Dietary amino acids, macronutrients, vaginal birth, and breastfeeding are associated with the vaginal microbiome in early pregnancy.

The vaginal microbiome is a key player in the etiology of spontaneous preterm birth. This study aimed to illustrate maternal environmental factors associated with vaginal microbiota composition and function in pregnancy. Women in healthy pregnancy had vaginal microbial sampling from the posterior vaginal fornix performed at 16 weeks gestation. After shotgun metagenomic sequencing, heatmaps of relative abundance data were generated. Community state type (CST) was assigned, and alpha diversity was calculated. Demography, obstetric history, well-being, exercise, and diet using food frequency questionnaires were collected and compared against microbial parameters. A total of 119 pregnant participants had vaginal metagenomic sequencing performed. Factors with strongest association with beta diversity were dietary lysine (adj-R2 0.113, P = 0.002), valine (adj-R2 0.096, P = 0.004), leucine (adj-R2 0.086, P = 0.003), and phenylalanine (adj-R2 0.085, P = 0.005, Fig. 2D). Previous vaginal delivery and breastfeeding were associated with vaginal beta diversity (adj-R2 0.048, P = 0.003; adj-R2 0.045, P = 0.004), accounting for 8.5% of taxonomy variation on redundancy analysis. Dietary fat, starch, and maltose were positively correlated with alpha diversity (fat +0.002 SD/g, P = 0.025; starch +0.002 SD/g, P = 0.043; maltose +0.440 SD/g, P = 0.013), particularly in secretor-positive women. Functional signature was associated with CST, maternal smoking, and dietary phenylalanine, accounting for 8.9%-11% of the variation in vaginal microbiome functional signature. Dietary amino acids, previous vaginal delivery, and breastfeeding history were associated with vaginal beta diversity. Functional signature of the vaginal microbiome differed with community state type, smoking, dietary phenylalanine, and vitamin K. Increased alpha diversity correlated with dietary fat and starch. These data provide a novel snapshot into the associations between maternal environment, nutrition, and the vaginal microbiome. IMPORTANCE: This secondary analysis of the MicrobeMom randomized controlled trial reveals that dietary amino acids, macronutrients, previous vaginal birth, and breastfeeding have the strongest associations with vaginal taxonomy in early pregnancy. Function of the vaginal niche is associated mainly by species composition, but smoking, vitamin K, and phenylalanine also play a role. These associations provide an intriguing and novel insight into the association between host factors and diet on the vaginal microbiome in pregnancy and highlight the need for further investigation into the complex interactions between the diet, human gut, and vaginal microbiome.

Endometriosis specific vaginal microbiota links to urine and serum N-glycome.

Endometriosis is a chronic systemic disease, which results in endometrial-type tissue growing outside the uterus, and affects approximately 10% of reproductive-aged women worldwide. Its aetiology is poorly understood, and there is currently no long-term cure. Development and persistence of the disease depend on several coexisting factors including the vaginal microbiome. However, the role played by this important entity in endometriosis and its systemic involvement is not fully understood. Here, we investigated the vaginal microbiota, the serum and urine glycome, and antibody glycosylation in endometriosis patients. We reveal an endometriosis-specific vaginal microbiota in patients, being distinct from that present in a control group. Endometriosis patients were typified by a loss of the dominant Lactobacillus species, i.e. Lactobacillus iners, increased bacterial diversity and the presence of species such as Anaerococcus senegalensis, Prevotella jejuni, Porphyromonas bennonis and Anaerococcus octavius. The presence of trigalactosylated and triantennary serum glycans and urine core fucosylated mono-antennary glycans from IgG correlated with the vaginal presence of the bacterium A. senegalensis in endometriosis patients. Urine glycans did not differ in endometriosis, but urine IgG identified four novel sulfated glycans differing from serum IgG indicating functional relevance. Our findings contribute to understanding the relationships between the vaginal microbiota and the serum and urine glycome on the one hand, and endometriosis on the other. Further functional studies are warranted.

Impact of previous pregnancy and BMI on cellular and serum immune activity from early to late pregnancy.

