Lifestyle Intervention in Preparation for Pregnancy (LIPP)
Lifestyle Intervention in Preparation for Pregnancy (LIPP)
Studies evaluating lifestyle intervention in obese women during pregnancy have reported
 limited success in decreasing excessive gestational weight gain, and have failed to achieve
 the key outcome of breaking the obesity cycle and reducing neonatal adiposity or birth
 weight. Although some investigators advocate weight loss during pregnancy in obese women,
 these recommendations were based on extrapolation of retrospective epidemiological data. Of
 concern, we reported increased small for gestational age babies and decreased lean body mass
 in neonates of obese women with weight loss or inadequate gestational weight gain. Based on
 our research, optimal outcomes from lifestyle interventions are likely to be temporal and
 therefore must be initiated prior to conception to first improve maternal metabolic function,
 and subsequently, placental/fetal growth. Several large retrospective cohort studies support
 our hypothesis. For example, women who lost weight between pregnancies had fewer large for
 gestational age babies in contrast to women who increased interpregnancy weight. In addition,
 prospective randomized controlled trials have shown that postpartum weight loss is achievable
 without adverse maternal or neonatal outcomes, these studies include women who breastfed.
 Based on these observations, we propose a randomized control trial to determine the effect of
 lifestyle intervention initiated prior to a planned pregnancy on improving neonatal
 metabolism and adiposity. Our overarching hypothesis is that the maternal pre-pregnancy
 metabolic condition determines the obesogenic in-utero environment, which affects programming
 of placental mitochondrial function and metabolic pathways, promoting lipid accumulation and
 neonatal adiposity. Our rationale is based on the need to establish the most effective time
 to introduce an intervention that will break the obesity cycle in mothers and their children.
 Understanding how pregravid metabolic conditioning improves maternal physiology, and cellular
 and molecular function in pregnancy will provide the empirical data to support the
 intervention. We have a highly successful record of recruiting women who are planning a
 pregnancy, obtaining compliance in longitudinal studies, and in long-term follow-up of
 mothers and their offspring. Lifestyle intervention will be initiated prior to conception to
 decrease maternal body fat, inflammation, insulin resistance, and ?-cell dysfunction. Our
 transdisciplinary team has the required expertise in lifestyle interventions management of
 obesity, and in human physiology that is needed to determine the effects of these
 interventions on maternal metabolism and fetalplacental growth and function. We will recruit
 200 women to pursue the following specific aims:

 Specific Aim 1: To investigate the physiological significance of lifestyle intervention in
 preparation for pregnancy (LIPP) on maternal and neonatal metabolism and adiposity.

 Specific Aim 2: To determine the molecular effects whereby lifestyle intervention initiated
 before pregnancy can improve placental mitochondrial lipid oxidation and accumulation.
Specific Aim 1: To investigate the physiological significance of lifestyle intervention in
 preparation for pregnancy (LIPP) on maternal and neonatal metabolism and adiposity.

 Introduction/Rationale: Our preliminary data demonstrate that supervised lifestyle
 intervention leads to significant weight loss, improved insulin sensitivity, glucose
 tolerance and incretin secretion, together with healthier cardiovascular and body composition
 outcomes in overweight and obese adults. We expect that timing and implementation of the
 proposed lifestyle intervention will produce similar health benefits in overweight/obese
 women planning a second pregnancy, and lead to greater insulin sensitivity, reduced insulin
 secretion, and less inflammation. These improvements will result in the prevention of excess
 nutrient availability (glucose and lipids) from contributing to excess fetal
 growth/adiposity. The working hypothesis for this Aim is that in contrast to GWG, the
 decreased pre-pregnancy insulin sensitivity in obese mothers accounts for the greatest
 clinical variance in fat accretion in the infant. Although clinically we anticipate a
 decrease in weight and BMI in the LIPP group, the improvement in insulin sensitivity and
 metabolic profile are the key physiological measures related to the primary outcome of
 decreased neonatal adiposity, and not the weight loss per se.

