DNA methylation signatures associated with cardiometabolic risk factors in children from India and The Gambia: results from the EMPHASIS study.

BACKGROUND: The prevalence of cardiometabolic disease (CMD) is rising globally, with environmentally induced epigenetic changes suggested to play a role. Few studies have investigated epigenetic associations with CMD risk factors in children from low- and middle-income countries. We sought to identify associations between DNA methylation (DNAm) and CMD risk factors in children from India and The Gambia. RESULTS: Using the Illumina Infinium HumanMethylation 850 K Beadchip array, we interrogated DNAm in 293 Gambian (7-9 years) and 698 Indian (5-7 years) children. We identified differentially methylated CpGs (dmCpGs) associated with systolic blood pressure, fasting insulin, triglycerides and LDL-Cholesterol in the Gambian children; and with insulin sensitivity, insulinogenic index and HDL-Cholesterol in the Indian children. There was no overlap of the dmCpGs between the cohorts. Meta-analysis identified dmCpGs associated with insulin secretion and pulse pressure that were different from cohort-specific dmCpGs. Several differentially methylated regions were associated with diastolic blood pressure, insulin sensitivity and fasting glucose, but these did not overlap with the dmCpGs. We identified significant cis-methQTLs at three LDL-Cholesterol-associated dmCpGs in Gambians; however, methylation did not mediate genotype effects on the CMD outcomes. CONCLUSION: This study identified cardiometabolic biomarkers associated with differential DNAm in Indian and Gambian children. Most associations were cohort specific, potentially reflecting environmental and ethnic differences

Improving women’s diet quality pre-conceptionally and during gestation: effects on birth weight and prevalence of low birth weight; a randomized controlled efficacy trial in India (Mumbai Maternal Nutrition Project)

BACKGROUND: Low birth weight (LBW) is an important public health problem in undernourished populations.OBJECTIVE: We tested whether improving women's dietary micronutrient quality before conception and throughout pregnancy increases birth weight in a high-risk Indian population.DESIGN: The study was a nonblinded, individually randomized controlled trial. The intervention was a daily snack made from green leafy vegetables, fruit, and milk (treatment group) or low-micronutrient vegetables (potato and onion) (control group) from ? 90 d before pregnancy until delivery in addition to the usual diet. Treatment snacks contained 0.69 MJ of energy (controls: 0.37 MJ) and 10-23% of WHO Reference Nutrient Intakes of ?-carotene, riboflavin, folate, vitamin B-12, calcium, and iron (controls: 0-7%). The primary outcome was birth weight.RESULTS: Of 6513 women randomly assigned, 2291 women became pregnant, 1962 women delivered live singleton newborns, and 1360 newborns were measured. In an intention-to-treat analysis, there was no overall increase in birth weight in the treatment group (+26 g; 95% CI: -15, 68 g; P = 0.22). There was an interaction (P &lt; 0.001) between the allocation group and maternal prepregnant body mass index (BMI; in kg/m(2)) [birth-weight effect: -23, +34, and +96 g in lowest (&lt;18.6), middle (18.6-21.8), and highest (&gt;21.8) thirds of BMI, respectively]. In 1094 newborns whose mothers started supplementation ? 90 d before pregnancy (per-protocol analysis), birth weight was higher in the treatment group (+48 g; 95% CI: 1, 96 g; P = 0.046). Again, the effect increased with maternal BMI (-8, +79, and +113 g; P-interaction = 0.001). There were similar results for LBW (intention-to-treat OR: 0.83; 95% CI: 0.66, 1.05; P = 0.10; per-protocol OR = 0.76; 95% CI: 0.59, 0.98; P = 0.03) but no effect on gestational age in either analysis.CONCLUSIONS: A daily snack providing additional green leafy vegetables, fruit, and milk before conception and throughout pregnancy had no overall effect on birth weight. Per-protocol and subgroup analyses indicated a possible increase in birth weight if the mother was supplemented ? 3 mo before conception and was not underweight. This trial was registered at www.controlled-trials.com/isrctn/ as ISRCTN62811278<br/

Protocol for the EMPHASIS study; epigenetic mechanisms linking maternal pre-conceptional nutrition and children's health in India and Sub-Saharan Africa.

