R.D. Potdar

Improving women's diet quality preconceptionally 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 womens 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 β-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 < 0.001) between the allocation group and maternal prepregnant body mass index (BMI; in kg/m2) [birth-weight effect: −23, +34, and +96 g in lowest (<18.6), middle (18.6–21.8), and highest (>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.

A Daily Snack Containing Leafy Green Vegetables, Fruit, and Milk before and during Pregnancy Prevents Gestational Diabetes in a Randomized, Controlled Trial in Mumbai, India.

Background: Prospective observational studies suggest that maternal diets rich in leafy green vegetables and fruit may help prevent gestational diabetes mellitus (GDM). Objective: Our objective was to test whether increasing women’s dietary intake of leafy green vegetables, fruit, and milk before conception and throughout pregnancy reduced their risk of GDM. Methods: Project SARAS (“excellent”) (2006–2012) was a nonblinded, individually randomized, controlled trial in women living in slums in the city of Mumbai, India. The interventions included a daily snack made from leafy green vegetables, fruit, and milk for the treatment group or low-micronutrient vegetables (e.g., potato and onion) for the control group, in addition to the usual diet. Results for the primary outcome, birth weight, have been reported. Women were invited to take an oral-glucose-tolerance test (OGTT) at 28–32 wk gestation to screen for GDM (WHO 1999 criteria). The prevalence of GDM was compared between the intervention and control groups, and Kernel density analysis was used to compare distributions of 120-min plasma glucose concentrations between groups. Results: Of 6513 women randomly assigned, 2291 became pregnant; of these, 2028 reached a gestation of 28 wk, 1008 (50%) attended for an OGTT, and 100 (9.9%) had GDM. In an intention-to-treat analysis, the prevalence of GDM was reduced in the treatment group (7.3% compared with 12.4% in controls; OR: 0.56; 95% CI: 0.36, 0.86; P = 0.008). The reduction in GDM remained significant after adjusting for prepregnancy adiposity and fat or weight gain during pregnancy. Kernel density analysis showed that this was explained by the fact that fewer women in the treatment group had a 2-h glucose concentration in the range 7.5–10.0 mmol/L. Conclusions: In low-income settings, in which women have a low intake of micronutrient-rich foods, improving dietary micronutrient quality by increasing intake of leafy green vegetables, fruit, and/or milk may have an important protective effect against the development of GDM. This trial was registered at www.controlled-trials.com as ISRCTN62811278.

Effects of a food-based intervention on markers of micronutrient status among Indian women of low socio-economic status.

Intakes of micronutrient-rich foods are low among Indian women of reproductive age. We investigated whether consumption of a food-based micronutrient-rich snack increased markers of blood micronutrient concentrations when compared with a control snack. Non-pregnant women (n 222) aged 14–35 years living in a Mumbai slum were randomised to receive a treatment snack (containing green leafy vegetables, dried fruit and whole milk powder), or a control snack containing foods of low micronutrient content such as wheat flour, potato and tapioca. The snacks were consumed under observation 6 d per week for 12 weeks, compliance was recorded, and blood was collected at 0 and 12 weeks. Food-frequency data were collected at both time points. Compliance (defined as the proportion of women who consumed ≥ 3 snacks/week) was >85 % in both groups. We assessed the effects of group allocation on 12-week nutrient concentrations using ANCOVA models with respective 0-week concentrations, BMI, compliance, standard of living, fruit and green leafy vegetable consumption and use of synthetic nutrients as covariates. The treatment snack significantly increased β-carotene concentrations (treatment effect: 47·1 nmol/l, 95 % CI 6·5, 87·7). There was no effect of group allocation on concentrations of ferritin, retinol, ascorbate, folate or vitamin B12. The present study shows that locally sourced foods can be made into acceptable snacks that may increase serum β-carotene concentrations among women of reproductive age. However, no increase in circulating concentrations of the other nutrients measured was observed.

GWAS identifies population-specific new regulatory variants in FUT6 associated with plasma B12 concentrations in Indians

Abstract Vitamin B12 is an important cofactor in one‐carbon metabolism whose dysregulation is associated with various clinical conditions. Indians have a high prevalence of B12 deficiency but little is known about the genetic determinants of circulating B12 concentrations in Indians. We performed a genome‐wide association study in 1001 healthy participants in the Pune Maternal Nutrition Study (PMNS), replication studies in 3418 individuals from other Indian cohorts and by meta‐analysis identified new variants, rs3760775 (P = 1.2 × 10−23) and rs78060698 (P = 8.3 × 10−17) in FUT6 to be associated with circulating B12 concentrations. Although in‐silico analysis replicated both variants in Europeans, differences in the effect allele frequency, effect size and the linkage disequilibrium structure of credible set variants with the reported variants suggest population‐specific characteristics in this region. We replicated previously reported variants rs602662, rs601338 in FUT2, rs3760776, rs708686 in FUT6, rs34324219 in TCN1 (all P < 5 × 10−8), rs1131603 in TCN2 (P = 3.4 × 10−5), rs12780845 in CUBN (P = 3.0 × 10−3) and rs2270655 in MMAA (P = 2.0 × 10−3). Circulating B12 concentrations in the PMNS and Parthenon study showed a significant decline with increasing age (P < 0.001), however, the genetic contribution to B12 concentrations remained constant. Luciferase reporter and electrophoretic‐mobility shift assay for the FUT6 variant rs78060698 using HepG2 cell line demonstrated strong allele‐specific promoter and enhancer activity and differential binding of HNF4&agr;, a key regulator of expression of various fucosyltransferases. Hence, the rs78060698 variant, through regulation of fucosylation may control intestinal host‐microbial interaction which could influence B12 concentrations. Our results suggest that in addition to established genetic variants, population‐specific variants are important in determining plasma B12 concentrations.