 The rationale is that the optimal time to implement lifestyle intervention that effectively
 improves maternal health at the physiological, cellular and molecular level, and results in
 optimal adiposity in the baby, is prior to pregnancy. Women who lose weight postpartum,
 experience a decrease in neonatal birth weight (primarily adipose tissue) in subsequent
 pregnancy, whereas women who gain weight, experience an increase in neonatal birth weight and
 adiposity. We hypothesize that maternal pre-pregnancy metabolic condition determines the
 obesogenic in-utero environment, which in turn affects placental programming of mitochondrial
 and lipid pathways (Specific Aim 2), and body composition of the baby. An additional
 rationale is that there is a need to understand how improved pregravid metabolic condition
 affects maternal physiological and molecular function. We anticipate that obese women who
 complete the LIPP program will enter pregnancy with improved insulin regulated metabolism and
 reduced insulin resistance, thus facilitating a lower neonatal birth weight and adiposity. We
 will recruit mothers who delivered their first baby at MHMC. We recognize that these mothers
 represent a demographic that has limited access to exercise facilities or family support
 systems that would facilitate free time for exercise. In order to reduce barriers to
 participation, we will conduct the exercise sessions in local Community Recreation Centers.
 The Centers have child care facilities and we will support the cost so participants may bring
 their babies to the LIPP sessions. To further increase participation and maximize retention,
 we will provide transportation to and from the Recreation Centers. Support for transportation
 will be requested from the Cleveland Mt. Sinai Foundation. Lifestyle Intervention Weight Loss
 Phase: The LIPP program is designed to promote weight loss that is 5-10% of body weight. The
 4-month weight loss phase consists of aerobic exercise training with diet and behavioral
 counseling to induce weight loss as was successfully achieved in prior studies. Initially,
 supervised exercise will be prescribed at 55-60% of HRmax and gradually increased so that
 after 1-2 weeks, the subjects are exercising at 75-85% of HRmax (~65-70% VO2max). Supervised
 exercise will consist of walking/jogging on a treadmill and stationary cycling, 3 days/week,
 60 min/session (i.e., 500 kcal/session). The women will wear heart rate (HR) monitors (Polar
 Electro, Woodbury, NY) during each exercise session so that they have visual feedback of
 their personalized target heart rate goal. Participants will be advised to reduce caloric
 intake by ~500 kcal/d in order to support their weight loss goals. The recommended diet will
 provide ~55% of calories as carbohydrate, 25% as fat, and 20% as protein. Participants will
 be instructed to consume complex carbohydrates and to avoid simple sugars. Specific caloric
 needs will be estimated by indirect calorimetry and a sedentary (x1.3) physical activity
 correction factor. Energy intake will be estimated using the food photo diary app, Meal Snap.
 Digital photography provides an excellent estimate of energy intake (67). Participants who do
 not own a smart phone will have one provided through support from the Cleveland Foundation.

 Records covering a 72-hour diet period will be shared with the research team for
 determination of calorie and nutrient intake. Meal Snap has a food database of over 350,000
 items. However, some meals will not be in this database, therefore our Lifestyle Coaches will
 enter all foods eaten into our diet database (NDSR, Minneapolis, MN) to facilitate analysis
 of calorie, and macro/micro nutrient intake. Subjects will generate photos from before and
 after the meal in order to estimate the amount of food that was eaten. Data will be obtained
 at baseline, and at 2-week intervals during the initial 16-week supervised weight loss
 period.

