Abstract
Background. Human milk is the ideal food for nearly all healthy full-term infants and understanding factors that impact human milk composition is important for optimizing infant health. However, concentrations of many components of milk can be impacted by factors such as time of day, time postpartum, and time within a feed. In addition, laboratory methods (e.g., fraction of milk used) must be optimized to accurately assess milk composition. A particular component of interest to our group is the human milk microbiome (HMM), which varies greatly among women and populations. However, little is known about how collection and analysis methods impact the HMM. Additionally, whether variation in the HMM is associated with infant health, such as attained size and growth is understudied. The primary objectives of the studies described here were to investigate 1) variation in the HMM due to time of day, time within feed, and milk fraction analyzed and 2) the association between variation in the HMM and infant anthropometric measurements in a well described Bangladeshi population. We secondarily investigated factors impacting variation in other milk components, including macronutrients, somatic cell count (SCC), Na and K, and individual proteins and fatty acids. Materials and methods. In Study 1, we utilized previously collected data of Lackey 2017. We expanded on this previous work by investigating if characterization of the HMM was affected by whether DNA was extracted from the cell pellet (CP) or from whole milk (WM). In Study 2, we utilized standardized collection methods to systematically explore variation in the HMM due to time of day, time within feed, and left vs. right breast. Participants (n = 17) collected foremilk (FM), midmilk (MM), and hindmilk (HM) from a single breast expression. On a different day, they collected complete breast expressions in the morning, midday, evening, and night. At the midday time point, complete breast expressions were obtained from both breasts. DNA was extracted from the cell pellet (CP), cell pellet plus lipid (CPL), and lipid (L) fractions. Study 3 utilized the same milk samples to study whether time of day, time within feed, and breast side were related to variation in milk protein, lactose, and lipid concentrations, SCC, Na, and K and relative amounts of fatty acids and individual proteins. In Study 4, associations between the HMM and infant size and growth in a nutritionally at-risk population were investigated. Milk samples originated from a parent study designed to evaluate the effect of prenatal multiple micronutrient supplementation on infant morbidity and mortality in rural Bangladesh (West 2014). Milk was collected at 3 mo postpartum from mothers with exclusively breastfed infants. Infant anthropometrics were measured at birth and 3 mo of life. Results. Results of Study 1 indicated that, while no difference in alpha diversity was identified between CP and WM, beta diversity within CP samples was lower than within WM (p = 0.004). Of 234 genera identified, 102 were present in both CP and WM. Taxa (n = 67) unique to CP were all low prevalence and mean relative abundance (< 0.5%). Comparatively, of taxa (n = 65) unique to WM, 4 were present with a mean relative abundance ≥ 0.5% and high prevalence. Results of Study 2 found that Staphylococcus and S. epidermidis were more abundant in CP than CPL and L. No differences in diversity or relative abundances were observed within a feed, between breasts, or over the day, although bacterial richness at the genus and species levels in milk collected at midday was higher than that in samples collected during the night (p = 0.020 and 0.040, respectively). While some taxa were correlated with total milk lipid concentration, these correlations were weak and often driven by a few data points. Taxa shared across fractions, time within feed, between breasts, and over a day, respectively, represented > 98% overall relative abundance. As expected, results from Study 3 documented an increase in lipid concentration from FM to HM (p < 0.001). Milk lipids were also higher at midday than at night (p = 0.047). Surprisingly, however, protein concentration was also greater in HM than FM (p = 0.022) and increased from morning to evening and night (p = 0.016 and < 0.001, respectively). SCC was greater in HM than FM and MM (p = 0.007 and 0.011, respectively). Na concentrations were lowest (p < 0.01) at midday, while K was highest (p < 0.01) in samples collected at midday and evening. Unique proteins were detected in FM vs. HM samples and also in morning vs. night, though the majority were detected in both FM/HM and morning/night. Relative abundances of 10 proteins were nearly double in HM compared to FM. Proteome beta diversity differed between FM and HM (p = 0.013) but not between morning and night. Results from Study 4 identified Streptococcus, Staphylococcus, Corynebacterium, Kocuria, Veillonella, and Rothia as common bacteria in the milk analyzed. Shannon diversity of the milk bacterial communities was positively correlated with infant weight, length, and head circumference (HC) at birth and length and HC at 3 mo of life. Beta diversity of the HMM was also different by tertiles of infant weight, length, and HC at 3 mo of life and with infant weight gain from birth to 3 mo. In general, the HMM in milk consumed by the smallest and mid-sized infants were different from the those in milk consumed by the largest infants. Relative abundances of some taxa ware correlated with infant size at birth and 3 mo of life. Additionally, the predicted metabolic profiles of the bacteria in milk consumed by the heaviest infants at 3 mo of life were characterized by pathways involved in amino acid biosynthesis and central metabolism. Conclusions. These studies illustrate minimal differences in the HMM among milk fractions, between breast and over a day. However, some more highly abundant taxa unique to WM may be missed when only using the CP fraction. Whether the differences between breasts, within milk fraction, and over a 24-hr period exist in different geographical locations or by infant gestational age at birth should also be explored. Our studies also provide evidence that variation in the HMM is related to attained size at birth and 3 mo as well as growth over time in a population at nutritional risk. This finding will require additional research to understand – particularly in terms of what factors might be driving these relationships at birth. To understand the interplay between the maternal and environmental microbiomes and infant size, future studies should explore if maternal skin, gastrointestinal, and milk microbiomes during gestation and early life associate with infant size and growth outcomes. Results from the studies described in this dissertation also revealed higher protein concentration in HM compared to FM and in milk produced in the evening and night compared to morning. These findings support circadian variation in total milk protein concentration and suggest that current Adequate Intake values may need to be reevaluated.