
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

71950
10.1038/s41598-024-71950-x
Article
Glycomics of cervicovaginal fluid from women at risk of preterm birth reveals immuno-regulatory epitopes that are hallmarks of cancer and viral glycosylation
Wu Gang 12
Grassi Paola 12
Molina Belen Gimeno 234
MacIntyre David A. 23
Sykes Lynne 234
Bennett Phillip R. 23
Dell Anne a.dell@imperial.ac.uk

12
Haslam Stuart M. s.haslam@imperial.ac.uk

12
1 https://ror.org/041kmwe10 grid.7445.2 0000 0001 2113 8111 Department of Life Sciences, Imperial College London, London, UK
2 grid.7445.2 0000 0001 2113 8111 March of Dimes Prematurity Research Centre at Imperial College London, London, UK
3 https://ror.org/041kmwe10 grid.7445.2 0000 0001 2113 8111 Institute of Reproductive & Developmental Biology, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, UK
4 https://ror.org/01aysdw42 grid.426467.5 0000 0001 2108 8951 The Parasol Foundation Centre for Women’s Health and Cancer Research, St Mary’s Hospital, London, W1 2NY UK
6 9 2024
6 9 2024
2024
14 2081312 2 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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.

Subject terms

Biochemistry
Glycobiology
Glycomics
March of Dimes European Preterm Birth Research Centre at Imperial College Londonissue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Human pregnancy is a balancing act: the immune system is carefully steered to maintain an immune tolerance of the semi-allogeneic foetus1, whilst maintaining capacity to respond to pathogenic infection. During embryo implantation and placentation, the female reproductive tract is reflective of a pro-inflammatory state which subsequently transitions to an anti-inflammatory environment to facilitate foetal growth and tolerance of paternal antigens2. This is maintained in healthy pregnancies until late gestation and the onset of parturition. Labour is a pro-inflammatory state, in which leukocytes (macrophages and neutrophils in particular) infiltrate the human myometrium, foetal membranes and the cervix. Proinflammatory cytokines such as IL-1β, IL-6, IL-8 and TNF-α are released, contributing to the activation of uterine contractions, rupturing of the foetal membranes, cervical remodelling and dilation.3–5 (Supplementary Fig. S1a).

Untimely activation of inflammatory pathways in gestational tissues can lead to preterm birth, defined as delivery before 37 weeks of completed gestation. An estimated 40% of all preterm birth cases are linked with an infectious aetiology6, with pathogen colonisation of the cervicovaginal niche followed by ascension towards the maternal-foetal interface considered as a major route of infection7,8. Displacement of commensal Lactobacillus species from the vagina during pregnancy is associated with an increase in pro-inflammatory cytokines, early cervical remodelling and increased risk of spontaneous preterm birth7,9,10. This response is often characterised by recruitment of immune cells, in particular neutrophils11, to the foetal membranes and release of immune mediators at the cervicovaginal interface12. Consistent with these observations, the vaginal microbiome is now considered an important modifier of preterm birth risk13–15. Compositionally, the vaginal microbiome can be classified into five community state types (CSTs), of which four are primarily composed of a single dominant species of Lactobacillus (L. crispatus (CST-I), L. gasseri (CST-II), L. iners (CST-III) and L. jensenii (CST-V) respectively), and one diverse community (CST-IV) typically comprising Gardnerella, Prevotella, Sneathia, Atopobium, Molibuncus, Clostridium, Corynebacterium, Staphylococcus, Streptococcus, Enterococcus, and Mycoplasma7,16. CST I can be further divided into CST I-A and I-B based on the degree of L. crispatus dominance, and CST III can be divided into CST III-A and III-B reflective of the degree of L. iners dominance. CST IV can be further classified as CST IV-A, with a moderate relative abundance of Gardnerella, and CST IV-B reflective of a high relative abundance of Gardnerella. L. crispatus dominance (CST I) during pregnancy associates with term delivery, whereas L. iners (CSTIII) and high diversity (CST IV) associates with an increased risk of preterm births17–19.

The mechanisms underpinning compositional changes in the vaginal microbiome and subsequent host immune responses remain poorly characterised. However, many proteins with putative roles in mediating microbiota-host responses within the human female reproductive tract share several features including being heavily glycosylated with a rich repertoire of glycans20–22. These include important functional sequences (glycotopes), such as Lewis antigens, SLewis antigens, ABO blood groups, and poly N-acetyllactosamine (polyLacNAc) antennae (structures shown in Supplementary Fig. S1), which can serve as ligands for glycan binding proteins (GBPs; also called lectins) from both the immune system and the microbes23 (Supplementary Fig. S1 b–d). Glycan-lectin engagement is predicted to regulate key aspects of cell–cell recognition, adhesion, signalling and host–pathogen/host commensal interactions during human reproduction but the specific glycan epitopes involved have been challenging to identify24. To begin to address this, we recently described procedures and workflows that allow glycomic analysis of cervicovaginal fluid (CVF) during pregnancy25. In this study of a small cohort of 10 donors, high sensitivity mass spectrometric methods revealed a rich and complex CVF N-glycome, characterised by abundant paucimannose and high mannose glycans, as well as a remarkable diversity of complex bi-, tri- and tetra-antennary glycans whose antennae were extensively decorated with fucose and sialic acid25. A substantial portion of the complex-type N-glycome carried extended polylacNAc antennae. Quantitative profiling of fucosylation and sialylation of the smaller complex glycans provided the first physicochemical evidence that expression of specific glycan epitopes is related to the microbial community status and immune activity in the cervicovaginal region. In the present study, we substantially expanded the scope of our investigations to describe global and temporal N- and O-glycomics of CVF samples (n = 60) collected from 36 pregnant women at high risk of preterm birth and 4 non-pregnant women. Of the pregnant women, 15 delivered preterm and 21 at term (Supplementary Table S1). Our expanded glycomics analyses included identification of terminal glycotopes displayed on extended polylacNAc antennae, which are expected to act as more accessible ligands for lectins compared to counterparts on non-extended antennae. Many CVF samples were remarkably rich in glycan epitopes widely considered to be unique hallmarks of cancer and viral glycosylation. Complementary functional analyses revealed quantitative differences in glycan epitope expression associated with bacterial composition and concentrations of cytokines, matrix metalloproteinases (MMP) and complement proteins. Longitudinal glycomic studies of two women who delivered extreme preterm showed substantial changes in expression of immune-active glycan epitopes shortly before delivery. In contrast, glycomes remained stable into the third trimester for women who delivered at term. We also present an analysis strategy designed to address issues of overlapped isotopic peaks from multiple glycans commonly found in the high mass range of the complex CVF N-glycome. This approach facilitated accurate high-speed data annotation and quantitation enabling the construction of N-glycan, O-glycan and glycotope libraries, which are provided as open access repositories.

Results

Patient cohort and overview of CVF glycomic characterisation

A total of 60 CVF samples were collected from 36 pregnant donors at high risk of preterm birth between 10 and 34 weeks of gestation. Of these, 15 women subsequently delivered preterm (< 37 weeks gestation) and 21 delivered at term (≥ 37 weeks). Details of the sample cohort and gestational age at sampling is provided in Supplementary Fig S2. Of these women, 15 donors were blood group O, 13 were blood group A and 12 were blood group B. CVF samples were also collected from four non-pregnant donors to enable comparative analyses. All CVF samples were subjected to MALDI-TOF/TOF N-glycomic analysis. In addition, O-glycomics and profiling of glycotopes on extended antennae (derived from both N- and O-glycans) were performed on samples that were available in sufficient quantities for multiple experiments. The list of glycomic datasets obtained for each sample is provided in Supplementary Table S2 and the online repository (https://github.com/gw110/Glycomics-of-cervicovaginal-fluid-from-women-at-risk-of-preterm-birth.git).

Annotation and quantitation of N-glycans in overlapped mass spectrometry peaks

Due to the complexity of glycosylation in the CVF samples, overlapping molecular ion isotope clusters were commonly observed, especially in the high m/z region of the N-glycan spectra. An in-house bioinformatic tool was developed to accurately de-isotope overlapped peaks for accurate glycan assignment. A multinomial distribution model was first trained using standard permethylated glycan data and their molecular formulae. The model was then tested on poly hexoses and different types of N-glycans (Supplementary Fig. S1 a–b), both of which showed accurate prediction of mono-isotopic peak patterns. The optimized model was then used for deisotoping of an overlapped mono-isotopic peak cluster from a blood group A sample (Supplementary Fig. S3c). The deisotoped results showed an R-squared value as high as 0.99 for the peak cluster, and lack of a theoretical group B glycan at m/z 3955.0. Isotopic peak matchings for all glycomic data are freely accessible (https://github.com/gw110/Glycomics-of-cervicovaginal-fluid-from-women-at-risk-of-preterm-birth.git), accompanied by .csv tables summarising identifications of glycans from each CVF sample, including the sample name, sample collection time, mass, intensity, monosaccharide composition, and quality of monoisotopic peak pattern fit by R-squared values.

