Maternal Obesity, Breast Milk Composition, and Infant Growth
| Status: | Enrolling by invitation |
|---|---|
| Conditions: | Obesity Weight Loss, Women's Studies |
| Therapuetic Areas: | Endocrinology, Reproductive |
| Healthy: | No |
| Age Range: | Any - 45 |
| Updated: | 10/13/2018 |
| Start Date: | July 1, 2014 |
| End Date: | June 30, 2019 |
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.
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).
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
We found this trial at
2
sites
940 NE 13th St
Oklahoma City, Oklahoma 73190
Oklahoma City, Oklahoma 73190
(405) 271-6458
Phone: 405-271-8001
University of Oklahoma Health Sciences Center The OU Health Sciences Center is composed of seven...
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Minneapolis, Minnesota 55454
Principal Investigator: Ellen Demerath, PhD
Phone: 612-624-8231
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