Microbiome and Malnutrition in Pregnancy (MMiP)

Study Overview
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Brief Summary
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Nutritional status during pregnancy plays an important role in maternal health and birth outcomes. While few factors impacting nutritional status during pregnancy have been identified, studies of undernutrition in children have revealed a key role for the gut microbiome. Remarkably, studies examining the dynamics of the maternal gut microbiome before and during pregnancy and its impact on birth outcomes are limited. This study is being conducted to investigate how a mother's nutritional status and her gut microbiome during pregnancy contribute to the birth outcomes and health of her baby. The gut microbiome is the totality of microorganisms (e.g. bacteria, viruses, fungi) living in the gastrointestinal tract. This study will focus on married pregnant women 24 years and younger living in Matiari District in Pakistan. The focus is on younger women due to their vulnerability to undernutrition. Pregnant participants, and upon delivery, their newborns will be followed throughout pregnancy and for a year afterwards. Throughout this period, the investigators will collect stool samples, rectal swabs, blood samples, health assessments, nutritional and dietary assessments and birth/ labour details. The goal is to define the relationship between a mother's nutritional status and her microbiome dynamics during pregnancy and how they contribute to the birth outcomes and growth of her newborn. Investigators hypothesizes that alterations of the microbiota in the maternal gut (dysbiosis) is exacerbated by nutritional status or pathogen exposure during pregnancy. This impacts weight gain because of impaired nutrient absorption, and can lead to corresponding negative birth outcomes.

Detailed Description
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This project represents the first systematic investigation of the impact of the microbiome on nutritional status during pregnancy in young women and directly aligns with global health initiatives focused on this vulnerable cohort. The goal of the study is to define the relationships between host nutritional status and microbiome dynamics during pregnancy and how they contribute to birth outcomes. The gut microbiome has a profound influence on host nutritional status. Dysbiosis (loss of diversity/beneficial microbes and gain of pathobionts) has emerged as a major factor in the development of undernutrition. Despite the importance of nutrition during pregnancy, few studies have examined the role of the microbiome on maternal health and birth outcomes. Further, little is known concerning the influence of enteric eukaryotic microbes, such as parasites, on the bacterial microbiome and host nutrition. At the core of this study are two complementary cohorts of young women that provide an exceptional opportunity to obtain longitudinal samples to monitor the dynamic relationships between microbiome community structure and function with gut health and host nutritional status. This registration is for the Matiari, Pakistan cohort of the study, where there is known to be a high prevalence of undernutrition among young women. This cohort is expected to yield insights into the influence of eukaryotic microbes that are often viewed as asymptomatic. The target demographic of the study is young, married mothers, ≤24 years in Matiari District within the province of Sindh, Pakistan. Matiari District is representative of rural settings in Pakistan The investigators have identified this younger demographic due to the lack of knowledge on the microbiome of young women, and their vulnerability to undernutrition. A second complementary cohort will be based Toronto, Canada. This project will yield insights into the relationships between prokaryotic and eukaryotic microbes in the gut and their associations with maternal health and birth outcomes. The central hypothesis of the study is that alterations of the microbiota in the maternal gut (dysbiosis) exacerbated by nutritional status or pathogen exposure during pregnancy, impacts weight gain because of impaired nutrient absorption, leading to corresponding negative birth outcomes. The study will be a prospective, longitudinal, observational study to investigate the impact and relationship between prokaryotic and eukaryotic microbes in the gut and their association with maternal health and birth outcomes among married young women ≤24 years residing in Matiari District. . The study will aim to recruit 400 women into two groups based on BMI at time of recruitment (normal BMI will be defined as between 20 and 24.9 kg/m2 and low BMI will be defined as less than 20 kg/m2). With a goal of having 200 participants within the normal BMI group and 200 participants within the low BMI group. Although this is the recruitment aim, in the event that the investigators are unable to recruit 200 women with a low BMI, more women will be recruited that fall within the normal BMI range. The study will follow women and their infants over the course of their pregnancy and for a year postpartum, collecting stool, rectal and blood samples, nutritional information, heath assessments, anthropometric measurements and empowerment metrics at different time points.