Identification of a variety of distinctive N-glycan profile patterns in pregnant CVF

Detailed profiling of N-glycans of the 56 CVF samples from pregnant donors led to the discovery of unique global N-glycan profile patterns for each donor. Notably, complex-type glycans highly decorated with fucose and sialic acid on extended polylacNAc antennae were common to all the CVF samples. Nonetheless, samples could be categorised into one of six families, based on distinctive glycan signatures in the low to mid mass range of the N-glycomic spectra (https://github.com/gw110/Glycomics-of-cervicovaginal-fluid-from-women-at-risk-of-preterm-birth.git). Four of the families are illustrated in Fig. 1 and hierarchical clustering analysis of all CVF samples based on dominant glycans is shown in Supplementary Fig. S4. They are: (a) paucimannose dominant with the highest intensity peak at m/z 1141.5 corresponding to Fuc1Man2GlcNAc2 (Fig. 1a n = 10); (b) high mannose dominant, characterised by unusually high levels of Man8GlcNAc2 and Man9GlcNAc2 (Fig. 1b, n = 19); (c) spectra dominated by a single biantennary di-sialylated glycan without core fucose (m/z 2792.4, NeuAc2Gal2Man3GlcNAc4; Fig. 1c, n = 6); (d) spectra characterised by a high abundance of sialylated bisected glycans carrying one or 2 fucoses (at m/z 2850.4, 3024.5 and 3211.6, NeuAc1-2Fuc1-2Gal2Man3GlcNAc5) (Fig. 1d, n = 4). Linkage analysis showed a high level of 3,4,6-Man which confirms the presence of bisected glycan structures (Supplementary Table S3) The other two families exhibit combinations of (a-d). Thus family (e) has a strong signal at m/z 2792.4 together with abundant sialylated bisected glycans (mixture of c and d, n = 2), and family (f) displays characteristics of all families (a-d) with no clear dominance of any specific group of glycans (n = 19).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]+

Structural profiling of O-glycans

O-glycan data were obtained from 42 CVF samples (Supplementary Table S2). Both Core 1 and Core 2 structures were observed, as exemplified in Fig. 2. Unmodified Core 1 was identified at m/z 534 (GalGalNAc), while sialylated Core 1 structures were identified at m/z 895 (NeuAcGalGalNAc) and m/z 1256 (NeuAc2GalGalNAc). Fucosylated Core 1 carrying blood group epitopes H, A and B were identified at m/z 708 (FucGalGalNAc), 953 (FucGalGalNAc2) and 912 (FucGal2GalNAc) respectively (Fig. 2). A Core 1 structure modified by both fucose and sialic acid was detected at m/z 1069 (NeuAcFucGalGalNAc). In a small portion of samples, we identified structural isomers corresponding to elongated Core 1 structures, with the 3-linked arm modified by the addition of Gal, GlcNAc (eg. m/z 1432), Fuc and NeuAc residues (eg. m/z 1344). Most peaks with extended antennae were identified as Core2 O-glycans, with either or both the 3- and 6-linked antennae elongated by the addition of 1 to 4 LacNAc units (GalGlcNAc) and capped by a combination of up to 4 fucose and 2 sialic acid residues.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.

Several glycan epitopes of functional significance were found on O-glycans, including Lewis and SLewis epitopes. However, the most abundant epitopes identified on the O-glycans were the ABH blood group antigens, in contrast to the N-glycans where ABH blood group epitopes were only present on minor glycans. MS/MS analyses revealed different structural isomers carrying either Blood group H epitope in Blood Group O individuals or Lewis epitope in non-blood group O individuals (eg. m/z 1157). Interestingly, the blood group H epitope on the core 1 structure at m/z 708 is observed not only in Blood Group O donors but also in Blood Group A and B donors. Another interesting observation is the presence of internally fucosylated antennae carrying terminal blood group epitopes on several O-glycans (eg. m/z 1710, 1913).

Structural profiling of N- and O- glycan derived glycotopes on extended antennae

Glycotope analyses were performed on 37 CVF samples by incubating glycopeptides with endo-β-galactosidase, which cleaves internal β1-4 galactose linkages in unbranched, repeating poly-N-acetyllactosamine (GlcNAc-β1-3Gal-β1-4)n structures of both N- and O-glycans (Supplementary Fig S29, Supplementary Table S2). This included sample P34, whose preliminary glycan epitope analysis results were published in our earlier pilot study25. MALDI-MS spectra of glycotopes identified in three representative CVF samples are displayed in Fig. 3. The MS spectra are dominated by an unmodified tri-saccharide structure at m/z 739 corresponding to the sequence GalGlcNAcGal (the terminal cleavage product of endo-β-galactosidase digestion of uncapped polylacNAc antennae), and its fucosylated and sialylated variants at m/z 913 and 1100, respectively. After MS/MS analysis, the peak at m/z 913 was identified as a mixture of Lewis and Blood Group H epitopes. In blood group A donors, glycotopes carrying blood group epitopes were also identified at m/z 1158, 1782, 1956, 2405 and 2579 (Fuc1-5Gal2-3HexNAc2-3); glycotopes carrying blood group B epitopes were identified in blood group B donors at m/z 1566, 1740 and 1914 (Fuc1-3Gal4GlcNAc2) (Fig. 3). Several structures carrying multiple LacNAc units, multiple fucose residues and blood group epitopes were identified in the higher mass range, including structures carrying the Blood Group A epitope at m/z 2405, 2579, 3203 and 3228 which correspond to Fuc3-5Gal3-5HexNAc3-4, and structures carrying the Blood Group B epitope at m/z 2987, 3161 and 3610, which correspond to Fuc4-5Gal6-7GlcNAc4-5.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]+.

MS/MS analysis revealed the presence of the SdA epitope (NeuAcGal2GalNAcGlcNAc, m/z 1345), which was only identified on glycotopes but not on intact O- or N-glycans in CVF samples (Supplementary Fig. S5a). A di-sialylated glycotope at m/z 1461 (NeuAc2Gal2GlcNAc) was identified and characterized by MS/MS as a mixture of two structural isomers, one of which was derived from an extended antenna capped by a NeuAc-NeuAc moiety, whilst the other was derived from an extended antenna capped with a single NeuAc whilst the second NeuAc was linked to the GlcNAc residue (Supplementary Fig. S5b). Our observation of these glycotopes in this analysis, but not the intact O- or N-glycan analysis, indicates that they are present as very minor species. Several sialylated glycotopes were characterised: antennae with a backbone of 1 to 6 LacNAc units, combined with fucose to give SLewis X epitopes, spanning from smaller glycotopes at m/z 1274 (NeuAcFucGal2GlcNAc) and 1898 (NeuAcFuc2Gal3GlcNAc2) to larger glycotopes with m/z up to 4391 (NeuAcFuc6Gal7HexNAc6). MS/MS analysis of the structure at m/z 1724 (NeuAcFucGal3GlcNAc2) revealed that its fucose residue was on the internal LacNAc and not on the terminal LacNAc in most samples analysed by MS/MS (Supplementary Fig. S5c).

Correlation of fucosylation and sialylation with inflammatory status

Glycans with fucosylated antennae, which are the source of Lewis and SLewis antigens, were negatively correlated with pro-inflammatory factors involved in neutrophil migration and activation, such as IL-1β, MMP-8, C3a and C5a in the cervicovaginal niche (Fig. 4). The correlation was stronger for N- and O-glycans than glycotopes. Similar trends were observed for other common pro-inflammatory cytokines, MMPs and complement proteins (Supplementary Figs S6–S8). By contrast, N-glycans, O-glycans and glycotopes with only sialylated antennae showed an opposite trend and were mainly positively correlated to these factors (Supplementary Figs S9–S11).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.

Correlation of paucimannose glycans, ABO blood groups and LacdiNAc glycans with inflammatory mediators

Paucimannose glycans, which are known to be abundant in neutrophil granules26–28, showed a positive correlation to pro-inflammation, exemplified by IL-1β, IL-8, MMP-8 and C5a (Fig. 5a) and other pro-inflammatory factors (Supplementary Fig. S12). Blood group A and blood group B were identified on O-glycans, N-glycans and glycotopes (Fig. 5b). Patients within the same blood group type displayed variable levels of blood group antigen expression. For example, patients within blood group A type had blood group A antigen levels ranging from 10 to 60% in O-glycans. In addition, blood group antigens were found to be differentially expressed among N-glycans, O-glycans and glycotopes, with O-glycans having the highest expression. Further analysis implied that blood group A and blood group B could possibly have different correlation patterns to immune factors. For example, blood group A had a stronger correlation to C5a than blood group B on N-glycans (Fig. 5c). Analysis of other available data points showed that blood group A had negative correlation to MMPs and complement proteins but the correlations to cytokines was not strong (Supplementary Fig. S13). However, blood group B did not show clear correlation with complement proteins but showed a trend of negative correlation with cytokines (Supplementary Fig. S14). The LacdiNAc epitope was detected on N-glycans and confirmed by MS/MS analysis (see Supplementary Fig. S15). This structure was negatively correlated to MMPs, complement proteins, and inflammatory cytokines, with the exception of anti-inflammatory cytokine IL-4 (Supplementary Fig. S15).

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 %.

Glycosylation status and microbiota composition

Glycosylation features that showed the most notable relationship with microbial composition are outlined below. Higher levels of paucimannose glycans were observed in microbiota communities CST III-A, dominated by L. iners, and CST IV-B, which contains a high to moderate relative abundance of G. vaginalis and A. vaginae (Fig. 6). Lower levels of high mannose glycans were observed in L. crispatus dominated samples (Fig. 6). Communities characterised by mostly L. crispatus (CST I-A) colonisation had the highest levels of sialylated and bisected glycans.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.

