Glycomics of cervicovaginal fluid from women at risk of preterm birth reveals immuno-regulatory epitopes that are hallmarks of cancer and viral glycosylation

Abstract

During pregnancy the immune system needs to maintain immune tolerance of the foetus while also responding to infection, which can cause premature activation of the inflammatory pathways leading to the onset of labour and preterm birth. The vaginal microbiome is an important modifier of preterm birth risk, with Lactobacillus dominance during pregnancy associated with term delivery while high microbial diversity is associated with an increased risk of preterm birth. Glycans on glycoproteins along the lower female reproductive tract are fundamental to microbiota-host interactions and the mediation of inflammatory responses. However, the specific glycan epitopes involved in these processes are not well understood. To address this, we conducted glycomic analyses of cervicovaginal fluid (CVF) from 36 pregnant women at high risk of preterm birth and 4 non-pregnant women. Our analysis of N- and O-glycans revealed a rich CVF glycome. While O-glycans were shown to be the main carriers of ABO blood group epitopes, the main features of N-glycans were the presence of abundant paucimannose and high mannose glycans, and a remarkable diversity of complex bi-, tri-, and tetra-antennary glycans decorated with fucose and sialic acid. We identified immuno-regulatory epitopes, such as Lewis antigens, and found that fucosylation was negatively correlated to pro-inflammatory factors, such as IL-1β, MMP-8, C3a and C5a, while glycans with only sialylated antennae were mainly positively correlated to those. Similarly, paucimannose glycans showed a positive correlation to pro-inflammatory factors. We revealed a high abundance of glycans which have previously been identified as hallmarks of cancer and viral glycosylation, such as Man8 and Man9 high mannose glycans. Although each pregnant woman had a unique glycomic profile, longitudinal studies showed that the main glycosylation features were consistent throughout pregnancy in women who delivered at term, whereas women who experienced extreme preterm birth exhibited sharp changes in the CVF glycome shortly before delivery. These findings shed light on the processes underlying the role of glycosylation in maintaining a healthy vaginal microbiome and associated host immune responses. In addition, these discoveries facilitate our understanding of the lower female reproductive tract which has broad implications for women's health.

Fig. 1

Representative MALDI-TOF mass spectra of 4 global N-glycan profile patterns. (a) Paucimannose dominated spectrum, from a blood group O donor with CST III-A who delivered at term. CVF sample was collected at 23 weeks and 6 days (23w6d) of gestation. Peaks highlighted in green correspond to paucimannose glycan structures, (b) high mannose dominated spectrum, from a blood group B donor with CST II who delivered at term. CVF sample was collected at 14w4d. Peaks highlighted in orange correspond to high mannose glycan structures, (c) fully sialylated non-fucosylated dominated spectrum, from a blood group B donor with CST I-A who delivered preterm. CVF sample was collected at 20w3d. The peak highlighted in purple corresponds to a sialylated non fucosylated structure, (d) bisected dominated spectrum, from a blood group B donor with CST I-A who delivered at term. CVF sample was collected at 22w5d. Peaks highlighted in black correspond to bisected glycan structures, confirmed by GC–MS linkage analysis. Assignments are based on composition, tandem MS and knowledge of biosynthetic pathways. All molecular ions are [M + Na]⁺

Fig. 2

MALDI-TOF mass spectra (m/z 500–2000) of CVF O-glycans. (a) A blood group O donor with CST I-B who delivered at term; CVF was collected at 23 weeks and 3 days (23w3d) of gestation; (b) a blood group A donor with CST-II who delivered at term; CVF was collected at 22w5d; (c) a blood group B donor with CST IV-B who delivered preterm; CVF was collected at 23w5d; (d) a blood group B donor with CST I-A who delivered preterm; CVF was collected at 20w3d; (e) a blood group A non-pregnant donor. The O-glycans from CVF were released by reductive elimination and permethylated prior to MALDI-TOF and TOF-TOF profiling. Each spectrum is shown in a single panel, and all data are normalized to the most abundant component, which is designated as 100%. For clarity, a zoomed in panel is inserted for m/z above 900. Colour coding has been used to distinguish families of glycans: unmodified O-glycans are flagged as blue, peaks labelled in purple show sialylated O-glycans, while peaks labelled in red are fucosylated O-glycans. Structures of the colour coded peaks are displayed in the corresponding coloured rectangles in panel (f). Main structures are depicted. Assignments are based on composition, tandem MS and knowledge of biosynthetic pathways. All molecular ions are [M + Na]⁺. * Indicates non-glycan contaminant.

