N-glycosylation of cervicovaginal fluid reflects microbial community, immune activity, and pregnancy status

Human cervicovaginal fluid (CVF) is a complex, functionally important and glycan rich biological fluid, fundamental in mediating physiological events associated with reproductive health. Using a comprehensive glycomic strategy we reveal an extremely rich and complex N-glycome in CVF of pregnant and non-pregnant women, abundant in paucimannose and high mannose glycans, complex glycans with 2–4 N-Acetyllactosamine (LacNAc) antennae, and Poly-LacNAc glycans decorated with fucosylation and sialylation. N-glycosylation profiles were observed to differ in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome. Compared to CVF from women experiencing term birth, CVF from women who subsequently experienced preterm birth showed lower sialylation, which correlated to the presence of a diverse microbiome, and higher fucosylation, which correlated positively to pro-inflammatory cytokine concentration. This study is the first step towards better understanding the role of cervicovaginal glycans in reproductive health, their contribution to the mechanism of microbial driven preterm birth, and their potential for preventative therapy.


Results
N-glycan structural profiling. CVF samples from a total of 10 donors with diverse ethnicities were collected for analysis. 4 of the donors were non-pregnant women, while 6 of the 10 donors were pregnant women defined at high risk of spontaneous preterm delivery (sPTB). Of these, 3 delivered at term, while 3 delivered preterm (< 37 weeks of pregnancy) ( Table 1, Supplementary Table S1).
A detailed MALDI-MS based glycomic characterisation of permethylated N-glycans was undertaken. This produces [M + Na] + molecular ions. An overall relative quantitation of paucimannose, high mannose, hybrid and complex glycans is shown in Supplementary Fig. S1. More detailed structural characterization is illustrated in Fig. 1. Paucimannose gycans were identified in the low mass range at m/z 1141, 1171, 1345 and 1375, (Man 2-4 GlcNAc 2 Fuc 0-1 ). The glycans in the mid mass range were dominated by high mannose glycans (m/z 1579-2396, Man [5][6][7][8][9] GlcNAc 2 ) and bi-antennary complex glycans with fucosylation and/or sialylation (m/z 1835-3211, NeuAc 0-2 Gal 0-2 Man 3 GlcNAc 4-5 Fuc 0-1 ). The glycans at higher mass range contained multiple LacNAc units (Gal-GlcNAc) modified by different numbers of fucose and sialic acid (m/z 3402-4650, NeuAc 0-3 Gal 3-6 Man 3 GlcNAc 4-8 Fuc 0-5 ). The largest complex N-glycan observed was at m/z 6259 corresponding to a composition of Gal 9 Man 3 GlcNAc 11 Fuc 6 (Supplementary Table S2). More detailed N-glycan structural analysis was achieved by MS/MS analysis of selected molecular ions. This proved that the mono-fucosylated glycans can be both core or antenna fucosylated ( Supplementary Fig. S2a), and that peaks with composition consistent with www.nature.com/scientificreports/ multiple LacNAc units are a mixture of structural isomers with varying numbers and lengths of antennae. Complex glycans with 2-4 LacNAc units were identified in the medium mass range and glycans with poly LacNAc units in the high mass range (Fig. 1). Glycans above m/z 5000 included high numbers of LacNAc units exhibiting diverse modifications by Fuc and NeuAc (Supplementary table S2). The MS/MS analysis reveals that structures with extended antennae are often favoured over branched structures ( Supplementary Fig. S2b). These extended antennae were decorated by several fucose residues producing diverse poly Lewis antigens. LacdiNAc (GalNAcβ1-4GlcNAc) containing structures were identified at m/z 2285, 2326, 2500 and 2674, which were supported by MS/MS fragmentation analysis ( Supplementary Fig. S2c). Interestingly, sample P5, from a donor with CST IV-B who delivered preterm at 31 + 3 weeks, showed a distinct pattern of glycan abundances compared with other samples. Its N-glycome was dominated by non-sialylated, and non-/ mono-fucosylated glycans ( Supplementary Fig. S3). In addition, this sample had high levels of N-glycans with truncated antennae terminated by GlcNAc.
