Lacto-N-tetraose, fucosylation, and secretor status are highly variable in human milk oligosaccharides from women delivering preterm

Abstract

Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from 15 women delivering preterm and 7 women delivering at term using nanohigh performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene's test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2'-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments.

Figure 1

MALDI FT ICR (+) MS profiles of neutral human milk oligosaccharides isolated from human milk of mothers who delivered A) at term and B) pre term. Samples were analyzed in the positive ionization mode.

Figure 2

Line graphs of the percent fucosylation of human milk oligosaccharides (HMOs) in (A) term and (B) preterm milk during the course of lactation; percent abundance of 2′ fucosyllactose, an HMO containing an α 1,2 fucose in (C) term and (D) preterm milk; and percent sialylation of HMOs in (E) term and (F) preterm milk. Y values are expressed as percentage from the total HMO abundance normalized per sample. Each color represents a different mother. Averages are shown as black broken lines. Average percent values for preterm milk shown in B, D, and F include all preterm milk samples (N=41) listed in Table 1. Average percent values for term milk shown in A, C, and E include all term milk samples (N=23) listed in Table 2. Error bars are expressed as standard error of the mean (SEM).

Figure 3

Fucosylation in human milk of mothers who delivered preterm and full term infants. Preterm milk transitioning into full term milk, i.e. preterm milk collected after 36 weeks of corrected gestation were not included in the plot. Box whisker plots of fucosylation in HMOs by A) gestational age, B) birth weight, and C) corrected gestational age at the time of milk collection. N = number of samples. Error bars are expressed as standard error of the mean (SEM).

Figure 4

Bar graphs of mono, di, tri and tetrafucosylation of free HMOs in human milk of seven mothers who delivered prematurely. The line graph inset in each bar graph is the percent abundance of 2′ fucosyllactose present in the sample. Only mothers who had three or more samples were graphed. The height of each bar represents the total fucosylation in the sample. Error bars are expressed as standard error of the mean (SEM).

Figure 5

Bar graphs of mono, di, tri and tetrafucosylation of free HMOs in human milk of three mothers who delivered at term. The line graph inset in each bar graph is the percent abundance of 2′ fucosyllactose present in the sample. Only mothers who had three or more samples were graphed. The height of each bar represents the total fucosylation in the sample. Error bars are expressed as standard error of the mean (SEM).