BACKGROUND: Animal studies have shown that nutritional exposures during pregnancy can modify epigenetic marks regulating fetal development and susceptibility to later disease, providing a plausible mechanism to explain the developmental origins of health and disease. Human observational studies have shown that maternal peri-conceptional diet predicts DNA methylation in offspring. However, a causal pathway from maternal diet, through changes in DNA methylation, to later health outcomes has yet to be established. The EMPHASIS study (Epigenetic Mechanisms linking Pre-conceptional nutrition and Health Assessed in India and Sub-Saharan Africa, ISRCTN14266771) will investigate epigenetically mediated links between peri-conceptional nutrition and health-related outcomes in children whose mothers participated in two randomized controlled trials of micronutrient supplementation before and during pregnancy. METHODS: The original trials were the Mumbai Maternal Nutrition Project (MMNP, ISRCTN62811278) in which Indian women were offered a daily snack made from micronutrient-rich foods or low-micronutrient foods (controls), and the Peri-conceptional Multiple Micronutrient Supplementation Trial (PMMST, ISRCTN13687662) in rural Gambia, in which women were offered a daily multiple micronutrient (UNIMMAP) tablet or placebo. In the EMPHASIS study, DNA methylation will be analysed in the children of these women (~1,100 children aged 5-7 y in MMNP and 298 children aged 7-9 y in PMMST). Cohort-specific and cross-cohort effects will be explored. Differences in DNA methylation between allocation groups will be identified using the Illumina Infinium MethylationEPIC array, and by pyrosequencing top hits and selected candidate loci. Associations will be analysed between DNA methylation and health-related phenotypic outcomes, including size at birth, and children's post-natal growth, body composition, skeletal development, cardio-metabolic risk markers (blood pressure, serum lipids, plasma glucose and insulin) and cognitive function. Pathways analysis will be used to test for enrichment of nutrition-sensitive loci in biological pathways. Causal mechanisms for nutrition-methylation-phenotype associations will be explored using Mendelian Randomization. Associations between methylation unrelated to supplementation and phenotypes will also be analysed. CONCLUSION: The study will increase understanding of the epigenetic mechanisms underpinning the long-term impact of maternal nutrition on offspring health. It will potentially lead to better nutritional interventions for mothers preparing for pregnancy, and to identification of early life biomarkers of later disease risk

Healthcare choices in Mumbai slums: A cross-sectional study [version 2; referees: 1 approved, 2 approved with reservations]

Background: Informal urban settlements, known as slums, are the home for a large proportion of the world population. Healthcare in these environments is extremely complex, driven by poverty, environmental challenges, and poor access to formal health infrastructures. This study investigated healthcare challenges faced and choices made by slum dwellers in Mumbai, India. Methods: Structured interviews with 549 slum dwellers from 13 slum areas in Mumbai, India, were conducted in order to obtain a population profile of health-related socio-economic and lifestyle factors, disease history and healthcare access. Statistical tools such as multinomial logistic regression were used to examine the association between such factors and health choices. Results: Private providers (or a mixture of public and private) were seen to be preferred by the study population for most health conditions (62% - 90% health consultations), apart from pregnancy (43% health consultations). Community-based services were also preferred to more remote options. Stark differences in healthcare access were observed between well-known conditions, such as minor injuries, pulmonary conditions, and pregnancy and emerging challenges, such as hypertension and diabetes. A number of socio-economic and lifestyle factors were found to be associated with health-related decisions, including choice of provider and expenditure. Conclusions: Better planning and coordination of health services, across public and private providers, is required to address mortality and morbidity in slum communities in India. This study provides insights into the complex landscape of diseases and health providers that slum dwellers navigate when accessing healthcare. Findings suggest that integrated services and public-private partnerships could help address demand for affordable community-based care and progress towards the target of universal health coverage

How do fruit and vegetable markets operate in rural India? A qualitative study of impact of supply and demand on nutrition security