Fig. 3

MALDI-TOF mass spectra (m/z 700–2500) of glycotopes isolated form CVF of: (a) a blood group B donor with CST I-B who delivered preterm. CVF was collected at 31 weeks and 3 days (31w3d) of gestation; (b) a blood group A donor with CST I-A who delivered at term. CVF was collected at 21w2d; (c) a blood group O donor with CST III-A who delivered at term. CVF was collected at 24w1d ; Glycotopes were released by endo-beta-galactose digestion and were deutero-reduced and permethylated prior to MALDI-TOF and TOF-TOF profiling. Each spectrum is shown in a single panel, and all data are normalized to the most abundant component, which is designated as 100%. For clarity, a zoomed in panel is inserted for m/z above 1600. Colour coding has been used to distinguish families of glycotopes: unmodified glycotopes are flagged as blue, peaks labelled in purple show sialylated glycotopes, peaks labelled in orange are fucosylated glycotopes with one LacNAc unit, peaks labelled in red are fucosylated glycotopes with two LacNAc units and peaks labelled in brown are fucosylated glycotopes with three LacNAc units. Structures of the colour coded peaks are displayed in the corresponding coloured rectangles in panel (d). Main structures are depicted. Assignments are based on composition, MS/MS and knowledge of biosynthetic pathways. All molecular ions are [M + Na]⁺.

Fig. 4

Correlation of glycans with fucosylated antennae to IL-1β, MMP-8, C3a and C5a. Fucosylated antennae % was calculated as the intensities of glycans or glycotopes with fucosylated antennae relative to the total complex glycan intensities in the spectra.

Fig. 5

a Correlation of paucimannose glycans to IL-1β, IL-8, MMP-8 and C5a; b expression of blood group antigens; c correlation of blood group A and blood group B antigens to C5a. Paucimannose glycans % was calculated as the summed intensity of paucimannose glycans relative to the total intensity of paucimannose and high mannose glycans. Blood group A % in O-glycans is calculated as the summed intensity of blood group A on O-glycans in the total intensity of O-glycans in the spectrum. Blood group A in N-glycans % is calculated as the summed intensity of blood group A N-glycans in the total intensity of complex N-glycans in the spectrum. The same method was used to calculate Blood group B %.

Fig. 6

Relationship between CST status and paucimannose glycans, high mannose glycans, sialylated N-glycans and bisected glycans. Sialylation % was calculated as sialylated glycans in all N-glycans.

Fig. 7

Preterm samples showed unstable N-glycosylation changes during pregnancy. a change of N-glycomic spectrum patterns during pregnancy of the term donor P12, a blood group O donor with CSTI-B who delivered at term at 37w6d, and the preterm donor P4, a blood group B donor with CST I-A who delivered preterm at 27w3d. Donor P12 had 3 samplings and donor P4 had 2 samplings during pregnancy. b Pearson coefficient values of donors with longitudinal samplings with mass range from m/z 1500 to 3500. A donor with 2 sampling time points has one coefficient value, while a donor with 3 sampling time points can have three coefficient values.

Fig. 8

Change of bisected glycans in all longitudinal donors with samplings between 13 and 33 weeks. The preterm pregnancies showed a sharp decrease in bisected glycans. Relative intensity % was calculated as the summed intensities of bisected glycans in the total intensities of glycans at m/z range 1000–3500 of each spectrum.

Fig. 9

Longitudinal N-glycan spectra of sample P4 at 20w5d (upper panel) and 26w0d (lower panel), full spectra (m/z 1000–5000) are reported in panel a, while zoomed in sections are reported in panels b, m/z 3000–3500, and c), m/z 3500–4000. Cartoon structures of main peaks are depicted in panels b and c, Assignments are based on composition, tandem MS and knowledge of biosynthetic pathways. All molecular ions are [M + Na]⁺