The complexity of CVF N-glycans with their multiple fucosylation and sialylation led us to apply additional complementary analytical strategies to facilitate deeper characterization of CVF N-glycan structures. Glyco-epitope centric N-glycan analysis using an Orbitrap mass spectrometer confirmed the presence of diverse glycotopes in a selected sample P3 (Fig. 2). MS 2 fragments of N-glycans detected LacdiNAc structures at m/z 505 and 679, terminal Lewis antigens at m/z 638 and 812, internal Lewis antigens at m/z 624, and Sialyl Lewis antigens at m/z 999. In addition, poly Lewis antigens were observed at m/z 1261 and m/z 1435. The MS 2 data also showed terminal GlcNAc or GalNAc at m/z 260, predicted to arise from bisecting glycans, glycans with truncated antennae, or LacdiNAc structures. The MS 2 peak at m/z 812 was selected for further fragmentation to obtain MS 3 data, which mainly detected the Lewis Y antigen. However, the MS 3 analysis of the MS 2 peak at m/z 638 revealed a mixture of Lewis X, Lewis A and the blood group H antigen, again highlighting the structural complexity of CVF N-glycans.
Correlation of fucosylation and sialylation to pregnancy status. We next focused on quantitative analysis of the fucosylation and sialylation of the CVF N-glycans containing 2 or 3 LacNAc units (Fig. 3). Glycans with 2 LacNAc units were shown to have up to 5 Fuc/glycan (Fig. 3a) whereas glycans with 3 LacNAc units had up to 7 Fuc/glycan (Fig. 3c). In terms of sialylation, glycans with 2 LacNAc units had up to 2 sialic acids ( Fig. 3b) and glycans with 3 LacNAc units had up to 3 sialic acids (Fig. 3d). Variations of fucosylation and sialylation were observed among the samples. Quantitative analysis of Sample P5 showed extremely low levels of poly-fucosylation and sialylation for glycans with 2 or 3 LacNAc units (Fig. 3).
As shown in Fig. 4, differential fucosylation was observed between non-pregnant and pregnant samples. For glycans with 2 LacNAc units, the pregnant samples had higher levels of non-fucosylated glycans and monofucosylated glycans. However, this trend started to reverse for bi-fucosylated glycans and was completely reversed for tri-and tetra-fucosylated glycans. Similar results were observed for glycans with 3 LacNAc units, except that the reversing point was at the tri-fucosylated glycans. The proportion of tetra-fucosylated glycans decreased to almost zero in the pregnant samples, but was retained at about 12% for glycans with 2 LacNAc units and 30% for glycans with 3 LacNAc units in the non-pregnant samples. An initial trend of fucosylation and sialylation changes were detected between preterm and term samples (Fig. 5). CVF samples of women who delivered preterm showed higher levels of non-fucosylated glycans among glycans with 2 or 3 LacNAc units. The sialylation level among poly-fucosylated glycans tended to be lower for the preterm samples.

Correlation of fucosylation and sialylation to microbial composition. Community State Type
(CST) was used to characterise the vaginal microbial community status. Analysis of vaginal microbiota revealed that 3 of the pregnant samples had a vaginal bacterial community dominated by L. crispatus, consistent with Community State Type I (CST I) as described elsewhere 47 , while 3 had a CST IV-B, characterised by G. vaginalis and A. vaginae dominance. Of the CST I samples two (P2, P4) were CST I-A (almost completely dominated by L. crispatus), and one (P1) was CST I-B (mostly dominated by L. crispatus but also containing low abundance of L. gasseri, L. jensenii and G. vaginalis). Of the samples that were classified as CST IV-B, there was a high relative abundance of G. vaginalis in two (P5 and P6), and a moderate relative abundance of A. vaginae and L. acidophilus in the other (P3) (Supplementary Fig. S5). The correlation between fucosylation and vaginal CST was investigated in relation to the number of Fuc residues per glycan (Fig. 6). N-glycans with 2 LacNAc units and those with 3 LacNAc units showed the same trend: samples from CST IV-B donors had higher levels of non-fucosylated glycans and mono-fucosylated glycans, but lower levels of bi-fucosylated, tri-fucosylated and tetra-fucosylated glycans, compared with CST I-A/B donors. Detailed analysis of the correlation between sialylation and CST is shown in Fig. 7. Sialylation of glycans with different numbers of Fuc was analysed separately. The same trend was found regardless of the fucosylation status: CST IV-B samples consistently showed a lower level of sialylation compared to CST I-A/B samples. Our results suggest that the presence of a high relative abundance of Lactobacillus spp. is associated with a higher percentage of sialylation, bi-, tri-and tetra-fucosylated glycans, and lower levels of non-or mono-fucosylated glycans. www.nature.com/scientificreports/ Correlation of fucosylation and sialylation to pro-inflammatory cytokines. Strong correlation was found when fucosylation was mapped to levels of IL-1β and IL-18 (Figs. 8 and 9). The proportion of nonfucosylated glycans (Fig. 8a) and the proportion of mono-fucosylated glycans (Fig. 8b) to total glycans were positively correlated with cytokine concentration, while the proportion of poly-fucosylated glycans to total glycans ( Fig. 8c) and the proportion of highly-fucosylated glycans to poly-fucosylated glycans ( Fig. 8d) were negatively correlated with the cytokine concentrations. Similar correlations were observed for glycans with 3 LacNAc units ( Fig. 9). No statistically significant correlations were seen with IL-8 and IL-6, however similar trends were observed (Supplementary Figs. S6 and S7). We also investigated levels of glycan sialylation and pro-inflammatory cytokines, however, only weak correlations were observed (Supplementary Figs. S1-S11).