Official Title
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Elucidating the Dynamics and Impact of the Gut Microbiome on Maternal Nutritional Status During Pregnancy

Conditions
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Weight Gain, Pregnancy, Microbial Colonization, Pregnancy Related, Pregnancy Loss, Pregnancy Complications, Pregnancy; Parasitic Disease, Parasitic Disease, Microbial Disease, Infant Malnutrition, Malnutrition, Malnutrition in Pregnancy, Breastfeeding

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Consent provided Married female aged 17-24 years In good general health, with no comorbidities Absence of COVID19 symptoms Intend to comply with study procedures and follow up Exclusion Criteria: Women who do not meet the enrollment age criteria Women participating in interventional clinical trials Women who intend to leave the study area Women who cannot comply with study procedure's and follow-up Illness and other co-morbidities Signs of potential COVID19 infection BMI higher than 24.9 kg/m2 Women who already have a member of their household participating Women who have taken antibiotics within the past 3 months Women who are past 16 weeks post- conception

Ages Eligible for Study
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Minimum Age: 17 Years
Maximum Age: 24 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| To assess if alterations of the microbiota in the maternal gut (dysbiosis) are corelated with changes in maternal gestational weight gain | The primary endpoint will be the change in maternal gestational weight gain (GWG) during pregnancy, measured between the first (8-16 weeks post-conception) and second time point (30-34 weeks post-conception) | 8-20 weeks post-conception, 30-34 weeks post-conception | 
| To determine the correlation between maternal microbiome dysbiosis during pregnancy and birth weight. | The primary endpoint will be change in birthweight measured in kilograms. | At birth | 
| To determine the correlation between maternal microbiome dysbiosis during pregnancy and infant growth | The primary endpoint will be change in WHO z-scores during first year of infant's life. These z-scores will be calculated for weight (measured in kg), length and head circumference (measured in cm). | 3month, 6month and 12month postpartum | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Anthropometrics | Maternal BMI | 8-16 weeks post-conception, 30-34 weeks post-conception, delivery, 3-months post-partum, 6 months post-partum and 12 months post-partum ] | 
| Anthropometrics: Maternal middle upper arm circumference | Measured in cm | 8-16 weeks post-conception, 30-34 weeks post-conception, 3-months post-partum,and 12 months post-partum | 
| Anthropometrics: Maternal triceps skinfold thickness | Measured in mm | 8-16 weeks post-conception, 30-34 weeks post-conception, 3-months post-partum, and 12 months post-partum | 
| Anthropometrics: Maternal height | measured in cm | 8-16 weeks post conception, 30-34 weeks post conception, delivery, 3 months post-partum and 12 months post partum | 
| Anthropometrics: Maternal weight | measured in kg | 8-16weeks post conception, 30-34 weeks post conception, delivery, 3 months post-partum and 12 months post-partum | 
| Maternal blood biomarker-1 | Concentration of HB in g/dL, marker of anemia. | 8-16 weeks post-conception, 30-34 weeks post-conception, and 12 months post-partum | 
| Maternal blood biomarker-2 | Level of MCV in whole blood measured in femtoliters (fL). | 8-16 weeks post-conception, 30-34 weeks post-conception, and 12 months post-partum | 
| Maternal blood biomarker-3 | Concentration of Ferritin in serum measured in ng/mL, marker of iron stores in blood. | 8-16 weeks post-conception, 30-34 weeks post-conception, and 12 months post-partum | 
| Maternal blood biomarker-4 | Concentration of CRP in blood, measured in mg/dL, marker of inflammation . | 8-16 weeks post-conception, 30-34 weeks post-conception, and 12 months post-partum | 
| Infant blood biomarker-1 | Concentration of HB in g/dL, marker of anemia. | 1 year infant age | 
| Infant blood biomarker-2 | Level of MCV in whole blood measured in femtoliters (fL). | 1 year infant age | 