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

Abstract 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.

Candidate genes linking maternal nutrient exposure to offspring health via DNA methylation: a review of existing evidence in humans with specific focus on one-carbon metabolism.

Abstract Background Mounting evidence suggests that nutritional exposures during pregnancy influence the fetal epigenome, and that these epigenetic changes can persist postnatally, with implications for disease risk across the life course. Methods We review human intergenerational studies using a three-part search strategy. Search 1 investigates associations between preconceptional or pregnancy nutritional exposures, focusing on one-carbon metabolism, and offspring DNA methylation. Search 2 considers associations between offspring DNA methylation at genes found in the first search and growth-related, cardiometabolic and cognitive outcomes. Search 3 isolates those studies explicitly linking maternal nutritional exposure to offspring phenotype via DNA methylation. Finally, we compile all candidate genes and regions of interest identified in the searches and describe their genomic locations, annotations and coverage on the Illumina Infinium Methylation beadchip arrays. Results We summarize findings from the 34 studies found in the first search, the 31 studies found in the second search and the eight studies found in the third search. We provide details of all regions of interest within 45 genes captured by this review. Conclusions Many studies have investigated imprinted genes as priority loci, but with the adoption of microarray-based platforms other candidate genes and gene classes are now emerging. Despite a wealth of information, the current literature is characterized by heterogeneous exposures and outcomes, and mostly comprise observational associations that are frequently underpowered. The synthesis of current knowledge provided by this review identifies research needs on the pathway to developing possible early life interventions to optimize lifelong health.

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

BackgroundAnimal 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.MethodsThe 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 (~1100 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.ConclusionThe 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.

Processes involved in developing palatable micronutrient-rich snacks for a pre-conceptional and intra-pregnancy intervention trial: The Mumbai Maternal Nutrition Project

The objective of the current study was to develop palatable food supplements produced from locally-available vegetarian ingredients that would improve the quality of the diet of young Indian women living in Mumbai slums. The supplements were to be used as a daily intervention in a currently-ongoing randomised controlled trial testing the hypothesis that ‘enhancing the micronutrient content of women’s diets before and during pregnancy would improve fetal growth and development of their offspring’. A vehicle in the form of a cooked snack food, prepared by kitchen staff recruited from the local slum community in a low-tech kitchen, was developed. The snack was distributed to women 6 d/week and was designed to provide supplementary green leafy vegetables (GLV), fruit and milk Samples of the snacks were chemically analysed to measure target levels of nine ‘marker’ nutrients. Acceptability of the snack was assessed by holding sensory evaluation sessions with project staff and trial participants assigning scores to the snacks based on taste, texture and appearance. As an objective measure of acceptability the women’s compliance with consuming the intervention was recorded on a daily basis by project health workers throughout the trial and these data were used to assess the uptake of each snack as the trial progressed. Results of sensory evaluation, objective compliance assessment and nutrient analyses were used to develop a vehicle that met the requirements for improving micronutrient intake, while at the same time being palatable and acceptable to the women. Over the course of the trial a variety of recipes were distributed to the women to prevent monotony. Acceptability and some target micronutrient levels were achieved using combinations of fresh GLV, dried fruits and milk powder. All snacks contained ‡ 25 g one or more of a variety of fresh locally-sourced GLV e.g. coriander (Coriandrum sativum), colocasia (Colocasia esculenta) or radish leaf (Raphanus sativus), 12 g milk powder and ‡ 4 g dehydratedfruit. In addition, the snacks contained binding ingredients such as chickpea (Cicer arietinum) flour or jowar (sorghum) flour. Mean micronutrient levels of the final product (per 60 g serving) were: b-carotene 123 retinol equivalents; folate 68 mg; riboflavin 0.14 mg; Fe 4.9 mg; Ca 195 mg; vitamin B12 0.24 mg. These values are between 12 % and 43 % of the UK estimated average requirements (2) . Target vitamin C levels were not achieved. It has been demonstrated that it is possible to develop palatable culturally-acceptable and safe micronutrient-rich food supplements using a ‘low-tech’ approach, a local work-force and locally-available fresh and dehydrated ingredients. The effect of these supplements on the nutritional status of the mothers and the health outcomes of their babies is yet to be reported.

Dietary intakes of folate and vitamin B12 in female urban slum-dwellers in Mumbai, India

Aims: To estimate dietary intakes of folate and vitamin B12 (vital nutrients during the early stages of human development) in women of childbearing age (15 40 years). Study design: Baseline data were collected from 1668 women recruited to an intervention trial. Materials: An interviewer-administered food frequency questionnaire (101 items, 15 food-groups) was developed to obtain intakes in the past month. Data were recorded as frequencies ranging from three times/day to once/month or less. Daily folate and B12 intakes were calculated, assuming standard portion sizes, and using published nutrient-content tables and (for GLVs) laboratory analysis. Outcome measures: Adequacy of intakes according to ICMR Recommended Daily Allowances (RDAs; folate 100 mg/day; B12 1 mg/day).