 Lifestyle Intervention - Weight Management/Maintenance: The pre-pregnancy weight management
 program (phase 2A, 2B and 2C) is designed to facilitate personalized weight loss goals using
 lifestyle behaviors that include, exercise, diet and behavioral modification, and is based in
 part on the Look AHEAD trial. The intervention includes a toolbox concept to help meet weight
 loss goals. The Lifestyle Coaches will provide personalized instruction on physical
 activity/exercise - 10,000 steps/day, and the participants will use FitBit Flex (Fitbit.com),
 to track step count and exercise time. During the first phase of weight maintenance (2A) the
 women will attend 2 supervised group sessions/week. These sessions include structured
 exercise (e.g., Zumba, jazzercise, stroller walking), review of dietary photo records, where
 meals have been eaten (home or away; recorded on smart phone app), and behavioral counseling
 (with their Lifestyle Coach). Participants will be encouraged to eat 1,200-1,500 kcal/d (~55%
 carbohydrate, 25% fat, 20% protein) if below 113 kg, or 1,500-1,800 kcal/d if >113 kg.
 Dietary data will be analyzed at 4-week intervals during the weight maintenance period.
 Importantly, to minimize subject burden and to maximize retention and data acquisition, the
 Lifestyle Coach will track calorie intake using the participant's photo diary. Behavioral
 strategies to motivate healthy lifestyle decisions will include: self-monitoring (food,
 activity, and weight, using FitBit online resources), goal setting (steps/day, weight loss),
 stimulus control (i.e., social eating, fast food, sitting vs. standing), problem solving
 (have snacks available, exercise at home), and relapse prevention (i.e., holidays, alcohol,
 fast food, sweets, problem foods, compulsive eating). After 4 months and the desired weight
 loss, subjects move to Phase 2B. During this phase subjects maintain an exercise goal of
 10,000 steps/day, but will be required to attend only 1 supervised session/week. If a subject
 fails to maintain weight loss, defined as weight gain of 3% of current body weight,
 participants will return to Phase 2A for more supervised weight management. Alternatively, if
 weight loss is maintained after 3 months, subjects will progress to Phase 2C until the
 subsequent pregnancy. Phase 2C consists of no supervised exercise sessions. However, the
 subject and Lifestyle Coach will converse weekly by phone to review progress, including
 Fitbit exercise data and diet. Data suggest that weight management programs delivered by
 phone are comparable to clinically delivered programs. During the calls participants will be
 counseled to continue to exercise at the intensity and duration prescribed during Phase 2B.
 They will be provided with language-specific food and exercise log books. These will be used
 to guide calorie intake and will provide another record of compliance. Participants
 randomized to the Control group will receive information on post-pregnancy diet/weight loss
 from the CRU nutritionist as distinct from the LIPP nutritionist (HB) to decrease cross
 contamination between groups. Weight Management during Pregnancy: All LIPP and usual
 care/control groups will be followed by their primary Obstetrical provider. The OB/GYN
 department at MHMC has recently revised its clinical guidelines for the management of the
 overweight/obese women based on the December 2015 ACOG practice bulletin (2). All
 overweight/obese women will be offered nutrition counseling early in pregnancy by a
 registered dietician from the MHMC Nutrition Department with follow-up visits as needed to
 support GWG within the IOM guidelines. Nutritional therapy will consider maternal pregravid
 BMI, ethnic, cultural and social factors in individualizing healthy eating. The electronic
 health record (EPIC) includes a graphic GWG nomogram and so GWG will be monitored at each
 visit. All subjects will be encouraged to increase physical activity for at least 30
 minutes/day (primarily walking). Clinical management such as, ultrasounds to estimate fetal
 growth, and fetal surveillance will be based on the ACOG recommendations. The Lifestyle Coach
 will continue to follow-up only with the LIPP subjects as described in the maintenance phase
 of the research design.

 Metabolic Evaluations: Metabolic evaluations will be performed at baseline (3 months ? 2
 weeks) postpartum. Follow-up evaluations will be performed after 4 (+/-2 weeks) and 12 months
 (+/-2 weeks), and then every 6 months (+/-2 weeks) prior to pregnancy up to a maximum of 24
 months. Once a subject has pregnancy dating and viability confirmed by ultrasound, metabolic
 evaluations will continue at 12-16 and 32-36 weeks of gestation.

 Maternal Body Composition: Anthropometry measurements will include height, weight, and hip
 and waist circumferences. Total body fat will be measured by whole body plethysmography (Bod
 Pod; Cosmed, Rome, Italy). We will use a hydration constant of 76% for fat free mass during
 late pregnancy.