Background:Diets in rural India are cereal based with low intakes of micronutrient-rich foods. The value chains for nutrition approach aims to study supply and demand of such foods. This may aid in development of interventions to improve diets and livelihoods.Objectives:(1) To identify how fruit and vegetables are accessed, (2) to describe and map the structure of value chains for exemplar foods, (3) to understand how foods are priced, and (4) to explore factors that affect decisions about which crops are grown, marketed, and sold.Methods:After stakeholder consultation, we identified 2 fruits (mango and guava) and 2 vegetables (shepu and spinach) as exemplar foods. Criteria for these exemplar foods were that they should be known to participants and there should be variability in intakes. We held 24 interviews with value chain actors including farmers, wholesalers, and vendors of the exemplar foods. Data collection was stopped when no new information emerged. We used inductive thematic coding for our analysis.Results:The value chains for each of the exemplar foods were relatively simple and involved farmers, middlemen, and vendors at either city or village level. The main themes identified as being factors considered when making decisions about which foods to grow and sell were (1) farming resources and assets, (2) quality of produce, (3) environmental conditions, (4) financial factors, (5) transport availability, and (6) consumer demand.Conclusions:There are opportunities to intervene within fruit and vegetable value chains to increase availability, affordability, and access to produce in rural India. Future research is required to determine which interventions will be feasible, effective, and acceptable to the community and other stakeholders

Barriers and facilitators to fruit and vegetable consumption among rural Indian women of reproductive age

Background: Micronutrient insufficiencies are a serious public health problem among women of reproductive age in Low and Middle Income Countries including India, adversely affecting maternal health and economic productivity, and child growth and educational outcomes. Fruit and vegetables are important sources of micronutrients and consumption of these foods is less than recommendations. Objective: To identify perceived barriers and facilitators to fruit and vegetable consumption among women of reproductive age living in rural communities in Eastern Maharashtra, India.Methods: We used qualitative methods and held 9 focus group discussions and 12 one to one interviews. The data collection was stopped when no new information emerged. We used inductive thematic coding to analyse the data. Women aged 18-40 years were recruited from eight villages surrounding the city of Wardha, Maharashtra, India.Results: Women knew that fruit and vegetables were beneficial to health and expressed that they wanted to increase intakes of these foods for themselves and their children. Seven main themes were identified as being barriers or facilitators to fruit and vegetable consumption: 1) Personal factors; 2) Household dynamics; 3) Social and Cultural Norms; 4) Workload; 5) Time pressures; 6) Environmental Factors; 7) Cost. Conclusions: Rural Indian women consumed fruit and vegetables infrequently and said they would like to consume more. Several potentially modifiable factors affecting intakes were identified. Value chain analyses of fruit and vegetables in these communities are important to identify opportunities to intervene to increase consumption. <br/

Effect of a daily snack containing green leafy vegetables on women's fatty acid status: a randomized controlled trial in Mumbai, India

Background and Objectives: There are few data on the fatty acid status of non-pregnant Indian women. Our objective was to investigate the effect of a snack containing green leafy vegetables (GLVs) on women’s erythrocyte long chain polyunsaturated fatty acid status (LCPUFA). Methods and Study Design: Non-pregnant women (n=222) aged 14-35 years from Mumbai slums were randomized to consume a snack containing GLVs, fruit and milk (treatment) or a control snack containing foods of low micronutrient content such as potato and onion, daily under observation. One treatment snack contained a mean (SD) of 54.1 (33.7) mg alpha-linolenic acid (ALA) and one control snack contained 4.1 (3.4) mg ALA. Blood was collected at baseline (0 weeks) and after 12 weeks of supplementation. Erythrocyte fatty acids were analyzed using gas chromatography and expressed as g/100g fatty acids. Plasma malondialdehyde, homocysteine, and erythrocyte superoxide dismutase and glutathione peroxidase were measured. The effect of the treatment on 12 week LCPUFA was assessed using ANCOVA models. Results: Median (IQR) erythrocyte DHA in the treatment group increased from 1.50 (1.11, 2.03) at baseline to 1.86 (1.50, 2.43) (p&lt;0.001) at 12 weeks, and fell in controls from 1.78 (1.37, 2.32) to 1.60 (1.32, 2.04) (p&lt;0.001). The total n-3 fatty acids increased in the treatment group. There was no effect on malondialdehyde and antioxidant enzyme levels. Plasma homocysteine at 0 and 12 weeks was inversely associated with erythrocyte DHA at 12 weeks. Conclusion: Daily consumption of a snack containing GLV improved women’s erythrocyte DHA levels without increasing oxidative stress