Longitudinal glycomic profiling of CVF during pregnancy

CVF was sampled longitudinally (2 to 3 time points) throughout the pregnancies of 13 women, two of whom delivered extremely preterm. Analysis of N-glycan data indicated high stability of profiles for most term patients, but for both preterm patients this was not the case. For example, the CVF N-glycan profile for donor P12 remained highly stable across three sampling timepoints spanning the second and third trimesters of pregnancy (Fig. 7a). In contrast, donor P4, who delivered preterm, exhibited a major shift in the N-glycan profile between 20 and 26 weeks of pregnancy (Fig. 7a). Pearson correlation analysis indicated that women subsequently delivering preterm had very low coefficient values, indicative of large changes in their glycan profiles during pregnancy, whereas 8 of the 11 women delivering at term had high coefficient values (Fig. 7b, Supplementary Fig. S16). We also observed 3 term donors, which showed different coefficient values (Supplementary Fig. S17). Preterm donors also showed very high levels of bisected glycans at mid-trimester. Remarkably, the levels dropped dramatically shortly before the preterm birth (Fig. 8). In contrast, for women who went on to deliver at term, the values remained more stable. Interestingly, for preterm pregnancy P4, there was also a substantial increase in glycans carrying SLewis antigens, glycans carrying blood group B antigens, and glycans carrying both SLewis antigens and blood group B antigens just prior to preterm birth (Fig. 9, Supplementary Fig. S18).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]+

Discussion

This study reports the detailed characterisation of a broad range of glycan structures (N-glycans, O-glycans and their glycotopes) from CVF and their relationship with pregnancy status, microbiota composition and inflammatory and immune phenotypes. The work expands extensively on our previous description of N-glycosylation of CVF sampled during pregnancy, which was limited to 10 patients25. Validating our earlier findings, we observe a strong positive correlation between CVF sialylation and microbiota composition, and a negative correlation between high levels of fucosylation in CVF glycans and inflammatory cytokine concentrations (Supplementary Fig. S19–S26). The integration of O-glycan, N-glycan and glycotope datasets expands our characterisation of the CVF glycome and illuminates its complexity, characterised by diverse structures modified by different numbers of fucose and sialic acid, generating important antigens such as Lewis antigens, SLewis antigens and ABO blood groups. It should also be noted that the complexity of the CVF glycome does provide challenges for structural characterisation and that complementary analytical approaches, particularly those that also incorporate chromatography separation and ion mobility mass spectrometry could be of value for isomer separation and characterisation29,30. Functional correlation analysis of overall levels of glycan antenna sialylation and fucosylation also showed similar biological trends for N-glycans, O-glycans and glycotopes (Fig. 4). However, we did observe that CVF O-glycans had higher levels of blood group A and blood group B antigens than N-glycans and glycotopes (Fig. 5b). In addition, blood group A and blood group B appear to be correlated to immune factors in different ways (Fig. 5c).

Rich repertoires of high molecular weight fucosylated and sialylated glycans were found in all the pregnant CVF N-glycomes. In contrast, smaller N-glycans, notably paucimannose, high mannose and bisected biantennary complex-type, were found to be highly variable in their abundance amongst samples from pregnant donors (Fig. 1). The dominance of a particular family of low molecular weight glycans is most likely due to changes in the levels of specific glycoproteins in the CVF. CVF glycans can be derived from multiple sources, including epithelial cells in both vaginal and cervical regions, secreted mucins, antibodies, immune cells and liver derived glycoproteins abundant in amniotic fluid and serum31. Therefore, changes of individual protein levels could contribute specific glycan structures to the CVF glycome.

We found that a substantial number of pregnant CVF N-glycomes are dominated in the low mass region by minimally-processed high mannose glycans (Man9GlcNAc2 and Man8GlcNAc2; Fig. 1b, Supplementary Fig S27). These findings are consistent with our earlier results for non-pregnant CVF25. An abundant expression of high mannose N-glycans, and in particular the limited processing of Man9GlcNAc2 and Man8GlcNAc2 in the N-glycan biosynthetic pathway, is widely considered to be a feature of human cancer glycosylation32–35. This “abnormal” cancer high mannose glycosylation pattern is currently being targeted in the development of new cancer treatments by linking high mannose specific lectins to peptide toxin36, human IgG1 Fc37 or by generating lectin CAR-T cells38. A similar approach is being pursued for developing novel antiviral immunotherapies39, because again it is known that pathogenic human viruses such as SARS-CoV-240 and Zika41 and HIV42 also express glycoproteins carrying high levels of Man-9 and Man-8 N-glycans. Our findings that high mannose glycosylation is a common characteristic of the CVF glycome stresses the need for careful assessment of potential off-target effects of anti-cancer or anti-viral therapeutics targeting such glycomic features.

Liver derived glycoproteins are considered to be major contributors to sialylated non-fucosylated N-glycans in the CVF43. There are two likely sources of these glycoproteins in the CVF: serum44 and amniotic fluid45. Therefore, high levels of sialylated non-fucosylated glycans could indicate leaking of serum proteins and/or amniotic fluid proteins to the cervicovaginal niche. Quantitative analysis showed sialylated non-fucosylated N-glycans were positively correlated to MMP levels, suggesting they are inflammation related, which was consistent with the correlation analysis to complement factors and cytokines (Supplementary Fig. S9–S11). MMPs are zinc-dependent proteases that degrade various proteins in the extracellular matrix and play important roles in vascular remodelling46. In the cervicovaginal region, however, MMPs can be a risk factor of pregnancy complications. Activation of extracellular matrix metalloproteinase causes the degradation of mucin, the disruption of the immune barrier to pathogens, eventually resulting in upper genital tract infections, inflammation and pregnancy complications47,48.

N- and O-glycans with fucosylated antennae were found to be negatively linked to inflammation (Fig. 4, Supplementary Fig. S6–S8). Fucosylated glycans are important immune regulators, which are involved in immune cell development, leukocyte adhesion and antigen presentation49. They are known to regulate host-microbiome interactions50, and studies on intestinal epithelial cells have found that they support the maintenance of symbiotic microorganisms and resistance to pathogens such as Citrobacter rodentium, Salmonella typhimurium, and Enterococcus faecalis. It is possible that the fucosylated glycan structures play similar roles in regulating the immune system and the microbiome in the CVF.

While ABO blood groups are important antigens on red blood cells, they are also expressed by epithelial cells, suggesting multiple biological roles for these antigens. Studies on human intestinal epithelium have found that ABO blood group antigens are genetically determined host factors that can be recognized by bacterial adhesins and modulate the composition of microbiota51,52. Studies on the human genital tract have discovered that blood group B can be a risk factor in the development of neonatal group B streptococcal disease53, and loss of blood group A can be linked to the progression of squamous intraepithelial lesions54. In the present study, we used a glycomic strategy to directly investigate ABO blood group antigens and identified these antigens on N-glycans, O-glycans and extended antennae (Figs. 2, 3, supplementary Fig. S18). More importantly, we discovered that patients with the same blood group genotype can have very different expressions of blood group antigens. Moreover, blood group antigen levels are different among N-glycans, O-glycans and glycotopes, with O-glycans having the highest blood group antigen levels (Fig. 5b). Affinity enrichment using anti-blood group antibodies followed by quantitative proteomics could be used in future studies to identify the key glycoproteins that carry the blood group antigens.

The activity of the host immune system must be delicately balanced during pregnancy. A loss of balance can cause pregnancy complications. Our longitudinal glycomic data showed stable glycosylation during pregnancy in most term birth donors (Fig. 7), which could be an indicator for good maintenance of immune balance. By contrast, in the preterm group, there was a big glycosylation change within a short time, which could indicate a disruption of immune homeostasis (Fig. 7).

Bisected glycans have a distinctive structural feature: a non-extended GlcNAc β1-4 linked to the core mannose, which has a big impact on the conformation of the glycan structure55. Bisected glycans have been found to be involved in immune tolerance by suppressing NK cells56,57. NK cells are the major type of immune cells in the maternal-foetal interface58, where bisected glycans are also highly expressed, and are believed to protect the foetus from immune rejection59. However, the major immune cell population in the cervicovaginal region are neutrophils60–62, not NK cells, indicating additional functionality. We observed large variations in the levels of bisected glycans, which dominated the spectra of some CVF samples, but were not detected in some other samples, suggesting they could be derived from specific glycoprotein carriers. It is known that bisected glycans are not abundant in neutrophils63, liver derived proteins64 or antibodies65,66. Therefore, these constituents of CVF are unlikely to be the source of the abundant bisected glycans that are observed in some samples. On the other hand, mucins are amongst the most abundant glycoproteins in the CVF. A large-scale identification of mucins in endocervical mucus previously identified three gel-forming mucins (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1)67. The gel forming mucins and MUC1 are predominantly glycosylated with O-glycans and have only a limited number of N-glycosylation sites. Thus, they are not good candidates as carriers of the abundant bisected glycans. In contrast, MUC16, which is the largest mucin with a ~ 22,152 amino acid sequence, has as many as 250 potential N-glycosylation sites68. It has been found to be highly expressed in the cervix and its extracellular glycosylated domains can be shed from the epithelial cells by proteolysis69. In addition, the proteolytically cleaved soluble form of ovarian MUC16, which is also known as CA125 and is used for cancer screening, is characterised by high levels of bisected glycans70. These observations suggest that a potential source of the bisected glycans we identified in the CVF is cervical MUC16.