Discussion
Despite the importance of human CVF in shaping microbiota-host responses in the lower reproductive tract, to our knowledge, a detailed profiling of CVF N-glycans has not been reported. Our study characterises CVF as a glycan-rich environment with distinct and different N-glycosylation in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome. Our findings are based on a small cohort at present. This is due to a combination of factors including the collection device being more invasive than a simple swab and the labour intensive and high cost of sample processing. However, some initial trends have already been consistently observed. We also acknowledge that factors such as the stage of menstrual cycle or pregnancy, age, cervical length, blood group and ethnicity could influence the CVF glycosylation profile. We aim to study these factors with larger cohorts. The N-glycome of CVF is highly complex and abundant in paucimannose glycans and high mannose glycans in the low mass range. Complex glycans with 2-4 antennae in the medium mass range and glycans with poly LacNAc units in the high mass range are readily detectable. Antennae structures of detected N-glycans included terminal fucosylation of mono-fucosylated glycans, glycans with LacdiNAc structures, as well as sialylation and fucosylation patterns of extended poly LacNAc structures. Identification of a higher prevalence of glycan isomers carrying extended poly LacNAc chains compared to multi-antennary structures, suggests a biological role of the elongated antennae protruding from the mucin protein backbone 49,50 . While extended poly-N-acetyllactosamine glycans can serve as spacers and carriers of additional glycotopes through their modification with fucose and sialic acid, poly LacNAc itself has been identified as a recognition motif of galectins 51 , an important class of mammalian GBP involved in regulations of immune cell activities and microbial recognition as part of the innate immune system [52][53][54] . N-Glycan structures carrying extended poly LacNAc chains have recently been reported as a characteristic feature of neutrophil granules 55 , which suggests these structures in CVF might partially be due to leukocyte infiltration. High mannose glycans can be recognized by DC-SIGN, the mannose receptor and other mannose binding GBP, which have been found to be involved in inflammation and infection 56 .
Modifications of genital tract epithelia, mucus, and immune status occur throughout the menstrual cycle to accommodate conception and implantation 29 . At the time of conception, Sialyl Lewis X glycan epitopes on the human oocyte facilitates sperm-egg binding 57 , and glycans have also been implicated in the mediation of implantation 58 and immune homeostasis at the maternal-fetal interface 59 . Once implantation is successful, the cervicovaginal interface undergoes further modification, with the primary aim of forming a cervical barrier to infectious microbes to protect the developing fetus. Our study shows that this physiological switch to a pregnancy phenotype also involves marked changes to cervicovaginal glycan profiles. In both non-pregnant and pregnant women, N-glycans were found to be a mixture of paucimannose and high-mannose structures in the lower mass range, biantennary complex poly-fucosylated structures in the mid mass range, and large PolyLacNAc glycans the higher mass range. However, spectra of non-pregnant women CVF are clearly dominated by high mannose glycans in the lower mass range, which could be a sign of reduced glycan maturation and differentiation 7 or a different microbiota composition 25 compared to pregnant women. Oligosaccharides carrying terminal mannose residues are the main host receptors for bacteria such as E. coli. 60 A loss of high mannose residues on glycoproteins of the CVL from women with BV was observed by Wang et al. 61 , suggesting that native high mannose residues found on glycoproteins in fluids of the lower reproductive tract may act as natural inhibitors of pathogenic interactions, helping to prevent bacterial adhesion in women with normal microflora.   We also demonstrated differences in glycan profiles between women who subsequently delivered preterm and women who delivered at term. The preterm samples showed higher levels of non-fucosylated glycans among glycans with 2 or 3 LacNAc units and lower sialylation among poly-fucosylated glycans. Desialylation of glycoproteins and glycolipids by sialidase leads to loss of or exposure of new glycan epitopes and interference with host immune recognition and binding of microbes 62 . It is feasible that this could contribute to the mechanism of microbial driven preterm birth. In support of this, high sialidase concentrations have been associated with late miscarriage and preterm delivery 63,64 . Further, we and others have reported an association between vaginal microbial composition and preterm birth risk 9 . High diversity vaginal microbial composition (referred to as community state type IV, CST IV), or dominance of the niche by L. iners, is associated with higher rates of preterm birth, whereas L. crispatus (CST I) dominance is protective 26,65,66 . In this study N-glycans with 2 and 3 LacNAc units isolated from CVF of women with CST IV have lower levels of both poly and highly-fucosylated glycans and sialyated glycans compared to women with CST-I.
