Choline Supplementation on Fetal Growth in Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM), characterized by maternal high blood sugar, affects up to 15% of pregnancies worldwide. GDM doubles the risk of fetal growth (i.e. macrosomia, large fetus,) which further predisposes the affected fetus to elevated risks of obesity, diabetes and cardiovascular disease later in life. The placenta mediates macrosomia by increasing fat and glucose transport to the fetus during GDM. Effective methods to normalize placental nutrient transport and prevent macrosomia are lacking. Choline is a semi-essential nutrient found in egg yolks, meat, fish and cruciferous vegetables. The demand for this nutrient increases substantially during pregnancy. Previous research demonstrated that maternal choline supplementation (MCS) normalized placental fat and glucose transport as well as fetal growth and fat mass in GDM mice. In this study, the investigators will recruit pregnant women with GDM and randomized them to receive a choline supplement (470mg/day, a dose with demonstrated safety), or a placebo for 8 weeks, starting after gestational week 28. The investigators will determine the effects of choline on fetal growth as well as the influence of choline on placental transport and epigenetic modifications in growth-related genes in the placenta and umbilical cord. Successful completion of this study will provide valuable insights into the use of choline supplement as a modifier of placental macronutrient homeostasis in GDM to improve fetal growth outcomes.

STUDY OBJECTIVES Specific Aim 1: Determine the effect of MCS during pregnancy on birth weight in GDM. Specific Aim 2: Determine the influence of MCS on macronutrient transport and epigenetic modifications in the placenta and cord blood. HYPOTHESIS Aim 1: The investigators hypothesize that the choline group would have lower birth weight and fewer LGA neonates than the non- choline group. Aim 2: The investigators hypothesize that the choline group would reduce placental transport and increase DNA methylation of metabolic genes. STUDY DESIGN Subjects: A convenient sampling methodology will be adopted. The investigators will recruit pregnant women diagnosed with GDM during the second trimester of pregnancy on a rolling basis. Block randomization will be conducted based on treatment methods (i.e. insulin therapy, oral hypoglycemics, or diet only) to randomly assign participants to the intervention and control groups. Recruitment will be conducted using the following methods: (i) study flyers will be posted at the prenatal clinic; (ii) the investigators will identify pregnant women who are potentially eligible by reviewing medical records. Eligibility Criteria: The inclusion criteria include GDM pregnant women before gestational week 28. GDM is diagnosed with the 2-step process: blood glucose higher than the 140 mg/dL cut-off value after 50 g non-fasting 1-hr and 100 g 3-hr oral glucose challenges. Pregnant women are qualified to participate if they are English- or Spanish- speaking, over 18 years of age, having singleton pregnancy, intending to deliver their babies at the institution and are without any of the conditions listed in the exclusion criteria. Exclusion criteria include pre-existing hyperglycemia, diabetes, cardiovascular conditions and liver disease prior to pregnancy. Patients may choose to participate in the study and not donate a biopsy of their placenta or umbilical cord. Patients who desire stem cell donation or storage will not be able to donate a biopsy of their placenta or umbilical cord. These patients will not be excluded from the primary analysis. Design and Data Collection Procedures: All interaction with participants will occur at the delivering hospital while sample analyses will occur at the College where the co-investigator's lab is located. The coordinator for the study, employed by the co-investigators (Brooklyn College), will not access or store data remotely and all data collection and analysis will happen on hospital campus. The investigators will obtain written informed consent from the pregnant women before study entry. Randomization will be done by pharmacy after patient signs consent. It will be block randomization of 6 patients. After randomization, the investigators will provide the participant in the intervention group with choline supplement pills containing 500 mg of choline bitartrate (235 mg choline, Douglas Lab). The participant will consume 2 pills per day to obtain 470 mg choline for 8 weeks. The recommended intake of choline in the form of Adequate Intake (AI) is 450 mg/d for pregnant women and the Tolerable Upper Intake Level (UL) is 3500 mg/d. Since less than 10% pregnant women reach the AI of choline intake and the upper quartile of choline intake is below 900 mg/d. The dosage that the investigators provide plus the habitual intake of choline in these pregnant women will fall between the AI and UL, i.e. sufficient but not exceeding the limit. The participants in the control group will receive a placebo. Both groups will also receive standard prenatal multivitamins. The investigators will provide the participants with 5 weeks supply of supplements and the investigators will verify their compliance during the time with daily check-off logs of supplement consumption and leftover pill count during their next visit. The participants will come in for a total of 3 visits at times of routine clinical care. The first visit will be between 24 and 28 weeks of gestation when the investigators enroll the participants, the second visit will be between 28 and 32 weeks of gestation, and the third visit will be between 32 and 36 weeks. The investigators will collect a baseline questionnaire at the first visit (approximately 20 minutes), conduct 3-day dietary recalls after the first visit (approximately 20 minutes, each), and check current weight and collect 20 mL blood samples at all visits. At delivery, the investigators will collect placenta samples and cord blood from the participants. Baseline questionnaire: The questionnaire includes demographic and medical information such as age, due date, ethnicity, parity, marital status, education level, occupation, household income, medical insurance, substance use, medication use, nutrition supplement, family history of chronic diseases, and self-reported pre-pregnancy weight. Dietary assessment: Three 24-hr dietary recalls, 2 on