Correlation analysis showed that bisected glycans had a higher relative abundance in CST I-A than other CST subtypes (Fig. 6). In addition, expression of bisected glycans was generally stable in women experiencing term births whereas a sharp decrease in their levels was observed before delivery in women experiencing preterm birth (Fig. 8). These results suggest that MUC16 levels could be correlated to the cervicovaginal microbiome and preterm birth. A knockdown of MUC16 in an epithelial cell line was shown to decrease all barrier functions, exemplified by increased dye penetrance and bacterial invasion, decreased transepithelial resistance, disruption of tight junctions, and greater apical surface cell area. By contrast, knockdown of MUC1 did not cause an obvious barrier loss71.

In summary, this study demonstrates important relationships between CVF glycans and microbiota-host immune interactions, which are risk modifiers of pregnancy outcome. Detecting changes in CVF glycosylation during pregnancy could provide a new, non-invasive method for identifying women who are at risk of preterm birth and therefore implementation of personalised interventions. Additionally, understanding the role of glycans during pregnancy will help uncover the causes of preterm birth in women without a known aetiology. This knowledge can lead to the development of new interventions and glycan-mimicry based therapies that have the potential to reduce levels of preterm birth and improve the health outcomes of mothers and their babies.

Materials and methods

Patient recruitment and sampling

The study was conducted with approval of the NHS National Research Ethics Service (NRES) Committees London—Stanmore (REC 14/LO/0328), and in accordance with relevant guidelines, regulations, and the Declaration of Helsinki. All pregnant and non-pregnant women provided written informed consent. Recruitment and sampling were performed at Queen Charlotte’s and Chelsea and Westminster Hospitals, Imperial College Healthcare NHS Trust, London, UK. Non pregnant women were eligible if they were of reproductive age and aged 18 or over. Pregnant women at risk of preterm birth were eligible. Risk factors included having a short (cervical length of ≤ 25 mm) or open cervix, a previous preterm delivery, mid trimester loss, history of preterm premature rupture of the membranes, previous fully dilated caesarean section, or previous excisional cervical treatment. Exclusion criteria included women under 18 years of age, those who had sexual intercourse within 72 h of sampling, vaginal bleeding in the preceding week, HIV or Hepatitis C positive status. Detailed maternal clinical metadata and birth outcome data was collected for all pregnant participants (Supplementary Table S1). CVF was sampled using the BBL™ CultureSwab™ MAXV liquid Amies swabs (Becton, Dickinson and Company, Oxford UK) for assessing microbial composition. Supernatant from the culture swab was also used for immune profiling assays. CVF was then collected using a menstrual cup (SoftdiscTM, The Flex Company, USA) by placing it against the cervix for 20 min. After removal, material from both sides of the cup was retrieved by repeated pipetting of phosphate buffer saline (PBS) over each side leading to resuspension of material in a 1:5 weight: volume ratio within 30 min of collection. The suspension was distributed into separate aliquots to prevent unnecessary free thaw cycles. Samples were stored at − 80 °C until analysis.

Glycan profiling of CVF samples

Methanol, acetonitrile, ammonia, chloroform, DMSO, propan-1-ol, sodium hydroxide and acetic acid were from Romil (Cambridge, UK). Iodoacetic acid, sodium chloride, iodomethane, ammonium bicarbonate, EDTA, trypsin, Tris, potassium hydroxide, potassium borohydride, sodium acetate, sodium borodeuteride and Dowex 1-X8 beads were from Merck (Poole, UK). PNGase F (cloned from Flavobacterium meningosepticum and expressed by E. coli), CHAPS and DTT were from Roche Applied Science (East Sussex, UK). 8 M guanidine hydrochloride (GuHCl) and Slide-A-Lyzer™ G2 Dialysis Cassettes, 3.5 K MWCO were from Thermo Scientific (Loughborough, UK). Endo-Beta-Galactosidase (F. keratolyticus) was from R&D Systems (Abingdon, UK).

The workflow for glycomic sample processing is illustrated in Supplementary Fig. S28, following the protocols detailed previously72,73. Briefly, CVF samples were sonicated in 25 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1% CHAPS, pH 7.4, dialysed in dialysis cassettes, reduced by DTT, carboxymethylated by IAA, and digested by trypsin.

N-Glycans

N-glycans were released by PNGase F, separated from O-glycopeptides by C18 Sep-Pak chromatography and permethylated following the NaOH procedure. The permethylated N-glycans were cleaned by C18 cartridges and freeze dried before mass spectrometry analysis.

O-Glycans

Four hundred microliters of 0.1 M potassium hydroxide containing potassium borohydride (54 mg/ mL) was added to dried samples and incubated at 45 °C for 14–16 h. The reaction was terminated by adding a few drops of 5% (v/v) acetic acid followed by purification with Dowex 1-X8 desalting column. Excess borates in the samples were subsequently removed by co-evaporating with 10% (v/v) acetic acid in methanol under a stream of nitrogen at room temperature. The purified native O-glycans were then permethylated following the NaOH procedure.

Glycotopes

Parallel to full-size N- and O-glycan analysis, glycotopes from extended antennae on N- and O-glycans were removed from glycopeptides by incubating with 8 ul (4 ug) endo-β-galactosidase (Supplementary Fig. S29) in 400 ul 50 mM sodium acetate buffer, pH 5.5–6, at 37 ˚C for 24 h. After digestion, 3 drops of acetic acid were added to the samples. After C18 Sep-Pak chromatography, the isolated, freeze-dried glycotopes were deuteroreduced by incubating with a 10 mg/ml solution of sodium borodeuteride in 2 M ammonia for 2 h at room temperature. The reaction was stopped by addition of 5 drops of acetic acid and the samples were dried under nitrogen. Excess borates were removed by co-evaporating with 10% (v/v) acetic acid in methanol under a stream of nitrogen at room temperature. The deuteroreduced glycotopes were then permethylated following the NaOH procedure and analysed by MALDI-TOF–MS and MS/MS analysis. It should be noted that in our previous publication25 glycotopes were analysed on an advanced Orbitrap Fusion Tribrid MS platform with nanoLC-MS2-product dependent-MS3 data acquisition workflow. This allowed isomeric glycotopes such as Lewis X/A to be differentiated. Such levels of differentiation are not possible with the MALDI-TOF–MS and MS/MS analysis.

GC/MS linkage analysis

GC–MS linkage analysis of partially methylated alditol acetates was carried out on a Bruker SCION 456-GC Gas Chromatograph, fitted with a BR-5 ms fused capillary column (15 m × 0.25 mm internal diameter, Bruker), coupled to a Bruker SCION SQ Mass Spectrometer. Partially methylated alditol acetates were prepared from permethylated samples as described previously71. The permethylated glycans were hydrolyzed with 2 m trifluoroacetic acid for 2 h at 121 °C, reduced with 10 mg/ml sodium borodeuteride in 2 m aqueous ammonium hydroxide at room temperature, and acetylated with acetic anhydride at 100 °C for 1 h. The sample was dissolved in hexanes and injected onto the column at 60 °C. The column was maintained at this temperature for 1 min and then heated to 300 °C at a rate of 8 °C/min.

MALDI-MS and MS/MS analysis

Glycan profiling was done on an AB Sciex 4800 MALDI-TOF/TOF mass spectrometer. The methylated glycans and glycotopes were dissolved in 10 ul methanol. 1ul of sample was mixed with 1 ul of 10 mg/ml DABP matrix in 75% ACN. The mixture was spotted on a MALDI plate for MALDI-TOF–MS and MS/MS analysis.

Glycomic data analysis

The data were analysed using Data Explorer™ version 4.6 from AB Sciex, Glycoworkbench74 and MALDIquant75. The glycomic data were annotated based on monosaccharide composition derived from the molecular ion m/z value, knowledge of glycan biosynthetic pathways, the isotopic peak cluster patterns, the glycosylation patterns in the low and medium mass range, and MS/MS derived fragmentation. An R programme (https://github.com/gw110/Glycomics-of-cervicovaginal-fluid-from-women-at-risk-of-preterm-birth.git) was developed to quantify glycans in overlapped mono-isotopic peak clusters of multiple glycans. A multinomial distribution model was first trained using standard individual permethylated glycans and the molecular formula of these glycans. The optimised model was then used for deisotoping of overlapped mono-isotopic peak clusters by matching the predicted monoisotopic peak patterns of individual glycans to the overlapped peaks. An overdetermined linear system was constructed, and the intensities of individual glycans were calculated by solving the linear system with least squares. Glycan matches with R-squared values above 0.9 were selected for further analysis.

The correlation analysis of glycans and data visualisation were done using Python 3.10.9 on Linux Debian 11, and the script is available on (https://github.com/gw110/Glycomics-of-cervicovaginal-fluid-from-women-at-risk-of-preterm-birth.git). The correlation of glycan relative intensities with MMPs, Complements and cytokines were done using the linear regression model. Pearson product-moment correlation coefficient and p value were calculated to evaluate the strength of the correlation.