| Infant blood biomarker-3 | Concentration of Ferritin in serum measured in ng/mL, marker of iron stores in blood. | 1 year infant age | 
| Infant blood biomarker-4 | Concentration of CRP in blood, measured in mg/dL, marker of inflammation . | 1 year infant age | 
| Infant sex | Male Female | At birth | 
| Infant morbidity | Assessed through infant health assessment questionnaire | at 3 months, 6 months and 12 months | 
| Maternal morbidity | Assessed through health assessment questionnaire | 8-16 weeks post-conception, 30-34 weeks post-conception, 3 months post-partum, 6 months post-partum and 12 months post-partum | 
| Infant growth: weight | Measured in kg | within 72 hours of birth, 3 months, 6 months and 12 months | 
| Infant growth: length | Measured in cm | within 24 hours of birth, 3 months, 6 months and 12 months | 
| Infant growth: head circumference | Measured in cm | within 72 hours of birth, 3 months, 6 months and 12 months | 
| Infant growth: mid upper arm circumference | Measured in cm | within 72 hours of birth, 3 months, 6 months and 12 months | 
| Infant growth: triceps skinfold thickness | Measured in mm | within 72 hours of birth, 3 months, 6 months and 12 months | 
| Gestational age at birth | Measured in weeks | Within 72 hours of birth | 
| Maternal age | Age between 17-24 years documented through national ID card, school certificate or through maternal recall | 8-16 weeks post conception | 
| Breast feeding | amount and initiation of breast feeding, top milk, formula milk and complementary feeding Based off of WHO 2010 Guidelines: Indicators for assessing infant and young child feeding practices (Part 2 Measurement) | at birth within 72 hours, 3 months, 6 months and 12 months | 
| Reported Maternal medicinal use | Questionnaire | 8-16 weeks post-conception, 30-34 weeks post-conception, 3-months post-partum, 6 months post-partum and 12 months post-partum | 
| Reported Infant medication use | Questionnaire | at birth within 72 hours, 3 months, 6 months and 12 months | 
| Maternal dietary intake Assessed through ASA 24 HR Dietary Recall system, completed 2x each time point | Assessed through ASA 24 HR Dietary Recall system | Baseline 8-16 weeks post conception, 30-34 weeks post conception and 12 months post partum | 
| Dietary diversity | Minimum Dietary Diversity Score for Women (MDD-W) | Baseline 8-16 weeks post conception, 30-34 weeks post conception, 3 months post-partum and 12 months post partum | 
| Household annual food insecurity | Food insecurity will be assessed using the Household Food Insecurity Access Scale (HFIAS) | 3 months post-partum and 12 months post-partum | 
| Generalized Self-efficacy | Self-efficacy will be measured using the Generalized Self-Efficacy scale, developed by Schwarzer and Jerusalem. A 10 item psychometric scale. | 3 months post-partum and 12 months post partum | 
| Perceived decision making | Questions pertaining to perceived decision-making are from the Pakistan Demographic and Health Survey (PDHS) | 3 months post-partum and 12 months post partum | 
| Perceived social support | Perceived social support will be measured using the Multi-dimensional Scale of Perceived Social Support (MSPSS), developed by Zimet et al. | 3 months post-partum and 12 months post partum | 
| Maternal demographics | Questions pertaining to demographic data are adapted from the Pakistan Demographic and Health Survey (PDHS) | Baseline 8-16 weeks post-conception | 
| Food insecurity | Questionnaire developed by Hager, E.R., et al., Development and validity of a 2-item screen to identify families at risk for food insecurity. | Baseline 8-16 weeks post conception, 3 months post partum and 12 months post partum | 
| Perceived parental stress | Perceived parental stress will be measured using the Perceived Stress Scale (PSS-10) | 3 months post-partum and 12 months post partum | 
| Preterm Births | Gestational age at birth in weeks | At birth within 72 hours | 
| Small for gestational age | Small-for-gestational-age (<10th percentile of weight-for-gestational-age and sex as defined by Intergrowth standards) | At birth within 72 hours | 
