Maternal Obesity, Breast Milk Composition, and Infant Growth
Maternal Obesity, Breast Milk Composition, and Infant Growth
Today the majority of pregnant women in the United States are either overweight or obese at
 conception with their offspring having greater adiposity at birth, a 2-fold greater risk of
 later obesity, and neonatal insulin resistance. It was long thought that breast milk
 composition was fairly uniform among women, having been optimized through evolutionary time
 to provide adequate sole nutrition for the growing infant regardless of the environmental
 circumstances. However, recent evidence shows that breast milk is a highly complex fluid with
 significant inter-individual variation in hormonal and cytokine concentrations. Pervasive
 maternal obesity is an evolutionarily novel condition for the human species but little effort
 has yet been made to systematically examine how this novel condition is associated with
 breast milk adipose-tissue derived hormone and cytokine (adipocytokine) variation, or whether
 that variation relates to infant metabolic status. The objective of this study is to
 comprehensively assess the "lactational programming" hypothesis, that is, whether or not
 recently documented variation in breast-milk composition is related to both maternal
 adiposity and to infant metabolic status. The central hypothesis is that a graded,
 dose-response relationship between maternal adiposity and adipocytokine concentrations in
 breast milk exists and that milk adipocytokine concentrations are associated with altered
 body composition in their exclusively breast-fed offspring. The results of the study will be
 used to design interventions to reduce maternal weight during pregnancy and lactation and to
 augment lactation education materials to focus on the needs of obese breast-feeding women.
General Study Design A prospective observational pregnancy cohort of 360 non-diabetic
 mothers, who exclusively breastfeed their infants to at least 1 month post-partum, will be
 recruited in the second trimester of pregnancy, across a wide range of maternal pre-pregnancy
 BMI, with equal numbers of normal weight, overweight, and obese women. The women will be
 recruited at two study centers (University of Oklahoma Health Sciences Center in Oklahoma
 City, OK and Health Partners Research Foundation in Saint Paul/Minneapolis, MN), with
 maternal and infant diet, body composition and metabolic health followed prospectively at 1,
 3 and 6-months post-partum in Specific Aims 1 and 2. In Specific Aim 3, a subset of 60 of
 these mother/infant dyads "Mothers in Focus" (30 OB and 30 NW mothers) will be recruited for
 additional biospecimen collection, including milk samples at 2 and 6-months, maternal body
 composition changes using DXA, infant DXA, metabolomic comparison of serial milk samples, and
 follow-up to 12 rather than 6-months.

 Data Collection Elements:

 Maternal Adiposity. Maternal pre-pregnancy BMI status (NW, OW, OB) is the primary exposure
 used to power the study. Pre-pregnancy BMI will be determined using first (baseline) clinic
 weights from the electronic medical records (EMR) (no more than 90 d. after last menstrual
 period), and stature measured by research staff at the mother's first visit to our research
 centers. BMI will be categorized as underweight (BMI <18.5), normal weight (BMI 18.5 - <25),
 overweight (BMI 25 - <30) and obese (BMI ≥30 kg/m2).

 Gestational weight gain (GWG) and postpartum weight loss/retention:Total GWG will be
 calculated as the difference between delivery weight and baseline weight (as documented
 above). If weight at delivery is not available, the last prenatal weight in the EMR, ≤14 days
 from birth, will be used. Trimester specific GWG will be calculated from the EMR. GWG will be
 used as a continuous and as a categorical exposure (excessive, appropriate, and insufficient,
 according to the IOM 2009 guidelines. Early PP weight loss (from delivery to 1-month
 postpartum), and later PP weight loss from 1-month to 3-months PP will be calculated.
 Concurrent changes in maternal weight and milk adipocytokine levels from 0 - 1 month and 1 -
 3 months will be examined, hypothesizing that greater PP weight loss (lower weight retention)
 is associated with lower levels of milk adipocytokines.

 Milk Collection: Changes in milk composition will be assessed from 1 to 3 months. Definition
 of exclusive breast feeding: Study staff will confirm that all infants have been exclusively
 breast milk fed (i.e. no formula, no liquid other than water and no solids) at least 14 days
 prior to the 1-month visit and plan to continue exclusively breastfeed to 3 months. Exclusive
 breast milk feeding will include any combination of expressed breast milk and breast milk fed
 via the breast.

 Breast Milk Collection Protocol: Mother and child will report to the study site at 1 and
 3-months (± 5 days) postpartum, between 8:00-10:00 am, at least 1½ hours since the last
 feeding and while the mother has fasted at least 1 hour. A pre-feeding infant weight will be
 obtained using a high sensitivity scale, the mother will be encouraged to feed the child ad
 libitum from both breasts until volitionally satisfied, and a postfeeding infant weight will
 be obtained (difference = milk output). Questionnaire information, maternal anthropometry,
 and infant body composition data will be collected, and 2 hours later, the mother will
 provide a single breast expression sample using an electric breast pump (Medela, Inc.,
 provided by the study team). Breast milk will be mixed, aliquoted and stored at -80°C.

 Milk Adipocytokine Assays: Milk samples will be thawed in the refrigerator and vortexed. Milk
 fat will be separated from the aqueous phase by centrifugation at 3,000g for 10 minutes at
 4°C. The resulting skimmed milk will be assayed using commercially available immunoassay kits
 for insulin, IGF-1/BP, high molecular weight adiponectin, visfatin, leptin, IL-1β, IL-6,
 IL-10, and TNF-α. Glucose will be measured by the glucose oxidase method. To examine
 additional cytokine and growth factor concentrations (e.g., interleukins, interferons, TGF-
 β) in a manner that conserves sample, a multiplex assay (EMD Millipore, Billerica, MA) and
 the Luminex 200 Multiplex analyzer will be employed.

