Fetal Programming of Immune Response and Body Fat by Maternal Obesity
Condition: Body Composition; Obesity
MeSH terms: Obesity, Maternal; Obesity
Study type: Observational

Summary: 
      According to the Chilean National Health Survey 2009-2010, 60% of woman in reproductive age
      are overweight or obese with detrimental consequences on women as well as offspring´s health
      at long term.

      New efforts are required to clarify how increased maternal body fat and obesity previous and
      during pregnancy impinge an increased cardiometabolic and obesity risk in the progeny.
      Nowadays it is clear that obesity in adults constitute a chronic state of sub-clinical
      inflammation characterized by an increased infiltration of monocytes in the adipose tissue as
      well as an imbalance between increased pro- (M1) and decreased anti- (M2) inflammatory
      macrophage polarization. Increased inflammatory markers have been found in obese children as
      young as 3 years of age, but if these markers are present at birth is completely unknown.

      Therefore, unveiling the mechanisms implicated in the capability of monocytes to
      differentiate into pro-inflammatory macrophages at birth would contribute to establish early
      markers of the potential risk to develop cardio-metabolic diseases. In this context,
      modulation of M1-M2 polarization seems to be crucial for the development of altered immune
      response, and this process would be tightly regulated by epigenetic mechanisms.

      On the other hand, long chain polyunsaturated fatty acids (LCPUFAs) play a role as precursors
      of cellular membrane components and modifiers, and as precursors of a plethora of signaling
      molecules that participates in cardiovascular, metabolic and immune functions. Additionally,
      DHA regulates gene expression in monocytes and macrophages altering the M1/M2 polarization.
      The supplementation with DHA in a high risk population of pregestational obese mothers, with
      known low n-3 intake, would have an important impact on newborn and infant % body fat. An
      improvement in the n-6/n-3 LCPUFA ratio during pregnancy in humans could represent a primary
      prevention strategy to revert fetal and neonatal high body fat and a healthy immune system
      maturation.

      The hypothesis of this proposal is that neonates born from obese mothers supplemented with
      DHA during pregnancy show a reduction in specific markers of high-risk of obesity. These
      markers would be evidenced as a lower percent of body fat at birth and at 4 months of age, as
      well as the reversion of functional and epigenetic changes in neonatal monocytes at birth,
      compared to neonates from obese mothers with low DHA intake.
    

Detailed description: 
      AIMS:

      In neonates born from pregnant women with normal BMI (> 18.5 and <24,9) at the first prenatal
      visit (<14 weeks of gestation) (Reference Group) and with pre-gestational obesity (BMI >30
      Kg/m2) with low (customary) DHA intake (200 mg/day) (MO Group) and pre-gestational obese
      women supplemented with a high dose of DHA (800 mg/day)(MO+DHA Group) we will:

        -  Analyze whether maternal DHA supplementation modifies the body composition in neonates
           and children at 4 months of age.

        -  Determine whether maternal supplementation with DHA during pregnancy modifies the
           balance of circulating pro- and anti-inflammatory cytokines and metabolic markers in the
           mother during pregnancy, the fetus, the placenta and the child at 4 months of age

        -  Determine whether the response of macrophages to inflammatory mediators associates with
           altered epigenetic markers in pro- and anti-inflammatory obesity-related-genes in fetal
           monocytes from obese mothers and if DHA maternal supplementation reverts this phenotype.

        -  Determine whether maternal obesity translates into changes in the genome-wide DNA
           methylation profile in fetal monocytes at birth and the impact of maternal DHA
           supplementation on this epigenetic effect.

      METHODOLOGY. The EpiFat study, is a nested cohort in the Maternal obesity control ThrouGh
      Healthy nuTrition, MIGHT study (PI: Dr. Garmendia NCT02574767). MIGHT study randomized 1000
      patients that began their prenatal care before 14 weeks of gestation to one of four parallel
      study arms: 2 groups received 200 mg/day DHA (based on Schizochytrium oil, 100% DHA, DSM) and
      2 groups 800 mg/day DHA, each group had a group with and one without home-based diet and
      physical activity interventions.

      Recruitment and follow up: At admission to the pre-delivery unit, midwives of the Dr. Sótero
      del Río Hospital invite all the pregnant women who fulfill eligibility criteria to
      participate in this study and carries out informed consent process.

      After recruitment in the EpiFat study, at delivery moment, midwife collect cord blood samples
      and the placenta tissue. Between 24 to 48 hrs. after delivery, patients are visited by the
      research midwife to perform anthropometry to the newborn and collect sociodemographic data
      and clinical information about women health and delivery process.

      At 4 months the infants have a visit, in the Research Center in Eating Environment and
      Prevention of Associated to Nutrition Chronic Diseases (CIAPEC for his Spanish acronym) from
      the Institute of Nutrition and Food Technology (INTA for his Spanish acronym), to evaluate
      anthropometry and body composition. In this visit, infant blood samples (5 ml.) and infant
      health status information are collected. Furthermore, maternal anthropometry is performed,
      and infant feeding and food frequency surveys are applied to the mother.