Effect of a micronutrient-rich snack taken preconceptionally and throughout pregnancy on ultrasound measures of fetal growth: The Mumbai Maternal Nutrition Project (MMNP)

Improving micronutrient intakes of under-nourished mothers in low- and middle-income countries increases birth weight, but there is little data on the nature and timing during gestation of any effects on fetal growth. Ultrasound measures of fetal size were used to determine whether and when a food-based supplement affected fetal growth. Non-pregnant women living in Mumbai slums, India (N = 6,513), were randomly assigned to receive either a daily micronutrient-rich snack containing green leafy vegetables, fruit, and milk (treatment) or a snack made from lower-micronutrient vegetables (control) in addition to their usual diet from before pregnancy until delivery. From 2,291 pregnancies, the analysis sample comprised 1,677 fetuses (1,335 fetuses of women supplemented for ≥3 months before conception). First-trimester (median: 10 weeks, interquartile range: 9–12 weeks) fetal crown-rump length was measured. Fetal head circumference, biparietal diameter, femur length, and abdominal circumference were measured during the second (19, 19–20 weeks) and third trimesters (29, 28–30 weeks). The intervention had no effect on fetal size or growth at any stage of pregnancy. In the second trimester, there were interactions between parity and allocation group for biparietal diameter (p =.02) and femur length (p =.04) with both being smaller among fetuses of primiparous women and larger among those of multiparous women, in the treatment group compared with the controls. Overall, a micronutrient-rich supplement did not increase standard ultrasound measures of fetal size and growth at any stage of pregnancy. Additional ultrasound measures of fetal soft tissues (fat and muscle) may be informative.</p

Life course programming of stress responses in adolescents and young adults in India: Protocol of the Stress Responses in Adolescence and Vulnerability to Adult Non-communicable disease (SRAVANA) Study

Background: Early life nutrition may affect individuals’ susceptibility to adult non-communicable diseases (NCD). Psychological stress is a well-recognised NCD risk factor. Recent evidence suggests that impaired foetal nutrition alters neuro-endocrine pathways, and hypothalamic-pituitary-adrenal axis feedback systems, resulting in abnormal stress responses, and NCD risk. This study aims to examine adolescent cortisol and cardiovascular stress responses in relation to maternal nutrition and contemporaneous NCD risk markers.Methods: The study sample will be drawn from three well-established birth cohorts in India; the Parthenon cohort, Mysore (N=550, age~20y), the SARAS KIDS prenatal intervention cohort, Mumbai (N=300, age~10-12y) and the Pune Rural Intervention in Young Adults/ PRIYA cohort, Pune (N=100, age~22y). We will perform the ‘Trier Social Stress Test (TSST)’, a well-accepted stress-test module which involves participants performing 5-minutes each of public speaking and mental arithmetic tasks in front of unfamiliar ‘judges’ (stressor). Repeated measures of salivary cortisol and autonomic cardiovascular outcomes relative to the stressor will be assessed. Measures of psychological stress, cognitive function, blood pressure, glucose-insulin metabolism and depression will be carried out. Mechanistic studies including DNA methylation in gluco-corticoid receptor (NR3C1) and 11β-HSD2 gene loci and neuroimaging will be carried out in a subsample. Qualitative interviews and focus group discussions in a subsample of the Parthenon cohort will explore the perception of stress and stressors among the youth.We will convert repeated measures into time-weighted averages before analysis. We will carry out multivariable regression analysis to test the associations. We will further refine the analyses using the mixed-model regression and conditional analyses for the association with repeated measures. Ethics and dissemination: This study has been approved by the research ethics committee of CSI Holdsworth Memorial Hospital, Mysore. The findings will be disseminated locally and at international meetings, and reports will be submitted to open access peer reviewed journals