Bacterial DNA extraction and metataxonomic profiling

Extraction of DNA from CVF samples and sequencing of 16S rRNA hyper variable regions was performed as previously described76. Briefly, V1-V2 hyper variable regions of bacterial 16S rRNA genes were amplified using a mixed forward primer set (28f.-YM) consisting of the following primers mixed at a 4:1:1:1 ratio; 28F-Borrellia GAGTTTGATCCTGGCTTAG; 28F-Chlorflex GAATTTGATCTTGGTTCAG; 28F-Bifido GGGTTCGATTCTGGCTCAG; 28F GAGTTTGATCNTGGCTCAG. The reverse primer consisted of; 388R TGCTGCCTCCCGTAGGAGT77. Amplified products were then pooled equimolar and each pool was size selected in two rounds using Agencourt AMPure XP (BeckmanCoulter, Indianapolis, Indiana) in a 0.7 ratio for both rounds. These were then quantified using the Quibit 2.0 Fluorometer (Life Technologies) and loaded on an Illumina MiSeq (Illumina, Inc. San Diego, California) 2 × 300 flow cell at 10 pM. All sequencing was performed at Research and Testing Laboratory (Lubbock, TX, USA).

Trimming of primer sequences was performed using Cutadapt78 and QC performed using FastQC79. Resulting amplicon sequence variant (ASV) counts were calculated for each sample using the Qiime2 pipeline80. DADA2 was used for denoising81 and taxonomic classification of sequences to species level was performed using the STIRRUPS reference database82. Samples were classified into vaginal community state types (CSTs) using the VAginaL community state typE Nearest CentroId classifier (VALENCIA)83. Using this standardised approach, CST I communities are characterised by Lactobacillus crispatus dominance and can be further subdivided into subtypes CST I-A (almost complete dominance by L. crispatus) and CST I-B (less L. crispatus, but still the majority). CST IV communities have a low relative abundance of Lactobacillus spp. and include subtypes CST IV-A, CST IV-B, and CST IV-C, depending on the relative abundances of G. vaginalis, A. vaginae, and BVAB1, respectively.

Immune profiling of CVF samples

The stored supernatant of CVF from the menstrual cup was thawed on ice and used for Luminex® immunoassays to quantify cytokines levels of IL-2, IL-4, IL-18 using the Human Premixed Multi-Analyte Kit (R&D Systems). Further immune profiling was performed using supernatant extracted from the BBL™ CultureSwab™ to quantify the other immune mediators by multiplexed bead-based immunoassays. The concentrations of cytokines (IL-1β, IL-6) were measured on a multiplex plate Human Premixed Multi-Analyte Kit (R&D Systems), following manufacturer’s instructions. IL-8 was measured on a single-plex Human Premixed Analyte Kit (R&D Systems), following a tenfold dilution using Calibrator Diluent RD6-52. The concentrations of MMPs (MMP-2, MMP-8, MMP-9) were measured on a Human Premixed Multi Analyte Kit (R&D Systems) following a fivefold dilution using Calibrator Diluent RD6-52. Complement proteins were detected using the Human Complement Magnetic Bead Panel 1 (C2, C4b, C5, C5a, Factor D, Factor I, MBL) and Human Complement Magnetic Bead Panel 2 (C3, C3b, C4, Factor H) (Milliplex® Meck/Millipore). C3a was measured using the C3a Human ProcartaPlex™ Simplex Kit in conjunction with ProcartaPlex Human Basic Kit (both Invitrogen™). All standards and samples were run in duplicate.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71950-x.

Acknowledgements

We would like to thank all women who have participated in this study and members of the Women’s Health Research Centre who facilitated and coordinated study recruitment and sample collection. This work was funded by the March of Dimes European Preterm Birth Research Centre at Imperial College London and supported by the National Institute of Health Research (NIHR) Imperial Biomedical Research Centre (BRC), LS is supported by The Parasol Foundation Clinical Senior Lecturer scheme, and BGM is supported by The Parasol Foundation Fellowship Scheme and The Rosetrees Trust.

Author contributions

S.M.H., A.D., L.S., P.R.B. and D.A.M. conceptualised the study and developed the experimental design. Experiments were performed by G.W., P.G. and B.G.M. Data processing, analysis, and interpretation was performed by G.W., P.G., B.G.M. and S.M.H., A.D., L.S., P.R.B. and D.A.M. G.W. and P.G. prepared all figures and tables. G.W., P.G., A.D. and S.M.H. wrote the first draft of the manuscript. All authors critically reviewed, read and approved the final manuscript.

Data availability

The glycan libraries, glycan isotopic peak matches, glycan identifications, R script for deisotoping and Python script for data visualisation are available on Github: https://github.com/gw110/Glycomics-of-cervicovaginal-fluid-from-women-at-risk-of-preterm-birth.git The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Gang Wu and Paola Grassi.
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References

