjects’ right side with a calibrated Harpenden skinfold caliper (British Indicators, Sussex, UK), by methods and placements as described by Huston-Presley et al. [
52], to quantify tissue distribution. All skinfolds are assessed three times at each study time point; a mean value of the three is computed. In cases where two measurements differ by more than 1.0 mm, the skin fold is measured a fourth time and the mean value of the four values is averaged.
Body circumferences (upper arm, waist and hip) are obtained recognizing that waist measurements are limited in pregnancy and the immediate postpartum period. Two waist circumferences are measured: (1) midway between the lowest lateral border of the ribs and the top of the iliac crest as per WHO recommendations and (2) at the top of the iliac crest as per NIH standards, in relaxed subjects during expiration [
53]. Hip circumference is measured at the maximum circumference overlying the buttocks. Arm circumference measured midway between the axilla and the elbow. All circumferences measured 3 times and an average calculated.
Biologic specimens are collected over the course of the study with the goal of gaining insight into biologic pathways and epigenetic signatures affected by or contributing to weight outcomes. As the main aims of this study did not focus specifically on biomarkers and biosignature evaluation, but rather the acceptability of serial biospecimens collection, subjects could opt-out of contribution in part or completely.
Maternal venipuncture for blood collection is performed at each study visit for consenting subjects. Additionally, at the time of delivery, umbilical cord blood is collected after delivery of the baby but prior to delivery of the placenta. All plasma samples are aliquoted into 1 ml cryovials and stored at −80°C until assayed with freeze/thaw cycles limited to a maximum of two. All samples are analyzed in duplicate according to manufacturer instructions by a single investigator (NNZ) on the same day to minimize day-to-day variation. Samples are measured using a commercially available MagPix Milliplex® kit (EMD Millipore) with a minimum of 100 positive beads for each assay acquired with Luminex Magpix laser-based fluorescent analytic test instrumentation (Luminex Corporation, Austin, TX). Manufacture supplied controls are used to monitor coefficients of variation (<15% and 20% for intra-assay and inter-assay, respectively). Samples are diluted as per manufacturer instructions. Serum evaluations include adiponectin, c-reactive protein (CRP), interleukin-1b, interleukin-6, interleukin-8, insulin, leptin, PAI-1 total, and TNFα.
After delivery of the placenta, a 1x1cm segment of
placenta and a 1 cm length of
umbilical cord are placed in 4% para-formaldehyde and a second similar sized specimen of each are placed in normal saline. Prior to study completion, a single mouthwash sample of
buccal epithelial cells is collected with Scope brand mouthwash [
54].
For the subset of subjects who have Cesarean delivery for obstetric indications, and who consent, two 1x1 segments of omental (visceral adipose) and subcutaneous adipose tissue are sampled after the uterus is returned to the abdomen, and after the rectus fascia is re-approximated but prior to skin closure, respectively. From each site, one sample is placed in 4% para-formaldehyde and one in normal saline. The size and number of adiopocytes within the samples is determined by light microscopy. Capillary density is measured by counting number of cell profiles stained with endothelial specific markers (e.g. vWF) in 10–20 fields per biopsy specimen.
To quantify adipose deposition (visceral vs. subcutaneous) following maximal GWG [
55], a subset of eligible participants (whom either did not have a Cesarean delivery or whom had subcuticular stitches for skin closure at the time of Cesarean, and whom have no metal foreign bodies), are asked to undergo optional
magnetic resonance imaging (MRI) during the 2–4 day postpartum inpatient stay. Images are captured centrally which decreases effect of postpartum fluid shifts that most often settle peripherally in dependent regions.
Behavioral
At each of two study time points,
dietary intake is assessed using three telephone-administered 24-hour (24HR) dietary recalls (two weekdays and one weekend over a three-week window), with each call duration lasting 15–30 minutes. The first dietary assessment is conducted in pregnancy at baseline, and then again at the three-month postpartum assessment. 24HR dietary recalls are considered the gold standard dietary assessment method for population-based research, with three recalls per time point being appropriate to examine inter- and intra- dietary variation patterns [
56]. These assessments are administered by trained dieticians who elicit information on dietary intake utilizing the multiple-pass technique, allowing for several distinct opportunities to obtain information about participants’ food intake during the previous 24 hours. This approach, and associated software, allow for range and logic error checking, information prompting and corrections. 24HR recalls are analyzed using the University of Minnesota Nutrition Coordinating Center's (NCC) Nutrition Data System for Research (NDS-R-2010) software (updated annually) [
57]. The system consists of data entry and analysis software and comprehensive food nutrient databases. The database contains over 18,000 foods (including culturally unique foods) and 8,000 brand name products along with values for 155 nutrients, nutrient ratios and other food components, allowing for analysis of individual dietary variations including portion size, preparation methods, ingredients, assessments and timing of eating. A Dietary Supplement Module captures supplements used by participants.
Physical activity is assessed on the same phone call via a validated 24HR physical activity recall [
58,
59]. Similar to the dietary recalls, three physical activity recalls are administered at baseline (in pregnancy) and then at three months post-partum, each lasting 5–10 minutes. Detailed information about specific types and intensities of activities are summarized and metabolic equivalents (METS) are calculated using Ainsworth and colleagues compendium of physical activities [
60].
Additionally, the Pregnancy and Physical Activity Questionnaire (PPAQ) [
61], modified for administration in the post-partum period, is administered by research personnel at all assessment time points. The PPAQ is a self-administered measure of physical activity that has been validated using ActiGraph accelerometers. This measure provides physical activity data in various domains relevant to pregnant and postpartum women, including occupational activity, household/care-giving activities, and sports/exercise [
61].
Extent of
breastfeeding, breast pumping, formula and other infant feeding practices are assessed with select questions from the Center for Disease Control’s (CDC) maternal-infant feeding study [
62] and a modified version of the United Kingdom’s Medical Research Council Epidemiology Unit’s feeding and growth questionnaire [
63]. Questions probe for exclusivity of breastfeeding versus extent to which other liquid and food products are offered to infants so as to quantify maternal energy expenditure with regards to lactation efforts.
Sleep quantity and quality are assessed with the 19 item Pittsburgh Sleep Quality Index [
64], which has been used to assess sleep disturbance in pregnant [
65] and post-partum [
66] women.
Tobacco and alcohol use are queried at each study visit and recorded. Selected items from the Behavioral Risk Factor Surveillance System questionnaire [
67] are utilized to assess tobacco.
Cognitive/Psychological
Self-administered survey tools are used to assess psychological and cognitive/attitudinal variables at all study time points. Areas assessed include anxiety evaluated by the 20-item Spielberger State Anxiety Inventory(SSAI)) [
68], depression as measured by the the 10-item Edinburgh Postnatal Depression Scale (EPDS) [
69,
70], stress as assessed by the 10-item Perceived Stress Scale [
71], a measure of life events [
72], and quality of life assessed via the SF-12 [
73]. We also assessed positive affect using the Happiness Scale modified (4 questions) [
74]. Attitude towards pregnancy and weight gain were assessed using the modified Pregnancy and Weight Gain Attitude Scale (18 questions) [
75] and questions on pregnancy intendedness. Selected eating behaviors namely emotional eating, uncontrolled eating and cognitive restraint are evaluated via the 18-item Three-Factor Eating Questionnaire [
76], and a 28-item inventory assesses food cravings [
77].