 Resting Energy Expenditure: Resting Metabolic Rate (RMR) will be determined after an
 overnight fast using the Cosmed OMNIA metabolic cart (Cosmed, Rome Italy) with a canopy
 system. We will control for diet by providing a standardized energy balanced meal the evening
 prior to the test from the CRU. Participants will relax in a quiet low-light metabolic room
 for 30 min prior to obtaining a 30 min measure of exhaled breath. Oxidative and non-oxidative
 glucose metabolism will be estimated and urine samples will be obtained before and after the
 measure in order to calculate non-protein RQ (NPRQ). These data will be used in conjunction
 with Specific Aim 2, and will be correlated with change in mitochondrial function during
 pregnancy.

 Exercise Capacity: An incremental graded treadmill test will be performed at baseline, 4, and
 6 month time points in both groups. Oxygen consumption (Jaeger OxyCon Pro/Delta System,
 Hoechberg, Germany), heart rate, and ratings of perceived exertion will be performed as
 previously described.

 Insulin Sensitivity and ?-Cell Function: A 75 g oral glucose tolerance test (OGTT) will be
 used to assess postprandial glycemia, and insulin sensitivity and secretion. After an
 overnight fast, blood samples will be drawn at 10 min intervals for the first 60 min, and at
 20 min intervals thereafter. C-peptide data will be analyzed using a combined model approach
 to provide pre-hepatic insulin secretion rates, insulin sensitivity, disposition index, and
 relate insulin and Cpeptide kinetics (73). Plasma glucose will be measured using the glucose
 oxidase method (YSI; Yellow Springs, OH).

 Insulin will be assayed by RIA (Millipore, Billerica, MA). Diagnosis of GDM will be made
 using criteria recommended by the ACOG (74).

 Enteroinsular Axis Responses: Plasma samples (with appropriate additives) will be obtained to
 measure incretin hormones (glucagon-like peptide-1 (GLP-1), and glucose-dependent
 insulinotropic polypeptide (GIP), and satietyrelated gut peptides (cholecystokinin (CCK), and
 peptide YY (PYY). Measurements will be made under static (fasting) and dynamic
 (glucose-stimulated) conditions (10 min intervals up to 1 hour).

 Metabolic and Inflammatory Biomarkers: Fasting blood samples will be obtained to measure CBC,
 TSH, HbA1c, lipid panel, and total free fatty acids (FFA). Adipocytokines (adiponectin,
 leptin interleukin-6, interleukin- 8, TNF-? and hsCRP) will be measured using ELISA (R&D
 Systems, Minneapolis, MN). All samples from each subject will be stored at -80oC and run in
 the same assay at completion to decrease variability.

 Quality of Life Questionnaire: The SF-36 health survey will be used at baseline, 4 and 12
 months, and then at 6 month intervals until pregnancy to assess the subject's health-related
 quality of life. These data will provide a generic measure of physical and mental health
 through assessment of physical functioning, bodily pain, limitations due to physical,
 personal, or emotional problems and well-being, energy/fatigue, and general health
 perceptions. During pregnancy the questionnaire will be administered at 12-16 and 32-36
 weeks.

 Measuring Fat Mass in the Infant: We have extensive experience in estimating body composition
 in neonates, and were one of the first centers to procure a Pea Pod (pediatric air
 densitometry), which is housed in the CRU next to Labor & Delivery and the postpartum unit.

 Insulin Resistance at Birth: At birth we will obtain umbilical cord blood for insulin and
 glucose to estimate insulin resistance using HOMA. Full lipid profile, CRP, and the
 adipokines IL-6 and leptin (an excellent marker of neonatal fat mass) will be measured in
 cord blood as described above.

 Anticipated Results, Challenges, and Alternative Approaches:

 The primary outcome measure of this proposal is lower neonatal adiposity at birth in the LIPP
 group relative to: 1) the Control group and 2) with the subject's firstborn. As secondary
 outcomes we anticipate that prior to a subsequent pregnancy, LIPP will produce significant
 improvement (absolute and percent changes) in maternal weight and body composition, and more
 importantly improved insulin sensitivity, beta-cell function, incretin response to glucose,
 lipid and inflammatory biomarkers, compared to the Control group. We also expect reduced
 insulin resistance, cord lipids and inflammatory profiles in babies born to LIPP compared to
 Controls.