1. Challis JR Inflammation and pregnancy Reprod. Sci. Thousand Oaks Calif 2009 16 206 215 10.1177/1933719108329095
Challis, J. R. et al. Inflammation and pregnancy. Reprod. Sci. Thousand Oaks Calif 16, 206–215 (2009).10.1177/1933719108329095
2. Mor G Introduction to the immunology of pregnancy Immunol. Rev. 2022 308 5 8 10.1111/imr.13102 35635382
Mor, G. Introduction to the immunology of pregnancy. Immunol. Rev. 308, 5–8 (2022).35635382 10.1111/imr.13102
3. Thomson AJ Leukocytes infiltrate the myometrium during human parturition: Further evidence that labour is an inflammatory process Hum. Reprod. Oxf. Engl. 1999 14 229 236 10.1093/humrep/14.1.229
Thomson, A. J. et al. Leukocytes infiltrate the myometrium during human parturition: Further evidence that labour is an inflammatory process. Hum. Reprod. Oxf. Engl. 14, 229–236 (1999).10.1093/humrep/14.1.229
4. Osman I Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term Mol. Hum. Reprod. 2003 9 41 45 10.1093/molehr/gag001 12529419
Osman, I. et al. Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term. Mol. Hum. Reprod. 9, 41–45 (2003).12529419 10.1093/molehr/gag001
5. Sykes L MacIntyre DA Yap XJ Teoh TG Bennett PR The Th1: Th2 dichotomy of pregnancy and preterm labour Mediators Inflamm. 2012 2012 967629 10.1155/2012/967629 22719180
Sykes, L., MacIntyre, D. A., Yap, X. J., Teoh, T. G. & Bennett, P. R. The Th1: Th2 dichotomy of pregnancy and preterm labour. Mediators Inflamm. 2012, 967629 (2012).22719180 10.1155/2012/967629
6. Humberg A Preterm birth and sustained inflammation: Consequences for the neonate Semin. Immunopathol. 2020 42 451 468 10.1007/s00281-020-00803-2 32661735
Humberg, A. et al. Preterm birth and sustained inflammation: Consequences for the neonate. Semin. Immunopathol. 42, 451–468 (2020).32661735 10.1007/s00281-020-00803-2
7. Chan D Microbial-driven preterm labour involves crosstalk between the innate and adaptive immune response Nat. Commun. 2022 13 975 10.1038/s41467-022-28620-1 35190561
Chan, D. et al. Microbial-driven preterm labour involves crosstalk between the innate and adaptive immune response. Nat. Commun. 13, 975 (2022).35190561 10.1038/s41467-022-28620-1
8. Romero R The role of inflammation and infection in preterm birth Semin. Reprod. Med. 2007 25 21 39 10.1055/s-2006-956773 17205421
Romero, R. et al. The role of inflammation and infection in preterm birth. Semin. Reprod. Med. 25, 21–39 (2007).17205421 10.1055/s-2006-956773
9. Kindinger LM Relationship between vaginal microbial dysbiosis, inflammation, and pregnancy outcomes in cervical cerclage Sci. Transl. Med. 2016 8 350ra102 10.1126/scitranslmed.aag1026 27488896
Kindinger, L. M. et al. Relationship between vaginal microbial dysbiosis, inflammation, and pregnancy outcomes in cervical cerclage. Sci. Transl. Med. 8, 350ra102 (2016).27488896 10.1126/scitranslmed.aag1026
10. Fettweis JM The vaginal microbiome and preterm birth Nat. Med. 2019 25 1012 1021 10.1038/s41591-019-0450-2 31142849
Fettweis, J. M. et al. The vaginal microbiome and preterm birth. Nat. Med. 25, 1012–1021 (2019).31142849 10.1038/s41591-019-0450-2
11. Gimeno-Molina B Muller I Kropf P Sykes L the role of neutrophils in pregnancy, term and preterm labour Life Basel Switz. 2022 12 1512
Gimeno-Molina, B., Muller, I., Kropf, P. & Sykes, L. the role of neutrophils in pregnancy, term and preterm labour. Life Basel Switz. 12, 1512 (2022).
12. Adapen C Local innate markers and vaginal microbiota composition are influenced by hormonal cycle phases Front. Immunol. 2022 13 841723 10.3389/fimmu.2022.841723 35401577
Adapen, C. et al. Local innate markers and vaginal microbiota composition are influenced by hormonal cycle phases. Front. Immunol. 13, 841723 (2022).35401577 10.3389/fimmu.2022.841723
13. Gudnadottir U The vaginal microbiome and the risk of preterm birth: A systematic review and network meta-analysis Sci. Rep. 2022 12 7926 10.1038/s41598-022-12007-9 35562576
Gudnadottir, U. et al. The vaginal microbiome and the risk of preterm birth: A systematic review and network meta-analysis. Sci. Rep. 12, 7926 (2022).35562576 10.1038/s41598-022-12007-9
14. Huang C Meta-analysis reveals the vaginal microbiome is a better predictor of earlier than later preterm birth BMC Biol. 2023 21 199 10.1186/s12915-023-01702-2 37743497
Huang, C. et al. Meta-analysis reveals the vaginal microbiome is a better predictor of earlier than later preterm birth. BMC Biol. 21, 199 (2023).37743497 10.1186/s12915-023-01702-2
15. Golob JL Microbiome preterm birth DREAM challenge: Crowdsourcing machine learning approaches to advance preterm birth research MedRxiv Prepr. Serv. Health Sci. 2023 10.1101/2023.03.07.23286920
Golob, J. L. et al. Microbiome preterm birth DREAM challenge: Crowdsourcing machine learning approaches to advance preterm birth research. MedRxiv Prepr. Serv. Health Sci.10.1101/2023.03.07.23286920 (2023).10.1101/2023.03.07.23286920
16. De Seta F Campisciano G Zanotta N Ricci G Comar M The vaginal community state types microbiome-immune network as key factor for bacterial vaginosis and aerobic vaginitis Front. Microbiol. 2019 10 2451 10.3389/fmicb.2019.02451 31736898
De Seta, F., Campisciano, G., Zanotta, N., Ricci, G. & Comar, M. The vaginal community state types microbiome-immune network as key factor for bacterial vaginosis and aerobic vaginitis. Front. Microbiol. 10, 2451 (2019).31736898 10.3389/fmicb.2019.02451
17. Kindinger LM The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk Microbiome 2017 5 6 10.1186/s40168-016-0223-9 28103952
Kindinger, L. M. et al. The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk. Microbiome 5, 6 (2017).28103952 10.1186/s40168-016-0223-9
18. Callahan BJ Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women Proc. Natl. Acad. Sci. USA. 2017 114 9966 9971 10.1073/pnas.1705899114 28847941
Callahan, B. J. et al. Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women. Proc. Natl. Acad. Sci. USA. 114, 9966–9971 (2017).28847941 10.1073/pnas.1705899114
19. Brown RG Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin BMC Med. 2018 16 9 10.1186/s12916-017-0999-x 29361936
Brown, R. G. et al. Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin. BMC Med. 16, 9 (2018).29361936 10.1186/s12916-017-0999-x
20. Blois SM Role of galectin-glycan circuits in reproduction: From healthy pregnancy to preterm birth (PTB) Semin. Immunopathol. 2020 42 469 486 10.1007/s00281-020-00801-4 32601855
Blois, S. M. et al. Role of galectin-glycan circuits in reproduction: From healthy pregnancy to preterm birth (PTB). Semin. Immunopathol. 42, 469–486 (2020).32601855 10.1007/s00281-020-00801-4
21. Pang P-C Haslam SM Dell A Clark GF The human fetoembryonic defense system hypothesis: Twenty years on Mol. Aspects Med. 2016 51 71 88 10.1016/j.mam.2016.06.002 27349751
Pang, P.-C., Haslam, S. M., Dell, A. & Clark, G. F. The human fetoembryonic defense system hypothesis: Twenty years on. Mol. Aspects Med. 51, 71–88 (2016).27349751 10.1016/j.mam.2016.06.002
22. Zhong J The functional roles of protein glycosylation in human maternal-fetal crosstalk Hum. Reprod. 2023 10.1093/humupd/dmad024 37766497
Zhong, J. et al. The functional roles of protein glycosylation in human maternal-fetal crosstalk. Hum. Reprod.10.1093/humupd/dmad024 (2023).37766497 10.1093/humupd/dmad024
23. Krautter F Iqbal AJ Glycans and glycan-binding proteins as regulators and potential targets in leukocyte recruitment Front. Cell Dev. Biol. 2021 9 624082 10.3389/fcell.2021.624082 33614653
Krautter, F. & Iqbal, A. J. Glycans and glycan-binding proteins as regulators and potential targets in leukocyte recruitment. Front. Cell Dev. Biol. 9, 624082 (2021).33614653 10.3389/fcell.2021.624082
24. Zhou JY Cobb BA Glycans in immunologic health and disease Annu. Rev. Immunol. 2021 39 511 536 10.1146/annurev-immunol-101819-074237 33577348
Zhou, J. Y. & Cobb, B. A. Glycans in immunologic health and disease. Annu. Rev. Immunol. 39, 511–536 (2021).33577348 10.1146/annurev-immunol-101819-074237
25. Wu G N-glycosylation of cervicovaginal fluid reflects microbial community, immune activity, and pregnancy status Sci. Rep. 2022 12 16948 10.1038/s41598-022-20608-7 36216861
Wu, G. et al. N-glycosylation of cervicovaginal fluid reflects microbial community, immune activity, and pregnancy status. Sci. Rep. 12, 16948 (2022).36216861 10.1038/s41598-022-20608-7
26. Kr R Yh L van Fpj A Ab M Ajr H Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci- and phosphomannose glycans Commun. Biol. 2021 4 1012 10.1038/s42003-021-02555-7 34446797
Kr, R., Yh, L., van Fpj, A., Ab, M. & Ajr, H. Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci- and phosphomannose glycans. Commun. Biol. 4, 1012 (2021).34446797 10.1038/s42003-021-02555-7
27. Loke I Østergaard O Heegaard NHH Packer NH Thaysen-Andersen M Paucimannose-rich N-glycosylation of spatiotemporally regulated human neutrophil elastase modulates its immune functions Mol. Cell. Proteomics MCP 2017 16 1507 1527 10.1074/mcp.M116.066746 28630087
Loke, I., Østergaard, O., Heegaard, N. H. H., Packer, N. H. & Thaysen-Andersen, M. Paucimannose-rich N-glycosylation of spatiotemporally regulated human neutrophil elastase modulates its immune functions. Mol. Cell. Proteomics MCP 16, 1507–1527 (2017).28630087 10.1074/mcp.M116.066746
28. Thaysen-Andersen M Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum J. Biol. Chem. 2015 290 8789 8802 10.1074/jbc.M114.631622 25645918