      Sample Size:

      To calculate sample size for body fat percentage (primary outcome) were considered a mean
      difference of 2.5%, and 3.0 SD that were described by Catalano. Considering a power of 80%
      and p-value of 0.05, the sample size estimation was 45 subjects per group. The calculation
      was made using the software Statistics/Data Analysis STATA version 14.0 (Texas, USA).
      Considering a 20% of loss to follow up the sample size was adjusted to 54 subjects per group.

      For the secondary outcome "the epigenetic-driven inflammatory response of neonatal
      macrophages in vitro" there is no evidence published. However, previous studies from our
      group have shown that: For % of methylation in the IL-1β promoter, a sample size of 15
      newborns per group from normal weight and obese mother was enough to find a statistical
      difference between the groups, and for IL-10 in macrophages derived from neonatal monocytes
      10 subjects per group was needed. Therefore, for these outcomes it was considered a sample
      size of 15 subjects per group.

      NEWBORN AND MATERNAL VARIABLES OF THE STUDY.

      The variables included in the study are:

        -  Variables of newborn/infants: % of body fat & epigenetic-driven inflammatory response in
           neonatal macrophages.

        -  Independent variables of the mothers during pregnancy (week 14 and 24-28 of pregnancy):
           maternal age (years), BMI (kg/m2, classification), weight (kg), height (m), gestational
           age (week + days), IL-6, IL-1β, TNFα, MCP-1, IL-10 (pg/mL), triglycerides, cholesterol,
           LDL-cholesterol, VLDL, HDL (all in mg/dl), fasting glycaemia (mg/dL) and insulin
           (pg/mL), HOMA-IR, adiponectin (ug/mL), plasma levels of DHA (μmol/l).

        -  Independent variables of the mother at birth: gestational weight gain (kg,
           classification), type of delivery, hours of labor.

        -  Independent variables of the newborn: sex, age (weeks at birth, postnatal weeks), weight
           (g), length (cm), head circumference (cm), ponderal index (kg/m3), weight for
           gestational age (classification), IL-6, IL-1β, TNFα, MCP-1, IL-10 (pg/mL), plasma levels
           of DHA (μmol/l), skinfolds (mm) (biceps, triceps, subscapular, suprailiac and tight).

        -  Other maternal variables: parity, smoking habit, education (years), type of delivery,
           placental weight (g).

      EX VIVO & IN VITRO STUDIES UMBILICAL CORD BLOOD. Umbilical cord blood are collected after
      delivery and before placental expulsion in Terumo blood collecting bags (40-100 ml) and
      stored at 4°C until processing (within 1-5 hours after delivery).

      DHA LEVELS IN MATERNAL AND FETAL BLOOD: These will be measured in MIGHT Study by gass
      chromatography.

      MONOCYTE ISOLATION, CELL CULTURE AND MACROPHAGE DIFFERENTIATION. Peripheral blood mononuclear
      cells (PBMC) are separated shortly after birth by Ficoll density gradient using
      Histopaque-1077 (Sigma-Aldrich, Missouri, US) centrifugation and incubated in RPMI 1640
      medium (Sigma-Aldrich, Missouri, US) and seeded at density of 8 to 10 × 106 cells/mL in 100
      mm culture dishes at 5% CO2 for 2 h at 37°C. Monocyte identity is assessed by flow cytometry.
      Monocytes are cultured for 7 days to assess macrophage differentiation. Macrophage
      polarization is induced by exposing for 24 hours to 100 ng/mL of LPS (SigmaAldrich, Missouri,
      US) and 20 ng/mL of INFγ (R&D Systems, Minneapolis, US) for M1 or, alternatively, to 20 ng/mL
      of IL-4 (R&D Systems, Minneapolis, US) for M2 in RPMI medium supplemented with 5% FBS.

      Cells are seeded on plastic culture dishes and maintained in a controlled atmosphere
      incubator, at 37°C with different experimental conditions.

      QUANTIFICATION OF PRO- AND ANTI-INFLAMMATORY CYTOKINES, INSULIN, LEPTIN & ADIPONECTIN IN
      MATERNAL, NEONATAL AND INFANT PLASMA.

      These pro and anti-inflammatory mediators, leptin and insulin are quantified using a
      multiplexed (Bio-Plex platform) immunometric assay (MILLIPLEX xMAP High Sensitivity Human
      Cytokine Panel, and human metabolic panel, Millipore, Billerica, MA, USA). Quantification of
      total adiponectin in umbilical cord plasma is done with High Molecular Weight & Total
      Adiponectin (ALPCO) ELISA kit, according to the manufacturer's instructions.

      PREPARATION OF PLACENTAL HOMOGENATES. Placentas are collected at delivery and weighed after
      trimming of the cord and membranes, immediately placed on ice after delivery and dissected.
      The chorionic plate, amniotic sac, and decidua removed. Approximately 50 g of villous tissue
      is cut into small pieces and rinsed with ice-cold physiological saline. Tissue will be placed
      in ice-cold buffer D [250 mM sucrose, 0.7 μM pepstatin A, 1.6 μM antipain, 80 μM aprotinin, 1
      mM EDTA, 10 mM HEPES-Tris (pH 7.4)] at 4°C and homogenized on ice using a polytron. The
      homogenate is snap-frozen in liquid nitrogen and stored at -80°C until analysis or further
      processing.

