Specific Objectives:~AIM 1 - Human studies component Comparative assessment of low BMI & normal BMI small intestinal (SI) and fecal microbiomes plus feature of SI mucosal, plasma and fecal proteomes prior to intervention.~Intervention with MDCF-2 to access effect on EED microbiome and physiologic state of low BMI women.~Identify candidate mediators/surrogate biomarker of EED (fecal/plasma) that can be deployed in future clinical studies.~AIM 1A will compare the SI and fecal microbiota and the plasma, duodenal and fecal proteomes/ metabolomes of non-pregnant, young malnourished Bangladeshi women (BMI<18.5kg/m2) who have histopathologic evidence of SI enteropathy versus those with normal BMIs (20-24.9kg/m2) and no histopathologic evidence of enteropathy who have undergone routine endoscopic evaluation for dyspepsia.~AIM 1B The investigators will perform an intervention study, beginning at the end of the first trimester, in which low-BMI (<18.5 kg/m2) pregnant women (aged 18-30 years) will be randomly assigned to receive either MDCF-2 or RUSF for the duration of their pregnancy and during the first 3 postnatal months, in addition to standard antenatal care (n=30/arm). A parallel cohort of age-matched normal-BMI pregnant women who will not receive any nutritional intervention will serve as a reference control group.~AIM 2 - Preclinical Component This Aim has two parts - a therapeutic target identification component (AIM 2A) and a glycan therapeutic development component (AIM 2B).~Test of MDCF-2 and new maternal microbiome directed glycans to ameliorate enteropathy/Biomarker of EED.~Background of the Project including Preliminary Observations:~Undernutrition among women of reproductive age is more common in South Asia than in any other region. In South Asia, the prevalence of maternal undernutrition varies between 10 and 40%. Particularly in Bangladesh, the prevalence of undernutrition among women is much higher than in any other developing country, with more than 30% of women of reproductive age reported to be malnourished. Maternal undernutrition has persistently been described to be a major contributor to child morbidity, mortality, and poor birth outcomes, including low birth weight (LBW), neonatal mortality, and subsequent childhood undernutrition. Maternal undernutrition alone accounts for about 25-50% of intrauterine growth restriction. In such a way, under-nutrition can be transferred from one generation to other. Half of the under-five children in slums of Bangladesh are stunted having retarded linear growth compared to one-third in non-slum areas.~The prevention or treatment of intergenerational malnutrition represents a critical medical need that is yet to be addressed and remains a pressing global health challenge. There is a scarcity of data on the contribution of small intestinal (SI) microbiota to pathogenesis of Environmental Enteric Dysfunction (EED) of malnutrition, as it is difficult to obtain gut biopsy specimens from malnourished individuals, especially children. The Bangladesh Environmental Enteric Dysfunction (BEED) study, involving participants who live in an urban slum (Mirpur) in Dhaka, provided an opportunity to examine the role of the duodenal microbiota in the pathogenesis of EED in children and also performed esophagogastroduodenoscopy (EGD) on thirty-eight 18-45-year-old malnourished (BMI<18.5 kg/m2) women residing in the same resource-poor setting of Mirpur, Dhaka who failed to respond to a egg/milk/micronutrients-based nutritional intervention comparable to that given to children. It was observed that malnourished women of childbearing age living in Mirpur exhibit small intestinal enteropathy resembling that found in Mirpur children with EED. In this proposal, our primary aim is to test the hypothesis that the SI microbiota contributes to SI enteropathy and malnutrition (low-BMI) in young Bangladeshi women of childbearing age. Furthermore, there is some initial evidence that pregnancy outcomes can be predicted by the features of the gut microbiota of pregnant women. The maternal gut microbiota itself may influence the development of the offspring, both in prenatal and postnatal life. As maternal gut microbiota, directly and indirectly, influences the metabolism of the fetus and infants, it may be possible to optimize gestational weight gain (GWG), pregnancy outcomes, and subsequent growth and development of children through modulation of intestinal microbiota in women during pregnancy and lactation. Therefore, a corollary of our primary aim is that direct or indirect transmission of the gut microbiota of mothers with EED to their children perpetuates intergenerational undernutrition.