 Recruitment is a well-recognized problem for the successful implementation and completion of
 lifestyle intervention trials. However, given the novel strategy for recruitment detailed
 above, our unique access to the patient population, and our extensive experience in metabolic
 research in pregnancy, we do not anticipate recruitment to present an insurmountable
 challenge. We will recruit 200 subjects in the first 4 years and complete all mother/baby
 evaluations according to the proposed timeline. If necessary we will recruit subjects from
 Cleveland Clinic and University Hospitals, both affiliated with Case Western Reserve
 University (CWRU). Retention strategies include free transportation to the exercise sessions,
 free childcare service during the lifestyle sessions and consults with the Lifestyle Coach,
 cell phone apps to reduce subject burden with data entry, regular telephone contacts, and
 financial incentives, including an infant car seat at delivery.

 Our team has had outstanding success in retaining pregnant women in our previous metabolic
 research and lifestyle intervention studies in overweight and obese populations (48,64). In
 the current proposal, additional strategies are also in place to maximize retention and
 minimize dropout. These include phone calls and emails from the Lifestyle Coach to review and
 reinforce problem solving and self-monitoring skills as described for participants in the
 Diabetes Prevention Program. We will also establish a buddy system where each participant
 becomes a buddy to another participant, the two buddies may prepare meals together, exercise
 together, share problems, etc. The buddy system fosters the sense that staying in the study
 is important not just for the individual's health but also for the buddy's as well. If
 subjects in the LIPP program do not meet their target weight loss goal, we will implement a
 meal replacement strategy using the resources of the CRU/CTSC metabolic kitchen, with
 attention to the caloric and nutrient needs of the mother depending on breast feeding. Not
 all participants will conceive. Since the primary outcome is neonatal adiposity, subjects who
 do not become pregnant will not be included in the primary analysis but will be included in
 secondary analyses relating to pregravid metabolic improvement. By recruiting women with a
 previous pregnancy the risk of infertility is significantly reduced. We anticipate that
 approximately 20% of women will experience a spontaneous abortion, but they will be allowed
 to continue and resume their previous participation in either the LIPP or Control group. We
 anticipate that approximately 25% of subjects will drop out before becoming pregnant, and
 another 15% may drop out during pregnancy. We will enroll 100 subjects in each arm to account
 for the unlikely event of 40% dropout. Adopting an even more stringent strategy, we report
 statistical power is available for as low as 50 subjects per group as a worst-case scenario.
 If retention lags behind projections we will recruit additional subjects at MHMC and CWRU
 affiliated hospitals. The CRU staff will assist in collecting cord blood and placenta at
 delivery and performing the Pea Pod body measures. If there is a Pea Pod equipment
 malfunction we will estimate body composition using neonatal anthropometrics.

 Although LIPP subjects will be encouraged to delay a second pregnancy until they are in the
 maintenance phase of the program, we will exclude LIPP subjects only if they conceive during
 the first 4 month weight loss phase. For the Control subjects, exclusion will occur if a
 subject becomes pregnant before the 3 month postpartum CRU randomization visit. Use of birth
 control in this phase is an inclusion criterion. Not all LIPP subjects will conceive at
 similar times after the 4 month weight loss intervention. We will not adjust for the time
 between pregnancies between the LIPP and Control subjects. We will use the LIPP and control
 subject's metabolic status (body composition, insulin sensitivity and response, etc.) at the
 last metabolic evaluation in the CRU before becoming pregnant as their pregravid or baseline
 status for the subsequent pregnancy. Because this is a pregnancy study only women with be
 recruited. However, we will assess the effect of LIPP on males and female offspring, together
 and independently, based on sex.