      QUANTITATIVE PCR FOR IN VITRO EXPRESSION OF CYTOKINES. RNA is isolated from monocytes using a
      commercial kit (GenElute, Sigma), according to manufacturer instructions. RT is perform be
      with the ImProm-II Reverse.

      Transcription System (Promega). PCR is carried out using oligonucleotide primers for human
      TNFα, IL-6, Il-1β, MCP-1 and IL-10. The levels of mRNAs for GADPH, ATP5F1 and RPLP2 are used
      as internal references. Primers are validated for use: checking amplification, primer
      concentrations, efficiency primers-HK and stability of HK in experimental conditions.
      Relative expression of target genes is calculated using the 2-delta delta ct method.

      FLOW CYTOMETRY FOR MONOCYTES CHARATERIZATION. The expression of different surface markers is
      evaluated using flow cytometry, which is performed either in whole blood for identification
      of monocyte subtypes or in vitro differentiated macrophages for identification of the
      immunophenotype. Among circulating monocytes (CD86+ cells) classic and non-classic subtypes
      are assessed according to their surface expression of CD14 and CD16.

      Immunophenotype of macrophages (i.e. M1 and M2) are assessed according to the expression of
      specific markers (e.g. CCR2, CXCL13, CX3CR, CD62L, KLF4, TNFα, NO production). Samples are
      stained in the laboratory and sent to a Flow Cytometry Facility at Universidad de Chile
      (CyAnTM ADP analyzer (Beckman Coulter®), within 48 hours.

      GLOBAL DNA METHYLOME. The DNA Methylome analysis are done in collaboration with Dr. Graham
      Burdge and Dr. Karen Lillycrop from Southampton using the Infinium Methylation EPIC BeadChip
      kit from Illumina.

      DNA METHYLATION ANALYSIS. DNA methylation status of the promoter regions (approximately 400
      bp from the transcription start site) of the genes coding for TNF-α, IL-6, IL-1β, MCP-1,
      IL-10, PPARγ and PCG1α will be determined using bisulfite modification coupled to DNA
      sequencing. Briefly, total DNA extracts from cells will be treated with sodium bisulphite to
      convert non-methylated cytosine to uracil, and promoter regions will be amplified by PCR
      using specific according to the strategy used and published by our group. Briefly, one of the
      PCR primers will be designed including a 5'-biotin tag, which will allow purification the
      amplicon directly from the PCR mix. Then biotin-containing amplified DNA strand will be
      transferred to a PyroMark Q96 MD pyrosequencer (Qiagen) for sequencing. Site-specific CpG
      methylations will be determined as percentage, comparing the signal intensity of
      non-bisulfite-sensitive CpG (conserved C, methylated CpG) and bisulfite-sensitive CpG
      (converted C→T, unmethylated CpG).

      STATISTICAL ANALYSIS. For descriptive statistics will be applied mean ± SD or median and
      interquartile range for quantitative variables, and percentage with n for categories.
      Distributions will be verified with a Shapiro Wilk test. Differences in fat mass and
      circulating markers will be determined by ANOVA or Kruskal Wallis according to data
      distribution. Spearman (parametric) or Pearson (no parametric) correlation will be applied to
      determine the association between dependent and independent (maternal and neonatal)
      variables. Independent variables will be fitted to logistic and lineal regression models to
      evaluate interactions as well as to identify confounding factors. For in vitro studies,
      comparisons between two or more groups will be performed by means of Student's unpaired
      t-test and analysis of variance (ANOVA), respectively. If the ANOVA demonstrates a
      significant interaction between variables, post hoc analyses were performed by Dunns when
      comparing selected groups and multiple-comparison Bonferroni correction test. Data for in
      vitro responses to cytokines will be fitted to dose-response curves from which maximal
      response and drug potency (pD2 = -Log EC50) will be obtained. Comparison of curves and
      maximal responses under different conditions will be analysed by ANOVA. All the analyses will
      be carried out with the statistical software Graphpad Prism, STATA or SPSS considering a
      p<0.05 as cut-off for statistical significance.
    

Intervention name(s): DHA
Intervention type(s): Dietary Supplement
Intervention description(s): Women received supplementation of Based on Schizochytrium oil, 100% DHA (DSM) from 14 weeks of pregnancy until delivery.
Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Pregnant women of Puente Alto district of Santiago, Chile.
      
Biospecimen description: 
      Maternal blood (plasma & serum) Umbilical cord blood (plasma, serum and cord blood
      mononuclear cells) Placental tissue (biopsies and IHQ) Infant blood samples at 4 months
      (plasma & serum)
    
Keywords: Maternal obesity; Body composition; DHA; n-3 LCPUFA