Immunological adaptions during pregnancy play a crucial role in healthy fetal development. Aberrant immune modifications however contribute to adverse pregnancy outcomes, which may be driven by maternal factors such as previous pregnancies and BMI. This secondary analysis of the MicrobeMom2 RCT investigates the changes to maternal inflammatory biomarkers derived from serum and stimulated peripheral blood mononuclear cells (PBMCs) during pregnancy, and the effects of previous pregnancies (parity) and BMI on maternal immune responses. Changes in immune and metabolic biomarkers from early (11-15 weeks' gestation) to late (28-32 weeks' gestation) pregnancy were compared using paired t-tests. Participants were then split by parity (nulliparous, parous) and BMI (BMI < 25, BMI > = 25), and the relationship between parity and BMI with immune biomarker levels was examined using independent t-tests, paired t-tests, ANCOVA, and linear regression. Equivalent non-parametric tests were used for skewed data. Recruited women (n = 72) were on average 31.17 (SD ± 4.53) years of age and 25.11 (SD ± 3.82) BMI (kg/m2). Of these, 51 (70.8%) had a previous term pregnancy. Throughout gestation, PBMC cytokines displayed contrasting trends to serum, with a dampening of immune responses noted in PBMCs, and enhanced production of cytokines observed in the serum. Significant decreases in PBMC derived TNF-α, IL-10 and IFN-γ were seen from early to late pregnancy. Serum C3, IL-17A, IL-6, TNF-α, CD163, GDF-15 and leptin increased throughout gestation. First pregnancy was associated with higher levels of leptin in late pregnancy, while parous women showed significant decreases in PBMC derived TNF-α, IL10, and IFN-γ with gestation. Differences in levels of C3, IL-17A, TNF-α, GDF-15 and leptin were observed across BMI groups. Overall, serum-derived cytokines exhibit contrasting levels to those derived from stimulated PBMCs. Maternal immune responses undergo significant changes from early to late pregnancy, which are influenced by parity and BMI. These differences aid our understanding as to why first-time mothers are at greater risk of placental disease such as pre-eclampsia and fetal growth restriction.

Characterization of intestinal O-glycome in reactive oxygen species deficiency.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.

Changes in tissue protein N-glycosylation and associated molecular signature occur in the human Parkinsonian brain in a region-specific manner.

Parkinson's disease (PD) associated state of neuroinflammation due to the aggregation of aberrant proteins is widely reported. One type of post-translational modification involved in protein stability is glycosylation. Here, we aimed to characterize the human Parkinsonian nigro-striatal N-glycome, and related transcriptome/proteome, and its correlation with endoplasmic reticulum (ER) stress and unfolded protein response (UPR), providing a comprehensive characterization of the PD molecular signature. Significant changes were seen upon a PD: a 3% increase in sialylation and 5% increase in fucosylation in both regions, and a 2% increase in oligomannosylated N-glycans in the substantia nigra. In the latter, a decrease in the mRNA expression of sialidases and an upregulation in the UPR pathway were also seen. To show the correlation between these, we also describe a small in vitro study where changes in specific glycosylation trait enzymes (inhibition of sialyltransferases) led to impairments in cell mitochondrial activity, changes in glyco-profile, and upregulation in UPR pathways. This complete characterization of the human nigro-striatal N-glycome provides an insight into the glycomic profile of PD through a transversal approach while combining the other PD "omics" pieces, which can potentially assist in the development of glyco-focused therapeutics.

Impact of Bifidobacterium longum1714® on maternal cytokine response in peripheral blood mononuclear cells.

PURPOSE: The maternal immune system is implicated in adverse pregnancy outcomes. Manipulation of maternal immune response by probiotics holds potential to reduce pregnancy complications. The MicrobeMom2 study investigates the impact of probiotic supplementation on maternal immune responses to pathogen associated molecular patterns (PAMPs) in peripheral blood mononuclear cells (PBMCs) during pregnancy. METHODS: This double-blinded randomised-controlled trial involved oral supplementation of Bifidobacterium longum subsp. longum 1714® (B. longum 1714; daily ingestion of a minimum of 1x109 colony forming units) or placebo from 16 to 20-weeks' gestation until delivery in healthy pregnant women. The primary outcome was a change in IL-10 production, after stimulation with Lipopolysaccharide (LPS) or anti-CD3/28/2, in PBMCs isolated from blood samples taken at baseline (11-15 weeks' gestation) and late pregnancy (28-32 weeks' gestation) after 48 h incubation. 68 subjects were needed (34ineachgroup) for 80 % power at an alpha significance of 0.05 to detect differences in IL10. RESULTS: 72 women (mean ± SD age 33.17 ± 4.53 years and median (25th, 75th centile) body mass index 24.93 (21.93, 27.57 kg/m2)) were recruited with primary outcome data. Using LPS, late pregnancy fold change in IL-10 in PBMCs after 48 h incubation was median (25th, 75th centile) 88.45 (4.88, 488.78) in the intervention, 24.18 (6.36, 141.17) in the control group, p = 0.183. Using anti-CD3/28/2, values were 189.69 (425.96, 866.57),148.74 (31.67, 887.03) in intervention and control groups, respectively, p = 0.506. No significant differences were observed between the two groups. CONCLUSION: Maternal antenatal supplementation with B. longum 1714 did not alter cytokine production by maternal PBMCs in response to PAMPs or anti-CD3/28/2. TRIAL REGISTRATION NUMBER: ISRCTN registry ISRCTN43013285.

