Human milk oligosaccharides in milk of mothers with term and preterm delivery at different lactation stage

Abstract

Human milk oligosaccharides (HMOs) are structurally diverse unconjugated glycans, and play crucial roles in protecting infants from infections. Preterm birth is one of the leading causes of neonatal mortality, and preterm infants are particularly vulnerable and are in need of improved outcomes from breast-feeding due to the presence of bioactive HMOs. However, studies on specific difference in HMOs as a function of gestation time have been very limited. We established an approach to extract and analyze HMOs based on 96-well plate extraction and mass spectrometry, and determined maternal phenotypes through distinctive fragments in product-ion spectra. We enrolled 85 women delivering at different gestation times (25-41 weeks), and observed different HMOs correlating with gestation time based on 233 samples from the 85 donors. With the increase of postpartum age, we observed a regular changing trajectory of HMOs in composition and relative abundance, and found significant differences in HMOs secreted at different postpartum times. Preterm delivery induced more variations between participants with different phenotypes compared with term delivery, and more HMOs varied with postpartum age in the population of secretors. The sialylation level in mature milk decreased for women delivering preterm while such decrease was not observed for women delivering on term.

Keywords: Distinctive oligosaccharides; Duration of gestation; Human milk oligosaccharides (HMOs); Phenotype; Preterm delivery.

Graphical abstract

Fig. 1

MALDI-TOF profiles of HMOs extracted using GC columns (a) and GC 96-well plates (b), shown with the corresponding relative quantification results (c) (GC columns in blue, GC 96-well plates in red). Error bars represent the standard deviation (SD) of all the analysis (n = 10). *, 0.01 ≤ p < 0.05, **, 0.001 ≤ p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Product-ion mass spectra of HMOs with m/z 1100 generated using MALDI-TOF MS/MS. Distinctive fragment ions were observed at m/z 433 and 690 (a), at m/z 790 and 864 (b), at m/z 433, 690, 790 and 864 (c), and at m/z 820 and 894 (d).

Fig. 3

Relative abundance of HMOs from women with preterm delivery (<37 weeks) and women with term delivery (≥37 weeks) (a). Relative abundance of HMOs at different gestation times (b). *, 0.01 ≤ p < 0.05；**, 0.001 ≤ p < 0.01；***, p < 0.001.

Fig. 4

OPLS-DA predictive vs orthogonal scores for secretors and nonsecretors delivering preterm infants (a), and the identified significantly different HMOs (b). OPLS-DA predictive vs orthogonal scores for secretors and nonsecretors delivering term infants (c), and the identified significantly different HMOs (d). The OPLS-DA analysis was based on the 20 most abundant oligosaccharides in the milk.

Fig. 5

MALDI-TOF profiles of HMOs present in colostrum (a), transitional milk (b) and mature milk (c). HMO compositions at the same postpartum ages (the period of colostrum, transitional milk and mature milk respectively) for the enrolled participants delivering at different gestation times (d, left panel), and HMO levels during breast feeding as a function of gestation time (d, right panel). In the left panel of d, α refers to women delivering before 28 weeks, β refers to women delivering between 28 and 32 weeks, and γ refers to women delivering between 33 and 36 weeks, and θ refers to women delivering after 37 weeks. In the right panel of d, C refers to colostrum, T refers to transitional milk, M refers to mature milk. HMOs in annotation were arranged by the increase of molecular mass (bottom-up, from H3N1F2 to H6N4F2). 100 % in (a), (b), and (c) refers to the relative intensity of the most intensive peaks in the mass spectra, and 100 % in (d) refers to the sum of relative intensity after normalization for 20 most abundant HMOs in each group.

Fig. 6

Dynamic changes of HMOs with certain types during lactation for women delivering at different gestation times (a), and discriminant analysis on different lactation stages through HMOs (b), and analysis on the value of variable importance for the projection for specific HMOs (c). The OPLS-DA analysis was based on the 20 most abundant oligosaccharides in the milk.

Fig. 7

Distinctive HMOs for women delivering at different gestation times. Women delivering at 28–32 weeks (a), 33–36 weeks (b), and after 37 weeks (c). *, 0.01 ≤ p < 0.05；**, 0.001 ≤ p < 0.01；***, p < 0.001.

Fig. 8

OPLS-DA assay for differentiation of colostrum and mature milk from secretors (a) and nonsecretors (b), and the detected significantly different HMOs in different lactation stages for secretors and nonsecretors (c). Multiple comparisons were performed between colostrum, transitional milk and mature milk. No significant difference was detected for the relative intensity figure labeled with the same superscript, and p value was not provided. Significant difference was detected for the relative intensity figure labeled with different superscript, and p value was presented. In panel (c), superscripts containing the same letters represent that significant difference was not found between these two groups, while superscripts containing different letters represent significant difference was observed in these two groups. The OPLS-DA assay was based on the 20 most abundant oligosaccharides in the milk.