~Conventional nutritional interventions or low-cost water and sanitation interventions may be ineffective in reversing EED-related growth faltering in children, warranting microbiota/microbiome targeted food and other interventions. Prototypes for nutritional interventions that are composed of locally available, affordable, culturally acceptable complementary foods commonly consumed in Bangladesh have recently been developed. Microbiota-directed complementary food (MDCF) formulations were subsequently tested in a pre-proof-of-concept (POC) study involving 12-18-month-old Bangladeshi children with moderate acute malnutrition (MAM) living in the same slum (Mirpur). One of the MDCFs, MDCF-2, was distinguished from the other formulations based on its superior performance based on certain parameters including growth. With this in mind, the investigators propose to include an interventional component involving the administration of MDCF-2 in pregnant and non-pregnant low-BMI women. In this intervention component, beginning at the end of the first trimester, low-BMI (<18.5 kg/m2) pregnant women (aged 18-30 years) will be randomly assigned to receive either the MDCF-2 or Ready-use-supplementary food (RUSF) for the duration of their pregnancy and during the first 3 postnatal months, in addition to standard antenatal care. A parallel cohort of age-matched normal-BMI pregnant women who will not receive any nutritional intervention will serve as a reference control group.~The investigators will test the hypothesis that small intestinal microbiota contributes to small intestinal enteropathy and malnutrition in young Bangladeshi women of childbearing age. An interventional component will be included involving the administration of MDCF-2 in pregnant and non-pregnant low-BMI women. This will be based on the hypothesis that transmission of the microbiota of mothers with EED to their children perpetuates intergenerational undernutrition.~The overarching goals for the current study will be to:~Delineate mechanisms by which the SI microbial community obtained from low-BMI Mirpur women contributes to maternal malnutrition and identify surrogate biomarkers that can be applied to malnourished pregnant women~Test whether MDCF-2 can ameliorate EED as judged by these surrogate endpoints in low-BMI women (who are either pregnant or non-pregnant)~Develop a gnotobiotic mouse model of maternal EED using culture collections from malnourished low-BMI as well as normal BMI women, and identify microbial therapeutic targets in their SI microbiota, and~Perform preclinical tests of MDCF-2 and candidate therapeutic glycans in gnotobiotic mice to identify/develop candidate glycan/synbiotic therapeutics for future clinical studies. These preclinical models will also serve as a platform for testing candidate therapeutics arising from other BMGF-sponsored initiatives where there is good confidence in rationale.~To achieve the goals, in this proposed study the investigators will use two specific aims involving the human subjects, AIM 1A and AIM 1B. In AIM 1A the investigators will compare the SI and fecal microbiota and the plasma, duodenal and fecal proteomes/ metabolomes of non-pregnant, young malnourished Bangladeshi women (BMI<18.5kg/m2) who have histopathologic evidence of SI enteropathy versus those with normal BMIs (20-24.9kg/m2) and no histopathologic evidence of enteropathy who have undergone routine endoscopic evaluation for dyspepsia. In AIM 1B, the investiagors will perform an intervention study, beginning at the end of the first trimester, in which low-BMI (<18.5 kg/m2) pregnant women (aged 18-30 years) will be randomly assigned to receive either MDCF-2 or RUSF for the duration of their pregnancy and during the first 3 postnatal months, in addition to standard antenatal care (n=30/arm). A parallel cohort of age-matched normal-BMI pregnant women who will not receive any nutritional intervention will serve as a reference control group.