Thaysen-Andersen, M. et al. Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum. J. Biol. Chem. 290, 8789–8802 (2015).25645918 10.1074/jbc.M114.631622
29. Bechtella L Ion mobility-tandem mass spectrometry of mucin-type O-glycans Nat. Commun. 2024 15 2611 10.1038/s41467-024-46825-4 38521783
Bechtella, L. et al. Ion mobility-tandem mass spectrometry of mucin-type O-glycans. Nat. Commun. 15, 2611 (2024).38521783 10.1038/s41467-024-46825-4
30. Wang J Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS The Analyst 2022 147 2048 2059 10.1039/D2AN00010E 35311852
Wang, J. et al. Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS. The Analyst 147, 2048–2059 (2022).35311852 10.1039/D2AN00010E
31. Kim YE Kim K Oh HB Lee SK Kang D Quantitative proteomic profiling of cervicovaginal fluid from pregnant women with term and preterm birth Proteome Sci. 2021 19 3 10.1186/s12953-021-00171-1 33588889
Kim, Y. E., Kim, K., Oh, H. B., Lee, S. K. & Kang, D. Quantitative proteomic profiling of cervicovaginal fluid from pregnant women with term and preterm birth. Proteome Sci. 19, 3 (2021).33588889 10.1186/s12953-021-00171-1
32. Everest-Dass AV N-glycan MALDI imaging mass spectrometry on formalin-fixed paraffin-embedded tissue enables the delineation of ovarian cancer tissues Mol. Cell. Proteomics MCP 2016 15 3003 3016 10.1074/mcp.M116.059816 27412689
Everest-Dass, A. V. et al. N-glycan MALDI imaging mass spectrometry on formalin-fixed paraffin-embedded tissue enables the delineation of ovarian cancer tissues. Mol. Cell. Proteomics MCP 15, 3003–3016 (2016).27412689 10.1074/mcp.M116.059816
33. Möginger U Alterations of the human skin N- and O-glycome in basal cell carcinoma and squamous cell carcinoma Front. Oncol. 2018 8 70 10.3389/fonc.2018.00070 29619343
Möginger, U. et al. Alterations of the human skin N- and O-glycome in basal cell carcinoma and squamous cell carcinoma. Front. Oncol. 8, 70 (2018).29619343 10.3389/fonc.2018.00070
34. Dd P Metastasis of cholangiocarcinoma is promoted by extended high-mannose glycans Proc. Natl. Acad. Sci. USA. 2020 117 7633 10.1073/pnas.1916498117 32213588
Dd, P. et al. Metastasis of cholangiocarcinoma is promoted by extended high-mannose glycans. Proc. Natl. Acad. Sci. USA. 117, 7633 (2020).32213588 10.1073/pnas.1916498117
35. Butler W Rewiring of the N-glycome with prostate cancer progression and therapy resistance NPJ Precis. Oncol. 2023 7 22 10.1038/s41698-023-00363-2 36828904
Butler, W. et al. Rewiring of the N-glycome with prostate cancer progression and therapy resistance. NPJ Precis. Oncol. 7, 22 (2023).36828904 10.1038/s41698-023-00363-2
36. Kurhade SE Ross P Gao FP Farrell MP Lectin drug conjugates targeting high mannose N-glycans Chembiochem Eur. J. Chem. Biol. 2022 23 e202200266 10.1002/cbic.202200266
Kurhade, S. E., Ross, P., Gao, F. P. & Farrell, M. P. Lectin drug conjugates targeting high mannose N-glycans. Chembiochem Eur. J. Chem. Biol. 23, e202200266 (2022).10.1002/cbic.202200266
37. Ohi YJ Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans Mol. Ther. 2022 30 4 1523 35 10.1016/j.ymthe.2022.01.030 35077861
Ohi, Y. J. et al. Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans. Mol. Ther. 30(4), 1523–35 (2022).35077861 10.1016/j.ymthe.2022.01.030
38. McKenna MK Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma J. Immunother. Cancer 2023 11 e005891 10.1136/jitc-2022-005891 36653070
McKenna, M. K. et al. Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma. J. Immunother. Cancer 11, e005891 (2023).36653070 10.1136/jitc-2022-005891
39. Christodoulou I Glycoprotein targeted CAR-NK cells for the treatment of SARS-CoV-2 infection Front. Immunol. 2021 12 763460 10.3389/fimmu.2021.763460 35003077
Christodoulou, I. et al. Glycoprotein targeted CAR-NK cells for the treatment of SARS-CoV-2 infection. Front. Immunol. 12, 763460 (2021).35003077 10.3389/fimmu.2021.763460
40. Tian Y Parsons LM Jankowska E Cipollo JF Site-specific glycosylation patterns of the SARS-CoV-2 spike protein derived from recombinant protein and viral WA1 and D614G strains Front. Chem. 2021 9 767448 10.3389/fchem.2021.767448 34869209
Tian, Y., Parsons, L. M., Jankowska, E. & Cipollo, J. F. Site-specific glycosylation patterns of the SARS-CoV-2 spike protein derived from recombinant protein and viral WA1 and D614G strains. Front. Chem. 9, 767448 (2021).34869209 10.3389/fchem.2021.767448
41. Pralow A Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins Sci. Rep. 2021 11 5147 10.1038/s41598-021-84682-z 33664361
Pralow, A. et al. Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins. Sci. Rep. 11, 5147 (2021).33664361 10.1038/s41598-021-84682-z
42. Panico M Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding Sci. Rep. 2016 6 32956 10.1038/srep32956 27604319
Panico, M. et al. Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding. Sci. Rep. 6, 32956 (2016).27604319 10.1038/srep32956
43. Kita Y Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans Mol. Cell. Proteomics MCP 2007 6 1437 1445 10.1074/mcp.T600063-MCP200 17522412
Kita, Y. et al. Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans. Mol. Cell. Proteomics MCP 6, 1437–1445 (2007).17522412 10.1074/mcp.T600063-MCP200
44. Anderson NL Anderson NG The human plasma proteome: History, character, and diagnostic prospects * Mol. Cell. Proteomics 2002 1 845 867 10.1074/mcp.R200007-MCP200 12488461
Anderson, N. L. & Anderson, N. G. The human plasma proteome: History, character, and diagnostic prospects *. Mol. Cell. Proteomics 1, 845–867 (2002).12488461 10.1074/mcp.R200007-MCP200
45. Cho C-KJ Shan SJ Winsor EJ Diamandis EP Proteomics analysis of human amniotic fluid * Mol. Cell. Proteomics 2007 6 1406 1415 10.1074/mcp.M700090-MCP200 17495049
Cho, C.-K.J., Shan, S. J., Winsor, E. J. & Diamandis, E. P. Proteomics analysis of human amniotic fluid *. Mol. Cell. Proteomics 6, 1406–1415 (2007).17495049 10.1074/mcp.M700090-MCP200
46. Raffetto JD Khalil RA Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease Biochem. Pharmacol. 2008 75 346 359 10.1016/j.bcp.2007.07.004 17678629
Raffetto, J. D. & Khalil, R. A. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease. Biochem. Pharmacol. 75, 346–359 (2008).17678629 10.1016/j.bcp.2007.07.004
47. Bayar E Bennett PR Chan D Sykes L MacIntyre DA The pregnancy microbiome and preterm birth Semin. Immunopathol. 2020 42 487 499 10.1007/s00281-020-00817-w 32797272
Bayar, E., Bennett, P. R., Chan, D., Sykes, L. & MacIntyre, D. A. The pregnancy microbiome and preterm birth. Semin. Immunopathol. 42, 487–499 (2020).32797272 10.1007/s00281-020-00817-w
48. Witkin SS Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: Implications for protection against upper genital tract infections mBio 2013 4 e00460 13 10.1128/mBio.00460-13 23919998
Witkin, S. S. et al. Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: Implications for protection against upper genital tract infections. mBio 4, e00460-13 (2013).23919998 10.1128/mBio.00460-13
49. Li J Hsu H-C Mountz JD Allen JG Unmasking fucosylation: From cell adhesion to immune system regulation and diseases Cell Chem. Biol. 2018 25 499 512 10.1016/j.chembiol.2018.02.005 29526711
Li, J., Hsu, H.-C., Mountz, J. D. & Allen, J. G. Unmasking fucosylation: From cell adhesion to immune system regulation and diseases. Cell Chem. Biol. 25, 499–512 (2018).29526711 10.1016/j.chembiol.2018.02.005
50. Kononova S Litvinova E Vakhitov T Skalinskaya M Sitkin S Acceptive immunity: The role of fucosylated glycans in human host-microbiome interactions Int. J. Mol. Sci. 2021 22 3854 10.3390/ijms22083854 33917768
Kononova, S., Litvinova, E., Vakhitov, T., Skalinskaya, M. & Sitkin, S. Acceptive immunity: The role of fucosylated glycans in human host-microbiome interactions. Int. J. Mol. Sci. 22, 3854 (2021).33917768 10.3390/ijms22083854
51. Mäkivuokko H Association between the ABO blood group and the human intestinal microbiota composition BMC Microbiol. 2012 12 94 10.1186/1471-2180-12-94 22672382
Mäkivuokko, H. et al. Association between the ABO blood group and the human intestinal microbiota composition. BMC Microbiol. 12, 94 (2012).22672382 10.1186/1471-2180-12-94
52. Uchida H Lactobacilli binding human A-antigen expressed in intestinal mucosa Res. Microbiol. 2006 157 659 665 10.1016/j.resmic.2006.03.001 16631357
Uchida, H. et al. Lactobacilli binding human A-antigen expressed in intestinal mucosa. Res. Microbiol. 157, 659–665 (2006).16631357 10.1016/j.resmic.2006.03.001
53. Regan JA Chao S James LS Maternal ABO blood group type B: A risk factor in the developement of neonatal group B streptococcal disease Pediatrics 1978 62 504 509 10.1542/peds.62.4.504 362365
Regan, J. A., Chao, S. & James, L. S. Maternal ABO blood group type B: A risk factor in the developement of neonatal group B streptococcal disease. Pediatrics 62, 504–509 (1978).362365 10.1542/peds.62.4.504
54. Moro-Rodríguez E Alvarez-Fernández E Losses of expression of the antigens A, Lea and Lex and over-expression of Ley in carcinomas and HG-SIL of the uterine cervix Diagn. Pathol. 2008 3 38 10.1186/1746-1596-3-38 18786253
Moro-Rodríguez, E. & Alvarez-Fernández, E. Losses of expression of the antigens A, Lea and Lex and over-expression of Ley in carcinomas and HG-SIL of the uterine cervix. Diagn. Pathol. 3, 38 (2008).18786253 10.1186/1746-1596-3-38
55. Miwa HE Song Y Alvarez R Cummings RD Stanley P The bisecting GlcNAc in cell growth control and tumor progression Glycoconj. J. 2012 29 609 618 10.1007/s10719-012-9373-6 22476631