Fucosylation and sialylation on LacNAc units can potentially produce different glycotopes, such as Lewis antigens, Sialyl Lewis antigens and poly Lewis antigens. Several studies have shown how glycans and their epitopes play a role in infective conditions such as HIV 67 and Candida albicans 16 . G. vaginalis is one of the main bacteria associated with bacterial vaginosis and CST IV, which is characterised by depletion of Lactobacillus species and polymicrobial overgrowth of anaerobes 12,68 . BV is also associated with increased vaginal concentrations of glycosidases such as sialidase, α-galactosidase, β-galactosidase and α-glucosidase 69,70 and decreased binding of lectins to both high mannose and α-2, 6 sialic acid. This observation suggests that bacteria sialidases and host sialoglycan receptors can be linkage specific, as also reported by other studies 1,71 . Findings from previous studies suggest that changes in glycosidases are accompanied by changes in glycosylation patterns in the vaginal fluid. On the other hand, a recent discovery found that Lactobacillus spp. Can create a protective micro-ecological environment through regulating the core fucosylation of vaginal epithelial cells 72 . Although glycosylation of the CVF could be influenced by hormone levels, it is thought that microbiota have a greater influence on CVF glycosylation than hormonal status 61 .
Similarly to CVF, the human gastrointestinal (GI) mucosa consists of extensive glycoproteins overlaying epithelial cells, interspersed by immune cells, which form a protective physical barrier, and serve as first line of defence against microorganisms and harmful substances 73,74 . Hydrolases, including exo-and endoglycosidases, are encoded in the genomes of mucin-degrading gut bacteria 73 allowing them to metabolize sugars 73,75 , which are then exploited as nutrient source 76 . The mucins of the GI tract mucus layer also provide ligands for the attachment of bacteria 77 and may facilitate invasion; moreover, mucus-binding proteins such as adhesins and GBP have been described in many lactic acid bacilli 78 . The human gut microbiome and its role in both health and disease has been extensively studied and its involvement in human metabolism, nutrition, physiology, and immune function is now well established [79][80][81][82] . It is highly probable that similar interactions exist in the mucosa of the female lower reproductive tract, creating a tight interconnection between glycans, microorganisms colonising the female reproductive tract and cervicovaginal immune homeostasis. www.nature.com/scientificreports/ It is well established that local inflammation is a risk factor for preterm birth. However, only recently are we beginning to understand the relationship between inflammation, specific microbial communities, and preterm birth 21,23,46,83 . Here we observed that non-fucosylated and monofucosylated glycans positively correlated with pro-inflammatory cytokines, whereas poly and high-fucosylated glycans are negatively correlated, highlighting the important role of N-glycosylation in maintaining immune homeostasis in the female reproductive tract. An abundance of paucimannose glycans was also detected in the low mass range of CVF, which could originate from infiltrating neutrophils, monocytes and macrophages 48,55,[84][85][86] . These glycans could be derived from a recently described noncanonical truncation N-glycosylation pathway in human neutrophils 48 . Paucimannose glycans are found on proteins in azurophilic granules of neutrophils, which can be selectively secreted upon pathogen stimulation, and their glycosylation status is involved in modulating multiple immune functions central to inflammation and infection [87][88][89] . The unusually high abundance of paucimannose structures found in CVF is potentially indicative of neutrophil invasion in the lower female reproductive tract, which could be used as a defence mechanism against invading pathogens. It is plausible that glycan-GBP interactions between pathogenic microbes and neutrophils leads to neutrophil degranulation, an abundance of paucimannoses, pro-inflammatory cytokine release and subsequent preterm labour. Another possible explanation for the paucimannose glycans is that they are the result of degradation by microbial glycohydrolytic enzymes 69,70 .
It should be noted that CVF is also heavily O-glycosylated and we are undertaking similar analytic approaches to characterise these glycan structures.