~Methods:~In AIM 1A, for the healthy group, the investigators plan to recruit a cohort of normal-BMI Bangladeshi women who will undergo esophagogastroduodenoscopy (EGD) for evaluation of functional dyspepsia and our goal is to identify 30 normal BMI participants who have normal duodenal mucosal histology and collect duodenal biopsies, duodenal aspirates, plus plasma and fecal specimens from these participants at the time of endoscopy. In order to enrol these 30 participants with normal duodenal mucosal histology, the investigators are planning to perform EGD on 100 healthy women (BMI 20-24.9 kg/m2) of childbearing age who have been referred for evaluation of functional dyspepsia. Participants will be screened from women attending Gastroenterology OPD of Sheikh Russel National Gastroliver Institute and Hospital and also from the Bangladesh Specialized Hospital, Dhaka, Bangladesh, who meet the inclusion criteria. Undernourished low-BMI (<18.5kg/m2; 18-30 years) women of childbearing age will be enrolled from Bauniabadh and adjacent slum area of Mirpur, Dhaka and EGD will be performed among 60 women at icddr,b Dhaka Hospital, Sheikh Russel National Gastroliver Institute and Hospital, Dhaka or at Bangladesh Specialized Hospital, Dhaka. Participants will be screened through household surveys from the Bauniabadh and adjacent slum area of from Mirpur, Dhaka. The endoscopist is the same individual who performed EGD on the children as well as malnourished women in the BEED study. After EGD, the low-BMI women will be randomized into two groups and receive daily dietary supplementation with either MDCF-2 or RUSF for a period of 90 days, with a further 90 days of follow-up after cessation of the intervention and biological samples will be collected from the participants according to the schedule. Healthy women with normal BMI who underwent EGD will also be followed similarly for 180 days, without any nutritional intervention. For AIM 1B, beginning at the end of the first trimester, in which low-BMI (<18.5 kg/m2) pregnant women (aged 18-30 years) will be randomly assigned to receive either MDCF-2 or RUSF for the duration of their pregnancy and during the first 3 postnatal months, in addition to standard antenatal care (n=30/arm). A parallel cohort of age-matched normal-BMI pregnant women who will not receive any nutritional intervention will serve as a reference control group. The investigators are planning to test a minimal-risk device to collect tissue samples from the small intestine (e.g. trans-nasal introduction tube or TNIT) in a sub-sample of pregnant women under our Aim 1b of our proposed proposal. Therefore, a nested sub-study will be done under aim 1B to test tethered capsule endoscopy (TNIT) to study small-intestinal morphology for EED in a group of 15 pregnant women enrolled in the study based on the assumption that by the time this technology would be validated with conventional endoscopy in women of reproductive age under the Experimental Medicine Platform (Protocol # PR-22082) as well as safety data will be available on pregnant women from the study conducted elsewhere. The participants of this sub-study will be selected randomly from those who will provide their consent to participate in the sub-study. In addition, a preclinical component will be performed aimed at a therapeutic target identification component (AIM 2A) and a glycan therapeutic development objective (AIM 2B) at Washington University in St. Louis.~The investigators plan to recruit a cohort of normal-BMI Bangladeshi women who will undergo esophagogastroduodenoscopy (EGD) for evaluation of functional dyspepsia and our goal is to identify 30 normal BMI participants who have normal duodenal mucosal histology and collect duodenal biopsies, duodenal aspirates, plus plasma and fecal specimens from these participants at the time of endoscopy. Moreover, plasma and fecal samples will be collected according to the follow-up schedule.~Sites:~Participants will be screened from women attending gastroenterology out patient department (OPD) of Sheikh Russel National Gastroliver Institute and Hospital and also from the Bangladesh Specialized Hospital, Dhaka, Bangladesh, who meet the inclusion criteria.~Field Site:~Participants will be screened through household surveys from the Bauniabadh and adjacent slum area of from Mirpur, Dhaka (Fig.4). This area is densely populated and is located 7-8 km from the icddr,b Dhaka Hospital at Mohakhali. Mirpur is selected as the study site because it is inhabited by poor and middle-class families, residential and sanitary conditions are typical of any congested urban settlement, and the study investigators have ongoing research activities in the area. The site has a typical squatter settlement; the average family size of households is 4.5, with 48% females. About 20% of households have a monthly income of only US$62, 30% of mothers never attended school, and only 3% obtained secondary school education. The majority of the people are day laborers, garment workers, and transport workers. Mirpur has a population of about half a million in an area of 14.22 km2. More than 38, 000 people live in each square kilometer of the area compared with the mean of 8229/km2 in Dhaka district and 976/km2 in Bangladesh17.