An insight on the N-glycome of notochordal cell-rich porcine nucleus pulposus during maturation.

Degeneration of the intervertebral disc is an age-related condition. It also accompanies the disappearance of the notochordal cells, which are remnants of the developmental stages of the nucleus pulposus (NP). Molecular changes such as extracellular matrix catabolism, cellular phenotype, and glycosaminoglycan loss in the NP have been extensively studied. However, as one of the most significant co- and posttranslational modifications, glycosylation has been overlooked in cells in degeneration. Here, we aim to characterize the N-glycome of young and mature NP and identify patterns related to aging. Accordingly, we isolated N-glycans from notochordal cell-rich NP from porcine discs, characterized them using a combined approach of exoglycosidase digestions and analysis with hydrophilic interaction ultra-performance liquid chromatography and mass spectrometry. We have assigned over 300 individual N-glycans for each age group. Moreover, we observed a notable abundance of antennary structures, galactosylation, fucosylation, and sialylation in both age groups. In addition, as indicated from our results, increasing outer arm fucosylation and decreasing α(2,3)-linked sialylation with aging suggest that these traits are age-dependent. Lastly, we have focused on an extensive characterization of the N-glycome of the notochordal cell-rich NP in aging without inferred degeneration, describing glycosylation changes specific for aging only. Our findings in combination with those of other studies, suggest that the degeneration of the NP does not involve identical processes as aging.

N-glycans from serum IgG and total serum glycoproteins specific for endometriosis.

Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of endometrial tissue outside the uterine cavity. There are currently no definitive non-invasive diagnostic tools. Glycosylation is the most common posttranslational modification of proteins and altered glycosylation has been found in many diseases, including chronic inflammatory conditions and cancer. Sialylation and galactosylation on serum IgG have previously been found to be altered in endometriosis and serum sialylation changed after Zoladex (Goserelin Acetate) therapy. Using IgG and whole serum glycoproteins, we investigated N-glycosylation in two clinical cohorts of women with and without endometriosis. PNGase F-digested serum samples were fluorescently labelled and N-glycans were profiled by ultra-performance liquid chromatography. Clinical data was collected to link glycomic findings with metabolic and hormonal profiles. Total serum glycoprotein and IgG glycosylation differed in patients with endometriosis compared to control cases. The most significantly altered was glycan peak 3 from IgG, containing bisected biantennary glycans, which was decreased in the endometriosis cohorts (p = 0.0000005-0.018). In conclusion, this is the first pilot study to identify changes in N-glycans from whole serum glycoproteins associated with endometriosis. A larger validation study is now warranted and such studies should include the follow-up of surgically and pharmacologically treated patients.

Detailed mapping of Bifidobacterium strain transmission from mother to infant via a dual culture-based and metagenomic approach.

A significant proportion of the infant gut microbiome is considered to be acquired from the mother during and after birth. Thus begins a lifelong and dynamic relationship with microbes that has an enduring impact on host health. Based on a cohort of 135 mother-infant (F = 72, M = 63) dyads (MicrobeMom: ISRCTN53023014), we investigated the phenomenon of microbial strain transfer, with a particular emphasis on the use of a combined metagenomic-culture-based approach to determine the frequency of strain transfer involving members of the genus Bifidobacterium, including species/strains present at low relative abundance. From the isolation and genome sequencing of over 449 bifidobacterial strains, we validate and augment metagenomics-based evidence to reveal strain transfer in almost 50% of dyads. Factors important in strain transfer include vaginal birth, spontaneous rupture of amniotic membranes, and avoidance of intrapartum antibiotics. Importantly, we reveal that several transfer events are uniquely detected employing either cultivation or metagenomic sequencing, highlighting the requirement for a dual approach to obtain an in-depth insight into this transfer process.