 Statistical Approach:

 The primary analyses of Specific Aim 1a will be intent-to-treat comparisons of the LIPP and
 Control groups in regard to changes in maternal insulin sensitivity, BMI, and fat mass. The
 comparisons will be performed first with two-sample t-tests at p=0.05. Should any imbalance
 of confounding factors in the groups be recognized, linear regression models that include
 significant differences (e.g. GDM) will be used to perform covariate adjustments. Based on
 our 1 year postpartum follow-up studies (62,63), we will have 90% power to detect an absolute
 or covariate-adjusted improvement in insulin sensitivity of 30% and an 80% power to detect an
 improvement as small as 25% in the LIPP vs. Control group. Corresponding 95% confidence
 intervals (95% CI) for the absolute or covariate-adjusted differences or percent improvements
 in insulin sensitivity between groups will be reported. We estimate the standard deviation
 (SD) of change in BMI from randomization until subsequent pregnancy to be 5.1 kg/m2. We will
 have 90% power to detect an absolute or covariate-adjusted difference in BMI of 2.6 kg/m2 and
 80% power to detect a difference of 2.26 kg/m2 and 90% power to detect an absolute or
 covariate-adjusted difference in fat mass of 5.9 kg and 80% power to detect a difference of
 5.1 kg between groups prior to the second pregnancy. The primary analysis for Specific Aim 1b
 is an intent-to-treat comparison of LIPP versus Control neonates with respect to fat mass at
 birth. The comparison will be performed using a two-sample t-test at p=0.05. Linear
 regression will be performed, which will include weight (body composition measures) of the
 subject's first child as a covariate. Should any imbalance of confounding factors (for
 example gestational age) in the groups be recognized, linear regression models will be used
 to perform a covariate adjustment. Based on our preliminary data, we estimate the SD of
 neonatal fat mass between the LIPP and Control groups to be no more than 225g. With at least
 50 women in each group, (assuming a 50% dropout), the t-test or linear regression will have
 90% power to detect an absolute or covariate-adjusted difference of 146 g fat mass between
 groups. We have 80% power to detect an absolute or covariate-adjusted difference as small as
 126 g fat mass between groups. Corresponding 95% CI for the absolute or covariate-adjusted
 difference in neonatal fat mass between groups will be reported. For secondary analyses, we
 will be using the same statistical approach. Based on our preliminary data, we estimate the
 SD of birth weight to be 700 g; with 50 neonates in each group we will have 90% power to
 detect an absolute or covariate-adjusted difference of 455 g in birth weight and 80% power to
 detect a 393 g difference between groups. Additional secondary analyses will include
 umbilical cord cytokines. Comparisons will be performed using a two-sample t-test at a
 significance level of p=0.05; however, Mann- Whitney U tests or log transformations will be
 employed if data are not normally distributed. Linear regression models, including
 confounding factors, will be used to perform covariate adjustments. Based on our published
 data (80), we estimate the standard deviations of umbilical cord IL-6 and CRP to be 3.4 pg/ml
 and 7,900 ng/ml, respectively. With 50 women in each group, we will have 90% power to detect
 an improvement in IL-6 and CRP levels of 50% and 42%, and 80% power to detect an improvement
 of 42% and 36%, respectively.

 Specific Aim 2: To determine the molecular effects whereby lifestyle intervention initiated
 before pregnancy can improve placental mitochondrial lipid oxidation and accumulation.

 Introduction/Rationale: Our data suggest that in obese women, a mitochondrial defect in
 placental tissue is present early in pregnancy, inhibiting the placental capacity for fatty
 acid oxidation and shunting fatty acids to esterification pathways and lipid accumulation,
 potentially leading to increased nutrient availability to the fetus and higher adiposity at
 term. Our group has shown that other mitochondrial processes, such as cholesterol transport
 and steroidogenesis are impaired in placentas of obese, insulin resistant women at term.
 Placental mitochondrial content (assessed by mtDNA and citrate synthase activity) is not
 affected by maternal obesity and insulin resistance at term, suggesting that the observed
 defects in function are due to changes in mitochondrial activity, rather than number.
 Previous dietary interventions initiated during pregnancy were unable to alter placental
 ?-oxidation or fetal fat deposition, potentially because the intervention was begun after
 placental mitochondrial function was impaired. We anticipate that LIPP will improve placental
 mitochondrial fatty acid oxidation, which will be measurable at term and associated with
 lower fatty acid esterification and accumulation, and neonatal fat mass. The hypothesis is
 that the decreased insulin sensitivity and increased inflammatory environment in obese
 mothers impairs mitochondrial ?-oxidation in the developing placenta. It is the changes in
 placental metabolism beginning early in pregnancy that lead to altered nutrient delivery and
 increased fetal fat deposition. Our rationale is based on the need to understand how changes
 in placental lipid metabolism mediate the effects of improved pregravid metabolism on
 neonatal adiposity. We expect that placentas of obese women in the LIPP program will show
 improved fatty acid oxidation and decreased lipid esterification and accumulation at term.
 Furthermore, we expect that these changes will correlate with lower maternal inflammation and
 insulin resistance in early pregnancy and lower neonatal adiposity at term.