~Screening, recruitment and consenting Census, screening, enrolment of subjects (low-BMI women) will be done in Bauniabadh area of Mirpur in Dhaka city. Women who meet the inclusion-exclusion criteria will be approached about enrolment into this study. A trained Field Research Assistant (FRA) will explain the study in detail, answer any questions from the participant or her relatives, and invite her for enrolment in the study. If she is interested to volunteer in the study, the designated staff will proceed to screening and consenting.~Screening will consist of a review of the inclusion and exclusion criteria listed above. A detailed history will be obtained from the participants following clinical examination. Adult participants who fulfill the inclusion criteria and are not excluded through history and clinical examination will undergo following screening tests based on clinical judgement: Chest x-ray, urine for R/E, ultrasonography of whole abdomen, fasting blood glucose/ HbA1c, stool for occult blood test (OBT) , cancer markers (ie. CEA, CA 15.3, CA 19.9). Patients diagnosed with any abnormal lab test results will be treated by the study physician as per standard guidelines. Patients requiring expert opinion regarding any complicated clinical condition will be referred to a subject specialist accordingly.~If the subject is eligible to participate, the process will proceed to consenting, consisting of a thorough review of the written consent form in a manner appropriate for their literacy level. Prior to signing the consent form, she will have an opportunity to ask any questions about the study. If the FRA determines that participants have demonstrated adequate comprehension of the study, the consent form will be signed by the FRA and the participant. If the participant is not sufficiently literate to read and/or sign the consent form, consenting and a thumbprint signature will be obtained in the presence of a witness who is not associated with the study. The participant will be provided with a copy of the signed consent form.~EGD and biopsy sample collection Endoscopy will be performed at the Sheikh Rasel Gastro Liver Institute and Hospital and the Bangladesh Specialized Hospital. The endoscopist is the same individual who performed EGD on the children as well as malnourished women in the BEED study. Clinical metadata will be collected, including socio-demographic data, dietary history, and use of antibiotics and proton pump inhibitors (PPI) during 12-months prior to EGD [note that consumption of PPIs is common in Bangladesh; in one representative study of an outpatient clinic population the incidence was 72%, with >30% of individuals obtaining PPIs without a prescription]. A tissue transglutaminase (tTG) test will be used to rule out Celiac Disease.
Undernutrition among women of reproductive age is more common in South Asia than in any other region. In South Asia, the prevalence of maternal undernutrition varies between 10 and 40%. There is a scarcity of data on the contribution of small intestinal (SI) microbiota to pathogenesis of Environmental Enteric Dysfunction (EED) of malnutrition, as it is difficult to obtain gut biopsy specimens from malnourished individuals, especially children. The Bangladesh Environmental Enteric Dysfunction (BEED) study, involving participants who live in an urban slum (Mirpur) in Dhaka, provided an opportunity to examine the role of the duodenal microbiota in the pathogenesis of EED in children and also performed esophagogastroduodenoscopy (EGD) on thirty-eight 18-45-year-old malnourished (BMI<18.5 kg/m2) women residing in the same resource-poor setting of Mirpur, Dhaka who failed to respond to a egg/milk/micronutrients-based nutritional intervention comparable to that given to children. In this intervention component, beginning at the end of the first trimester, low-BMI (<18.5 kg/m2) pregnant women (aged 18-30 years) will be randomly assigned to receive either the MDCF-2 or Ready-use-supplementary food (RUSF) for the duration of their pregnancy and during the first 3 postnatal months, in addition to standard antenatal care. A parallel cohort of age-matched normal-BMI pregnant women who will not receive any nutritional intervention will serve as a reference control group.