 Data Analysis for Specific Aim 1: For each of the milk adipocytokines (continuous dependent
 variables), separate mixed effects regression models (to account for serial milk data) will
 be constructed, first testing crude associations of milk IL-6 at both 1 and 3-months with
 each maternal adiposity variable (excessive GWG, pre-pregnancy BMI status, total GWG,
 trimester-specific GWG, early and later PP weight loss), and then minimally adjusted models
 (also adjusting for infant sex and gestational age, study center (OK or MN), maternal age,
 parity, ethnicity, and maternal socioeconomic status) and finally fully-adjusted regression
 models including those variables determined to be potential confounders. In secondary
 analyses, possible effect modification of the maternal adiposity associations will be
 assessed by adding interaction terms. If the interaction term is significant, a stratified
 analyses will be conducted.

 Data Collection for Specific Aim 2 Infant Body Composition. Total body composition will be
 measured using air displacement plethysmography (ADP) i.e. Pea Pod (v 3.1.1), a rapid, valid,
 highly reliable (% fat CV%< 2.5% in our hands) method of total body volume estimation from
 which total body fat (FM) and fat-free mass (FFM) is determined using prediction equations.
 Both the University of Minnesota and the University of Oklahoma have the same ADP (same
 manufacturer, model and software versions).

 Infant insulin secretion and resistance at 6 months: C-peptide, or connecting peptide, as a
 marker of insulin secretion will be collected. Serum C-peptide is secreted in equimolar
 amounts by the pancreas as insulin, but is a more stable measure because it is not prone to
 degradation following hemolysis. C-peptide was used as a marker of fetal beta cell function
 in the HAPO (Hyperglycemia and Adverse Pregnancy Out-come) study to assess the effects of
 maternal obesity and gestational diabetes on adverse pregnancy and infant outcomes. Insulin
 resistance will be estimated using homeostasis model assessment (HOMA-IR). Blood will be
 obtained via heel-stick in the infant at the time of the 6 month visit to the study center,
 as is routine in our laboratories, which yields ~ 1.0 cc of blood. Whole blood will be kept
 on ice until centrifugation in our laboratories, and 0.5 mL of serum aliquoted at stored at
 -80 C until shipped to Dr. Fields' laboratory for analysis using ELISA (Millipore, Billerica,
 MA; Intra-assay CV = 5%; Inter-assay CV = 6%). Interpretation of C-peptide requires blood
 glucose level which will be measured in the same blood sample using the glucose oxidase
 method.

 Milk Macronutrient Content: Total fat content will be measured by the Mojonnier method Fatty
 acid composition will be assessed by gas chromatography using the International Dairy
 Federation (IDF) methods. Other standard methods will quantitate total nitrogen, ash,
 moisture, and carbohydrates.

 Infant Appetite, Satiety, and Diet: Shortened versions of the infant diet questionnaires used
 in the Infant Feeding Practices Study II at 1 month, 3-months, and 6-months will assess
 maternal report of infant dietary intake. Maternal perception of infant appetite will be
 measured using the Baby Eating Behavior Questionnaire. Infant Sleep: The Sadeh Brief Infant
 Sleep Questionnaire has been validated against actigraphy and will be used to assess sleeping
 behavior at each visit. Additional Potential Confounders: See Specific Aim 1.

 Data Analysis(Specific Aim 2) Statistical Analysis: The over-arching primary hypothesis is
 that milk adipocytokine concentrations will be associated with altered body composition from
 ages 1 to 6-months, and with elevated C-peptide or HOMA-IR at 6 months, independent of
 potential confounders. Example Hypothesis 2.a1: Higher levels of the appetite suppressing
 hormone leptin at 1 and/or 3-months will be associated with lower infant weight gain,
 reflecting specifically lower FFM gain from 1 - 6 months. We will use mixed effects linear
 and logistic regression as the primary analysis approach to address this and related
 hypotheses. Growth and body composition data will be serial (0 [for weight], 1, 3, and
 6-months), which will be examined using mixed effects models which models the covariance
 structure of the repeated measures within subject and allows for subject-specific intercept
 and slopes to be modeled. Milk adipocytokines are the independent variables of interest.
 Because their individual relationships to infant growth and other outcomes may not be linear,
 quintiles or quartiles of milk adipocytokines as well as continuously distributed variables
 will be examined. Multiple milk adipocytokines will be included simultaneously in final
 models, to test their independent effects on infant outcomes and to assess their aggregate
 effects (variance explained).
Inclusion Criteria:

 -  Pregnant females

 -  21 to 45 years of age at the time of delivery

 -  Pre-gravid BMI between 18.5 to 40 kg/m2

 -  Healthy pregnancy defined as <3 days in hospital following delivery

 -  Report during enrollment procedures that they have social support for and intention to
 exclusively breastfeed for at least 3-months

 -  If parity >1, report that they successfully breast fed a previous pregnancy for at
 least 3 months

 -  In positive energy balance

 -  Singleton gestation

 -  Term pregnancy (gestational age >37 but <42 weeks)

 -  Infant birth weight >2,500 grams but <4,500 grams.

 Exclusion Criteria:

 -  Alcohol consumption >1 drink per week during pregnancy/lactation

 -  Tobacco consumption during pregnancy/lactation

 -  History/current Type I, II or gestational diabetes

 -  Inability to speak/understand English

 -  Known congenital metabolic, endocrine disease, or congenital illness affecting infant
 feeding/growth