| Large for gestational age | >90th percentile of weight-for-gestational-age and sex as defined by Intergrowth standards) | At birth within 72 hours | 
| Delivery Assessment | Questionnaire, mode of delivery, place of birth and other description around delivery | at birth within 72 hours of birth | 
| Infant dietary intake | NutricheQ Questionnaire: a tool designed for toddlers aged 1 to 3 years of age, with a focus on markers for inadequate or excessive intake and dietary imbalances | Infant age 1 year | 
| Maternal stool biomarkers-1 | Level of Calprotectin in stool a marker of intestinal inflammation, measured in μg/g. | At baseline 8-16 weeks post conception and 30-34 weeks post conception | 
| Maternal stool biomarkers-2 | Concentration of Claudin 15 in stool a marker of intestinal permeability measured in ng/mL. | At baseline 8-16 weeks post conception, 30-34 weeks post conception | 
| Maternal stool biomarkers-3 | Concentration of Lipocalin in stool, a marker of gut inflammation, measured in μg/mL. | At baseline 8-16 weeks post conception and 30-34 weeks post conception | 
| Maternal: incidence of pathobionts | As identified through 16S, 18S and ITS rDNA surveys | Baseline, 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum | 
| Infant: incidence of pathobionts | As identified through 16S, 18S and ITS rDNA surveys | 3 and 12 month | 
| Maternal: metabolomic profile of stool [Metabolites involved in central metabolism as analysed by Mass Specttrometry] | Analysis of the core metabolites involved in central metabolism. These metabolites will be analysed through Mass Spec and include short chain fatty acids, amino acids, intermediates in energy metabolism and nucleotide biosynthesis | Baseline, 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum | 
| Maternal gut bacteria profile | Measured through 16S rDNA sequence surveys | Baseline, 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum | 
| Maternal: blood metallomics profile | Measured through ICP-MS (https://www.metabolomicscentre.ca/new_service/25) - TMIC platform of metabolmics | Baseline, 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum | 
| Infant: blood metallomics profile | as measured through ICP-MS (https://www.metabolomicscentre.ca/new_service/25) Through TMIC platform | Infant age 1 year | 
| Infant: gut bacterial profile | Measured through 16S rDNA sequence surveys | 3 month and 12 month | 
| Maternal metabolic pathway expression profile | as measured through whole microbiome RNASeq (metatranscriptomics) | Baseline 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum | 
| Infant eukaryotic microbiome profile | as measured through 18S and ITS rDNA sequence surveys | 3 & 12 Months | 
| Maternal eukaryotic microbiome profile | as measured through 18S and ITS rDNA sequence surveys | Baseline 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum ] | 
| Maternal bacterial gene expression profile | as measured through whole microbiome RNASeq (metatranscriptomics) - The output of these analyses are readouts of microbial gene expression detailing biochemical activities as well as the taxa responsible. | Baseline 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum ] | 
| Maternal: microbiome taxonomic alpha and beta diversity | To define taxonomic diversity, species profiles from 16S, 18S and ITS rDNA data will be clustered to identify differences in community structure across samples. Alpha diversity will be measured through indices such as Chao, Shannon and Simpson indices. Beta diversity will be measured through standard indices such as Bray-Curtis distances. | Baseline 8-16 weeks post conception, 30-34 weeks post conception, 3 months post partum and 12 months post partum ] | 
| Infant: microbiome taxonomic alpha and beta diversity | To define taxonomic diversity, species profiles from 16S, 18S and ITS rDNA data will be clustered to identify differences in community structure across samples. Alpha diversity will be measured through indices such as Chao, Shannon and Simpson indices. Beta diversity will be measured through standard indices such as Bray-Curtis distances. | 3 and 12 months postpartum | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Maternal Malnutrition, Gut Dysbiosis, Infant Health