Miwa, H. E., Song, Y., Alvarez, R., Cummings, R. D. & Stanley, P. The bisecting GlcNAc in cell growth control and tumor progression. Glycoconj. J. 29, 609–618 (2012).22476631 10.1007/s10719-012-9373-6
56. Pang P-C Expression of bisecting type and Lewisx/Lewisy terminated N-glycans on human sperm J. Biol. Chem. 2007 282 36593 36602 10.1074/jbc.M705134200 17913713
Pang, P.-C. et al. Expression of bisecting type and Lewisx/Lewisy terminated N-glycans on human sperm. J. Biol. Chem. 282, 36593–36602 (2007).17913713 10.1074/jbc.M705134200
57. Clark GF The role of glycans in immune evasion: The human fetoembryonic defence system hypothesis revisited Mol. Hum. Reprod. 2014 20 185 199 10.1093/molehr/gat064 24043694
Clark, G. F. The role of glycans in immune evasion: The human fetoembryonic defence system hypothesis revisited. Mol. Hum. Reprod. 20, 185–199 (2014).24043694 10.1093/molehr/gat064
58. Erlebacher A Immunology of the maternal-fetal interface Annu. Rev. Immunol. 2013 31 387 411 10.1146/annurev-immunol-032712-100003 23298207
Erlebacher, A. Immunology of the maternal-fetal interface. Annu. Rev. Immunol. 31, 387–411 (2013).23298207 10.1146/annurev-immunol-032712-100003
59. Chen Q Evidence for differential glycosylation of trophoblast cell types Mol. Cell. Proteomics MCP 2016 15 1857 1866 10.1074/mcp.M115.055798 26929217
Chen, Q. et al. Evidence for differential glycosylation of trophoblast cell types. Mol. Cell. Proteomics MCP 15, 1857–1866 (2016).26929217 10.1074/mcp.M115.055798
60. Mohd Zaki A Neutrophils dominate the cervical immune cell population in pregnancy and their transcriptome correlates with the microbial vaginal environment Front. Microbiol. 2022 13 904451 10.3389/fmicb.2022.904451 35774454
Mohd Zaki, A. et al. Neutrophils dominate the cervical immune cell population in pregnancy and their transcriptome correlates with the microbial vaginal environment. Front. Microbiol. 13, 904451 (2022).35774454 10.3389/fmicb.2022.904451
61. Hunter PJ Sheikh S David AL Peebles DM Klein N Cervical leukocytes and spontaneous preterm birth J. Reprod. Immunol. 2016 113 42 49 10.1016/j.jri.2015.11.002 26637953
Hunter, P. J., Sheikh, S., David, A. L., Peebles, D. M. & Klein, N. Cervical leukocytes and spontaneous preterm birth. J. Reprod. Immunol. 113, 42–49 (2016).26637953 10.1016/j.jri.2015.11.002
62. Farr C Increased cervical neutrophil survival during bacterial vaginosis in Canadian women from the THRIVE study J. Immunol. 2020 204 157 10.4049/jimmunol.204.Supp.157.13
Farr, C. et al. Increased cervical neutrophil survival during bacterial vaginosis in Canadian women from the THRIVE study. J. Immunol. 204, 157 (2020).10.4049/jimmunol.204.Supp.157.13
63. Babu P Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology Glycoconj. J. 2009 26 975 986 10.1007/s10719-008-9146-4 18587645
Babu, P. et al. Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology. Glycoconj. J. 26, 975–986 (2009).18587645 10.1007/s10719-008-9146-4
64. Harazono A Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry J. Chromatogr. B 2008 869 20 30 10.1016/j.jchromb.2008.05.006
Harazono, A. et al. Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry. J. Chromatogr. B 869, 20–30 (2008).10.1016/j.jchromb.2008.05.006
65. Anthony RM Ravetch JV A novel role for the IgG Fc glycan: The anti-inflammatory activity of sialylated IgG Fcs J. Clin. Immunol. 2010 30 Suppl 1 S9 14 10.1007/s10875-010-9405-6 20480216
Anthony, R. M. & Ravetch, J. V. A novel role for the IgG Fc glycan: The anti-inflammatory activity of sialylated IgG Fcs. J. Clin. Immunol. 30(Suppl 1), S9-14 (2010).20480216 10.1007/s10875-010-9405-6
66. Royle L Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems * J. Biol. Chem. 2003 278 20140 20153 10.1074/jbc.M301436200 12637583
Royle, L. et al. Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems *. J. Biol. Chem. 278, 20140–20153 (2003).12637583 10.1074/jbc.M301436200
67. Andersch-Björkman Y Thomsson KA Larsson JM Ekerhovd E Hansson GC Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle Mol. Cell. Proteomics. 2007 6 4 708 16 10.1074/mcp.M600439-MCP200 17220477
Andersch-Björkman, Y., Thomsson, K. A., Larsson, J. M., Ekerhovd, E. & Hansson, G. C. Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle. Mol. Cell. Proteomics. 6(4), 708–16 (2007).17220477 10.1074/mcp.M600439-MCP200
68. Taniguchi T N-glycosylation affects the stability and barrier function of the MUC16 mucin J. Biol. Chem. 2017 292 11079 11090 10.1074/jbc.M116.770123 28487369
Taniguchi, T. et al. N-glycosylation affects the stability and barrier function of the MUC16 mucin. J. Biol. Chem. 292, 11079–11090 (2017).28487369 10.1074/jbc.M116.770123
69. Wong NK Characterization of the oligosaccharides associated with the human ovarian tumor marker CA125 J. Biol. Chem. 2003 278 31 28619 34 10.1074/jbc.M302741200 12734200
Wong, N. K. et al. Characterization of the oligosaccharides associated with the human ovarian tumor marker CA125. J. Biol. Chem. 278(31), 28619–34 (2003).12734200 10.1074/jbc.M302741200
70. Haridas D MUC16: Molecular analysis and its functional implications in benign and malignant conditions FASEB J. 2014 28 4183 4199 10.1096/fj.14-257352 25002120
Haridas, D. et al. MUC16: Molecular analysis and its functional implications in benign and malignant conditions. FASEB J. 28, 4183–4199 (2014).25002120 10.1096/fj.14-257352
71. Gipson IK Spurr-Michaud S Tisdale A Menon BB Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function PLoS ONE 2014 9 e100393 10.1371/journal.pone.0100393 24968021
Gipson, I. K., Spurr-Michaud, S., Tisdale, A. & Menon, B. B. Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function. PLoS ONE 9, e100393 (2014).24968021 10.1371/journal.pone.0100393
72. Jang-Lee J Glycomic profiling of cells and tissues by mass spectrometry: Fingerprinting and sequencing methodologies Methods Enzymol. 2006 415 59 86 10.1016/S0076-6879(06)15005-3 17116468
Jang-Lee, J. et al. Glycomic profiling of cells and tissues by mass spectrometry: Fingerprinting and sequencing methodologies. Methods Enzymol. 415, 59–86 (2006).17116468 10.1016/S0076-6879(06)15005-3
73. North SJ Mass spectrometric analysis of mutant mice Methods Enzymol. 2010 478 27 77 10.1016/S0076-6879(10)78002-2 20816474
North, S. J. et al. Mass spectrometric analysis of mutant mice. Methods Enzymol. 478, 27–77 (2010).20816474 10.1016/S0076-6879(10)78002-2
74. Ceroni A GlycoWorkbench: A tool for the computer-assisted annotation of mass spectra of glycans J. Proteome Res. 2008 7 1650 1659 10.1021/pr7008252 18311910
Ceroni, A. et al. GlycoWorkbench: A tool for the computer-assisted annotation of mass spectra of glycans. J. Proteome Res. 7, 1650–1659 (2008).18311910 10.1021/pr7008252
75. Gibb S Strimmer K MALDIquant: A versatile R package for the analysis of mass spectrometry data Bioinforma. Oxf. Engl. 2012 28 2270 2271 10.1093/bioinformatics/bts447
Gibb, S. & Strimmer, K. MALDIquant: A versatile R package for the analysis of mass spectrometry data. Bioinforma. Oxf. Engl. 28, 2270–2271 (2012).10.1093/bioinformatics/bts447
76. Pruski P Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and preterm birth Nat. Commun. 2021 12 5967 10.1038/s41467-021-26215-w 34645809
Pruski, P. et al. Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and preterm birth. Nat. Commun. 12, 5967 (2021).34645809 10.1038/s41467-021-26215-w
77. Frank JA Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes Appl. Environ. Microbiol. 2008 74 2461 2470 10.1128/AEM.02272-07 18296538
Frank, J. A. et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl. Environ. Microbiol. 74, 2461–2470 (2008).18296538 10.1128/AEM.02272-07
78. Kechin A Boyarskikh U Kel A Filipenko M cutPrimers: A new tool for accurate cutting of primers from reads of targeted next generation sequencing J. Comput. Biol. 2017 24 1138 1143 10.1089/cmb.2017.0096 28715235
Kechin, A., Boyarskikh, U., Kel, A. & Filipenko, M. cutPrimers: A new tool for accurate cutting of primers from reads of targeted next generation sequencing. J. Comput. Biol. 24, 1138–1143 (2017).28715235 10.1089/cmb.2017.0096
79. Babraham Bioinformatics—FastQC A Quality Control tool for High Throughput Sequence Data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
80. Bolyen E Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 Nat. Biotechnol. 2019 37 852 857 10.1038/s41587-019-0209-9 31341288
Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37, 852–857 (2019).31341288 10.1038/s41587-019-0209-9
81. Callahan BJ DADA2: High-resolution sample inference from Illumina amplicon data Nat. Methods 2016 13 581 583 10.1038/nmeth.3869 27214047
Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).27214047 10.1038/nmeth.3869
82. Fettweis JM Species-level classification of the vaginal microbiome BMC Genomics 2012 13 Suppl 8 S17 10.1186/1471-2164-13-S8-S17 23282177
Fettweis, J. M. et al. Species-level classification of the vaginal microbiome. BMC Genomics 13(Suppl 8), S17 (2012).23282177 10.1186/1471-2164-13-S8-S17
83. France MT VALENCIA: A nearest centroid classification method for vaginal microbial communities based on composition Microbiome 2020 8 166 10.1186/s40168-020-00934-6 33228810
France, M. T. et al. VALENCIA: A nearest centroid classification method for vaginal microbial communities based on composition. Microbiome 8, 166 (2020).33228810 10.1186/s40168-020-00934-6
