Background: In addition to a diet including fortified dietary staples, the use of prenatal multivitamin supplements among women has been shown, in some cases, to lead to excessive micronutrient intake levels for nutrients such as folic acid (FA). It was therefore hypothesized that prenatal vitamin supplementation, in addition to a standard Canadian diet, would place pregnant Canadians at risk for excessive FA intake. With little available research on the potential negative impact of excess FA intake in pregnancy, it was further proposed that high concentrations of FA may adversely affect placental health and function. Thus, the aim of the current study was three-fold: 1) To determine micronutrient intake in a large Canadian cohort of pregnant women; 2) To determine the extent to which FA intake in this cohort may exceed the tolerable upper intake level (UL) after prenatal supplementation; and 3) To determine the effects of excessive FA exposure on placental health and function in vitro. Methodology: Second trimester 3-day food records of pregnant women (N=216) were analyzed for micronutrient intake using ESHA Food ProcessorTM. Nutrient intake values were compared to established Dietary Reference Intake (DRI) values. In a series of experiments, the effects of exogenous folic acid (2-4000 ng/ml) on placental health and function were examined in two placental cell lines [HTR-8/SVneo (N=3) and BeWo (N=3)], and a human placenta explant model (N=6). Following a 48-hour incubation period, the effects of excessive folic acid exposure on placenta cell proliferation, viability, and apoptosis were determined, along with evaluation of placenta cell function via cell invasion and B-hCG hormone release assays. Results: Through dietary sources alone, most pregnant women studied were consuming adequate levels of most micronutrients. However the majority of examined women (>50%) demonstrated a risk of dietary inadequacy for vitamin D, vitamin E, folate, and iron. In the examined cohort, 83% of study participants reported prenatal supplement usage. In vitro exposure of human placenta cells and explants to excessive FA concentrations resulted in no significant differences in cellular proliferation, apoptosis, invasion, or B-hCG hormone production. However, decreased cell viability was observed in BeWo cells at increased FA concentrations (200-2000 ng/mL). Conclusion: Food sources alone do not appear to provide women in Canada with adequate intake of all micronutrients recommended for a healthy pregnancy. Though a prenatal supplement containing FA may be necessary for most women, current FA levels in many prenatal supplements may lead to excessive FA intake above the established UL. Yet, as measured in this study, high FA concentrations do not seem to adversely affect most primary indicators of placental cell health or function.

<div>The first 1000 days of life, from conception until the child’s second birthday, constitute a critical window for child growth and development. During infancy and early childhood, significant and rapid physical changes occur, including increases in weight, height, and brain size and organ development accompanied by cognitive and psychomotor development. Adequate infant feeding, including breastfeeding and complementary feeding, that meets the infants’ energy and nutrient requirements can help protect against growth faltering, infant and child morbidity and mortality, and delayed mental and motor development. Adequate nutrition during this critical period can also protect against adverse health outcomes and chronic diseases later in life according to the hypothesis of developmental origins of health and disease.</div><div><br></div><div>A web of factors that are country- and culture- specific influence infant feeding practices and child growth. Further, the microbiome has been suggested as a strong potential player in the association between infant nutrition and child growth. Therefore, the overarching theme of the current dissertation is to investigate hypotheses that can provide evidence to inform the paradigm linking socio-demographic, maternal, and child determinants including prenatal intake to infant feeding, the breast milk and infant gut microbiome, and child growth within the first 1000 days of life. Specifically, aims one and two examine the socio-demographic, maternal, and child determinants of child growth and breastfeeding in a cross-sectional survey of mother-child dyads in Lebanon, a middle-income country undergoing nutrition transition in the Middle East. The third aim focuses on the CHILD cohort study, a multi-center longitudinal prospective birth cohort study, to examine the associations between prenatal diet and supplement intake and the breast milk microbiome. Finally, the fourth aim is to review the evidence for the potential of the infant gut microbiome as a promising target linking complementary feeding to child undernutrition in low- and middle- income countries (LMIC) with the highest burden of undernutrition.</div><div><br></div><div>The results for aim one revealed sex-specific determinants of child growth in Lebanon. The determinants examined through a hierarchical conceptual framework included: maternal and paternal education among boys and crowding index among girls at the distal sociodemographic level, and maternal obesity among girls at the intermediate maternal level. The proximal child determinants included birth length, number of children in the household and breastfeeding duration among girls, birthweight among boys and child’s age among boys and girls.</div><div><br></div><div>In the analysis for aim two, breastfeeding practices were suboptimal in Lebanon as less than half (41.5%) of the infants were exclusively breastfed during the 40-day rest period and 12.3% were exclusively breastfed during the 6-month duration recommended by the World Health Organization. Higher socioeconomic status, as reflected by a larger number of cars owned, and C-section delivery were consistently inversely associated with lower odds of exclusive breastfeeding for 40 days and 6 months. Belonging to a family with more children was associated with higher odds of exclusive breastfeeding for 40 days; while maternal overweight and obesity were associated with lower odds of exclusive breastfeeding for 6 months.</div><div><br></div><div>Findings from aim three suggested that prenatal supplement use, but not prenatal dietary quality and patterns, modulate the breast milk microbiota composition in the CHILD cohort in Canada. This project was exploratory and utilized one of the largest birth cohort studies with available breast milk microbiome data. Specifically, use of vitamin C and D supplements plus multivitamins during any trimester in pregnancy was consistently associated with milk microbial diversity and genus composition before and after adjustment for socio-demographic, maternal, and child covariates. Use of other supplements such as fish oil, folate, and calcium was less consistently associated with the breast milk microbiome.</div><div>The fourth aim of the review chapter focused on the infant gut microbiome. The effects of complementary feeding on the infant gut microbiome are less commonly studied than those of breastfeeding, with most research conducted in high-income countries but not LMIC. In contrast, associations between inadequate complementary feeding and undernutrition have been examined in LMIC where undernutrition is most prevalent. Further, a disrupted gut microbiota has been associated with child undernutrition. Indeed, animal studies have suggested a causal association although the direction of the causality is not clear and is potentially bi-directional depending on genetic and environmental conditions. In light of the current state of knowledge described in our review supporting the potential of the gut microbiota as a key player in the relation between complementary feeding and undernutrition, the development of microbiota-directed interventions during the complementary period offers a promising route for undernutrition management.</div><div><br></div><div>Findings from the studies presented in this dissertation highlight several culture-specific determinants of child growth and breastfeeding in Lebanon. The findings also highlight the need for future research using longitudinal prospective cohorts, intervention trails and animal models to provide evidence for the proposed links to enhance the understanding of the paradigm. Such a holistic view of the determinants of and pathways between infant feeding and child growth are of great public health significance to improve the health of children throughout their lives.</div>