 Anticipated Results and Endpoints: We anticipate that compared to the Control group,
 placentas of women in LIPP will have: 1) increased ?-oxidation, 2) decreased fatty acid
 esterification, 3) lower lipid content, 4) increased activity of mitochondrial CPT1B, the
 rate-limiting enzyme in ?-oxidation, and higher phosphor ACC, which, when phosphorylated,
 produces less malonyl CoA, the main inhibitory regulator of CPT1B, and 5) no difference in
 mitochondrial content (as measured by mtDNA and citrate synthase activity). We also
 anticipate that mitochondrial ?-oxidation and CPT1B activity will negatively correlate with
 early pregnancy maternal serum inflammatory cytokine markers and insulin resistance and
 neonatal adiposity.

 Experimental Design: We will accomplish the objectives of Specific Aim 2 by measuring changes
 in placental mitochondrial enzyme activity and lipid metabolism in women enrolled in the
 Control or LIPP groups described in Specific Aim 1. Placental tissue will be collected at
 delivery from all study participants and embedded in paraffin, or snap frozen in liquid
 nitrogen, and stored at -80oC for molecular analysis. In a subset of women delivering by
 scheduled Cesarean delivery (we estimate ~30% of our participants or N=15-18/group), we will
 also collect fresh placental tissue for lipid metabolism activity assays.

 Placental lipid metabolism: These assays are well established in the O'Tierney-Ginn lab.
 Mitochondrial fatty acid oxidation (FAO) and esterification into total lipid assays will be
 performed in placental explants as described previously, with some modifications. Freshly
 isolated placental explants will be incubated in the presence of 100 ?M cold palmitate and
 3H-palmitate (Moravek Radiochemicals) for 18 hrs. At the end of the incubation period, media
 will be collected to quantify the FAO rate by detection of 3H2O using the vapor phase
 equilibration method of Hughes. Esterificationinto total lipids will be determined by
 homogenizing the treated explants in HPLC-grade acetone and incubating with agitation at room
 temperature overnight. An aliquot of the acetone-extract lipid suspension will be used to
 determine the radioactive content by liquid scintillation counting. Oxidation and
 esterification rates will be defined as nmol palmitate/mg tissue/hr.

 Assessment of placental mitochondria: Mitochondria will be isolated from frozen placental
 tissue as previously described. Markers of lipid oxidation (CPT1B) and
 synthesis/esterification (phospho-ACC) activity will be measured using commercially available
 kits (Cell Signaling, Abcam) in isolated mitochondria from all samples. Markers of
 mitochondrial content (mtDNA and citrate synthase activity) will be measured in whole
 placental tissue in all samples as previously described.

 Placental Lipid Accumulation: Total placental lipid content will be measured using the Folch
 method.