In summary, this study provides evidence that cervicovaginal glycans are associated with microbial composition and immune response in pregnancy where they may influence clinical outcome. Modulation of cervicovaginal glycans could thus represent a novel therapeutic strategy to prevent microbial driven preterm birth.   www.nature.com/scientificreports/ ane, ammonium bicarbonate, EDTA, trypsin and Tris 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, 3.5K MWCO were from Thermo scientific (Loughborough, UK). Glycomic sample processing was done following the protocol detailed previously 90,91 . Briefly, CVF samples from aliquot one 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 were released by PNGase F and were permethylated. The permethylated glycans were cleaned by C18 cartridges and freeze dried before mass spectrometry analysis. Glycan profiling was done on AB Sciex 4800 MALDI-TOF/TOF mass spectrometer. The methylated glycans were dissolved in 10 ul methanol. One 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 analysis. The data were analysed using Data Explorer™ version 4.6 from AB Sciex, Glycoworkbench 92 and MALDIquant 93 . The glycomic data were annotated based on monosaccharide composition derived from the molecular ion m/z value, knowledge of N-glycan biosynthetic pathways, the isotopic peak cluster patterns, the glycosylation patterns in the low and medium mass range, and MS/MS derived fragmentation. For glycans with overlapped isotopic peak clusters, the dominant one was annotated.
The statistical analysis of the glycans was done using SQLite3 and R 3.6.3 on a Linux Ubuntu 20.04.4 LTS platform. The scripts are available on GitHub (Link: https:// github. com/ gw110/ CVF-glyco sylat ion. git). In order to have the least interference of overlapped isotopic peaks, Glycans with 2 or 3 LacNAc units were selected for quantitative analysis to assure high accuracy of data annotation and quantitation. The relative intensities of different glycan subgroups were calculated using SQLite3. The results were exported and visualized using ggplot2 in R 3.6.3. The correlation of glycan relative intensities with cytokines was done using the linear regression model. Pearson product-moment correlation coefficient and p value were calculated using the cor.test function in R. Box plot and two tailed Student's t test was used to compare glycosylation between sample groups.
Glycans were further analysed following a glycotope-centric glycomic analysis based on a nanoLC-MS 2product dependent-MS 3 data acquisition workflow, previously established using the advanced Orbitrap Fusion Tribrid MS platform 94 . In essence, this takes advantage of the high acquisition speed, high mass accuracy and high sensitivity of the MS instrument to perform a comprehensive MS 2 mapping of the terminal glycotope based on preferred cleavage at the HexNAc of permethylated glycans. The respective intensity of each characteristic oxonium ion measured at 5 ppm mass accuracy or less can be summed from all glycan MS 2 spectral acquired throughout the LC-MS/MS run to provide a relative abundance index for the glycotope it represents. In cases when isomeric glycotopes need to be differentiated, as for the Lewis and H epitopes in this work, the MS 2 ion can be coupled with a further MS 3 event. The diagnostic MS 3 ions resulting from specifically eliminating the C3-substituent of the HexNAc + is sufficient to resolve the isomeric difference and their relative intensity can in turn provide a semi-quantitative estimation of the relative abundance of each isomeric constituent of that particular glycotope.
Bacterial DNA extraction and metataxonomic profiling. Extraction of DNA from CVF samples and sequencing of 16S rRNA hyper variable regions was performed as previously described 46 . 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 GAG TTT GAT CCT GGC TTA G; 28F-Chlorflex GAA TTT GAT CTT GGT TCA G; 28F-Bifido GGG TTC GAT TCT GGC TCA G; 28F GAG TTT GATCNTGG CTC AG. The reverse primer consisted of; 388R TGC TGC CTC CCG TAG GAG T 95 . 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 Cutadapt 96 and QC performed using FastQC 97 . Resulting amplicon sequence variant (ASV) counts were calculated for each sample using the Qiime2 pipeline 98 . DADA2 was used for denoising 99 and taxonomic classification of sequences to species level was performed using the STIRRUPS reference database 100 . Samples were classified into vaginal community state types (CSTs) using the VAginaL community state typE Nearest CentroId classifier (VALENCIA) 47 . Using this standardised approach, CST 1 communities are characterised by Lactobacillus crispatus dominance and can be further subdivided into subtypes CST 1-A (almost complete dominance by L. crispatus) and CST 1-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 from the CVF (aliquot two) was thawed on ice and used for Luminex® immunoassays to quantify cytokines by multiplexed bead-based immunoassays. The concentrations of 10 cytokines (IL-1β, IL-18, IL-6, IL-2, IL-4, IL-5, IL-10, IFN-γ, GM-CSF and TNF-α) 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 10-fold dilution using Calibrator Diluent RD6-52. All standards and samples were run in duplicate. Concentrations of IL-2, IL-5, IFN-γ, and GM-CSF were undetectable in all samples. IL-4, IL-10 and TNF-α were only detectable in a proportion of samples.