weekdays and 1 on a weekend day, will be obtained from each participant by a trained research assistant (RA) via phone calls following the first visits to quantify dietary choline intakes. The multi-pass method of Harnack and colleagues will be used. The RA will record food identification, quantity, ingredient specification, preparation method and enter data into the Nutrition Data System for Research (NDSR) software to analyze choline intake. The RA will also ask participants about their physical activity (PA) during the past 24 hours using the PA recall instrument. Anthropometric measurements and blood collection: The investigators will measure the participants' weight and height using a medical weighing scale with mechanical height rods during the visits. To minimize food-related concentration differences in plasma biomarkers, participants will be instructed not to consume food for at least 4 hours before their blood draw in the morning. This is a standard recommendation for prenatal visits and is considered as safe. About 20 mL of fasting venous blood will be drawn from each participant during the visits by an investigator. Blood samples will be collected into one EDTA blood collection tube and one serum separator tube and centrifuged to obtain plasma, buffy coat, and serum. Samples will be processed and stored at -80oC at the lab until being used for analytical measurements. Blood glucose management: The investigators will obtain information about fasting glucose, hemoglobin A1C, medication and insulin therapy from medical charts of the participants. Birth information and sample collection at delivery: The participants will notify the investigators when they are admitted to the birth center by phone. The investigators will also send them reminders as their due dates are approaching. In addition, the birth center medical staff will be given a list of participant names, thus they will also contact the investigators if a participant checks in when none of the study staff is on site. Once the baby is delivered, the investigators will collect two tubes of cord blood to retrieve serum, plasma, and buffy coat. The investigators will measure the size and weight of the placenta and process the placenta using the following method: full-thickness placental biopsies will be obtained using a 6-mm Keyes punch and stabilized in RNAlater® (for RNA), or frozen in liquid nitrogen (for other purposes). Samples will be transported to the co-investigator's lab for long-term storage at -80C before use. Dietary intake assessment: As mentioned above, dietary intake data collected from 3-day dietary recalls will be analyzed by the NDSR software. Daily intakes of total choline will be calculated by multiplying the frequency of consumption by the sum of choline derivatives including free choline, phosphatidylcholine (PC), glycerophosphocholine (GPC), phosphocholine, and sphingomyelin of each food item. Daily intakes of betaine (the oxidation product of choline) will be calculated by multiplying consumption frequency and betaine content of each food. Average daily intakes will be calculated as the average daily consumption of total choline and betaine over the 3 days of dietary recalls. Measurements of choline metabolites: Free choline, betaine and other choline derivatives [e.g. PC, GPC, lysophosphatidylcholine, dimethylglycine, trimethylamine oxide (TMAO)] will be measured in maternal plasma using liquid chromatography (LC)-mass spectrometry (MS) /MS as was conducted in previous studies. To ensure the accuracy and reproducibility of data, stable isotope labeled standards will be used in extraction, all samples will be quantified based on a 6-point standard curve, and each sample will be run in duplicate. Samples with a coefficient of variation (CV) % higher than 5% within run or 10% between run will be rerun. Specific Aim 2: Determine the influence of MCS on macronutrient transport and epigenetic modifications in the placenta and cord blood. To achieve this aim, the investigators will determine expression of fat and glucose transporters, as well as the upstream regulating pathways AKT/mTOR in placental biopsies from the participants. The investigators will measure the site-specific DNA methylation of key growth and metabolism related genes in the placenta and cord blood. Analyses of macronutrient transport markers in placental biopsies: To analyze how choline metabolism may be related to placental fat and glucose transport, the investigators will use real-time PCR to quantify mRNA abundance of fat and glucose metabolic genes and transporters, such as glucose transporters GLUT1 and GLUT3, fatty acid metabolism-related genes lipoprotein lipase (LPL), fatty acid translocase (CD36), FATPs, and amino acid transporters SNAT2 and SNAT4. Abundance of respective proteins, as well as phosphorylation of AKT, and mTOR targets 4EBP1 and S6K (indicative of activation of the pathway) will also be measured by western blotting that is routinely conducted in the co-investigator's lab, which includes SDS-PAGE, membrane transfer, incubation with antibodies and visualization with a chemiluminescence imaging system. All primer efficiency and antibody quality will be tested before being used. Both real-time PCR and western blot will be run in triplicate. Epigenetic regulation of growth and metabolism: the investigators will examine site-specific DNA methylation of genes that are published by previous studies as susceptible to prenatal exposures and mediating long-term growth and metabolic programming of the offspring using bi-sulfite sequencing as was previously conducted. Briefly, genomic DNA will be treated with bisulfite reagent, after which the target genomic region will be amplified by PCR. A MassArray EpiTyper system will be used to quantify methylated sequences. Genes of interest include LEP, SREBP1C, and PPARG which mediates fat metabolism, IGF2 which promotes growth, corticotropin releasing hormone (CRH) which promotes cortisol secretion, and glucocorticoid receptor (GR) which receives cortisol signals.

Inclusion Criteria: GDM pregnant women before gestational week 28. English or Spanish speaking, over 18 years of age singleton pregnancy intending to deliver their babies at Maimonides Medical Center Exclusion Criteria: pre-existing hyperglycemia diabetes cardiovascular conditions and liver disease prior to pregnancy.

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