 Anticipated Challenges and Alternatives: 1) We will use only placentas delivered by scheduled
 cesarean delivery for in vitro oxidation and esterification assays, to avoid changes
 pertinent to labor. At our hospital, the rate of cesarean for obese women is ~40%. Our
 conservative estimate of 40-50% drop-out followed by 30% of subjects delivering by cesarean,
 results in N=15-18/group. Based on our preliminary data, this will leave us adequately
 powered to detect differences in placental lipid metabolism due to LIPP. We will collect
 placental samples from all study participants for mitochondrial enzyme activity assays,
 providing us with an additional assessment of metabolic activity in a larger number of
 participants. 2) Placental mitochondrial fatty acid oxidation capacity may be affected by
 mitochondrial number, oxidative phosphorylation activity, and energetic efficiency
 (coupling). Assessment of mitochondrial oxidative phosphorylation capacity or energetic
 efficiency requires freshly isolated mitochondria and/or living cells which, for our
 proposal, would be overly ambitious and costly considering the unpredictable nature of
 delivery and the large number of participants. Alternatively, we will measure markers of
 mitochondrial content and key enzymes in mitochondrial lipid metabolism in all placental
 samples to assess some potential mechanisms underlying changes in placental fatty acid
 oxidation. Additionally, frozen samples collected from all placentas can be used to measure
 enzymes involved in electron transport (e.g., ATP synthase) as a marker of mitochondrial
 oxidative phosphorylation.

 Statistical Analysis: The primary goal of Aim 2 is to determine the effect of lifestyle
 intervention prior to pregnancy on placental mitochondrial fatty acid oxidation at term. We
 hypothesize that placental ?-oxidation will be higher in the LIPP group. We will conduct an
 intent-to-treat analysis using two sample t-test or non-parametric Wilcoxon rank-sum test to
 assess differences between groups. Regression analysis will be used to assess the association
 of placental ?-oxidation and enzyme activity with maternal inflammatory cytokine levels and
 insulin resistance in early pregnancy, along with neonatal fat mass with adjustment for
 gestational age and gender. Descriptive statistics, such as mean, median and range will be
 calculated for all variables. Power and sample size analysis based on our preliminary
 mitochondrial ?-oxidation data in obese women (38?14 nmol/mg/hr) showed that a sample size of
 N=18/group achieves 80% power to detect a difference of 25% between groups using a two-sample
 t-test with a significance level of 0.05.
Inclusion criteria:

 All subjects will have had by the time of randomization at 3 months postpartum:

 1. Planning another pregnancy within the next 24 months

 2. Planning to deliver at Tufts medical center during their next pregnancy

 3. A previous full-term singleton pregnancy (gestational age > 37 weeks)

 4. 18 to 40 years of age at the time of enrollment into the study

 5. Vaginal or cesarean delivery

 6. Normal glucose tolerance or gestational diabetes (GDM), but without evidence of
 postpartum diabetes as defined by a 75 g 2-hr oral glucose tolerance test (OGTT)

 7. Normal blood pressure or mild preeclampsia but normal postpartum blood pressure

 8. Bottle or breast feeding

 9. Normal thyroid function (determined by TSH concentration in blood), normal cell blood
 count and normal kidney and liver functions. Lipid profile with triglyceride levels
 not higher than 400 mg/dl (fasting) and LDL levels less than 180 mg/dL

 10. No clinical signs or symptoms of cardiovascular disease or any other disease or
 condition that may contraindicate participation in exercise training (i.e. COPD,
 severe asthma, orthopedic abnormalities)

 11. Using contraception

 Exclusion criteria:

 1. Pre or post-delivery diabetes

 2. Post-delivery hypertension requiring medication

 3. asthma requiring more than occasional use of a sympathomimetic inhaler, but not
 chronic inhaled steroids

 4. Inflammatory bowel disease

 5. Need for assisted reproductive technologies to become pregnant

 6. Medical or obstetrical contraindication to the defined exercise program or diet

 7. Tobacco, excessive alcohol use (greater than 2 drinks/day) or illicit drug use

 8. Eating disorders such as bulimia

 9. Gastric surgery to lose weight including banding or bypass procedures

 10. Any psychological or psychiatric condition which may impair participation in the
 lifestyle intervention program

 11. Multiple pregnancy

 12. HIV, or hepatitis B or C

 13. If a LIPP subject becomes pregnant prior to 16 weeks after randomization before the
 weight- loss phase for the lifestyle intervention is completed or a control subject
 becomes pregnant before the 3 month postpartum randomization, i.e. no baseline
 measurement.