Age-Related Changes in Serum N-Glycome in Men and Women-Clusters Associated with Comorbidity.

(1) Aim: To describe, in a general adult population, the serum N-glycome in relation to age in men and women, and investigate the association of N-glycome patterns with age-related comorbidity; (2) Methods: The serum N-glycome was studied by hydrophilic interaction chromatography with ultra-performance liquid chromatography in 1516 randomly selected adults (55.3% women; age range 18-91 years). Covariates included lifestyle factors, metabolic disorders, inflammatory markers, and an index of comorbidity. Principal component analysis was used to define clusters of individuals based on the 46 glycan peaks obtained in chromatograms; (3) Results: The serum N-glycome changed with ageing, with significant differences between men and women, both in individual N-glycan peaks and in groups defined by common features (branching, galactosylation, sialylation, fucosylation, and oligomannose). Through K-means clustering algorithm, the individuals were grouped into a cluster characterized by abundance of simpler N-glycans and a cluster characterized by abundance of higher-order N-glycans. The individuals of the first cluster were older, showed higher concentrations of glucose and glycation markers, higher levels of some inflammatory markers, lower glomerular filtration rate, and greater comorbidity index; (4) Conclusions: The serum N-glycome changes with ageing with sex dimorphism. The N-glycome could be, in line with the inflammaging hypothesis, a marker of unhealthy aging.

Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera.

Currently, there are no reliable biomarkers for the diagnosis of pancreatic cancer (PaC). Glycoproteomic approaches that analyze the glycan determinants on specific glycoproteins have proven useful to develop more specific cancer biomarkers than the corresponding protein levels. In PaC, mesothelin (MSLN) is a neo-expressed glycoprotein. MSLN glycosylation has not been described and could be altered in PaC. In this work, we aimed to characterize MSLN glycans from PaC cells and serum samples to assess their potential usefulness as PaC biomarkers. First, we analyzed MSLN glycans from PaC cell lines and then we developed an enzyme-linked lectin assay to measure core fucosylated-MSLN (Cf-MSLN) glycoforms. MSLN glycans from PaC cells were analyzed by glycan sequencing and through Western blotting with lectins. All of the cell lines secreted MSLN, with its three N-glycosylation sites occupied by complex-type N-glycans, which were mainly α2,3-sialylated, core fucosylated and highly branched. The Cf-MSLN glycoforms were quantified on PaC serum samples, and compared with MSLN protein levels. The Cf-MSLN was significantly decreased in PaC patients compared to control sera, while no differences were detected by using MSLN protein levels. In conclusion, Cf-MSLN glycoforms were differently expressed in PaC, which opens the way to further investigate their usefulness as PaC biomarkers.

Distinct Glycosylation Responses to Spinal Cord Injury in Regenerative and Nonregenerative Models.

Traumatic spinal cord injury (SCI) results in disruption of tissue integrity and loss of function. We hypothesize that glycosylation has a role in determining the occurrence of regeneration and that biomaterial treatment can influence this glycosylation response. We investigated the glycosylation response to spinal cord transection in Xenopus laevis and rat. Transected rats received an aligned collagen hydrogel. The response compared regenerative success, regenerative failure, and treatment in an established nonregenerative mammalian system. In a healthy rat spinal cord, ultraperformance liquid chromatography (UPLC) N-glycoprofiling identified complex, hybrid, and oligomannose N-glycans. Following rat SCI, complex and outer-arm fucosylated glycans decreased while oligomannose and hybrid structures increased. Sialic acid was associated with microglia/macrophages following SCI. Treatment with aligned collagen hydrogel had a minimal effect on the glycosylation response. In Xenopus, lectin histochemistry revealed increased levels of N-acetyl-glucosamine (GlcNAc) in premetamorphic animals. The addition of GlcNAc is required for processing complex-type glycans and is a necessary foundation for additional branching. A large increase in sialic acid was observed in nonregenerative animals. This work suggests that glycosylation may influence regenerative success. In particular, loss of complex glycans in rat spinal cord may contribute to regeneration failure. Targeting the glycosylation response may be a promising strategy for future therapies.

Biochemical and molecular characterization of sialylated cervical mucins in sheep†.

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Higher Inflammation Is Associated with Cardiometabolic Phenotype and Biochemical Health in Women with Obesity.

INTRODUCTION: Metabolic or inflammatory markers may predict adverse outcomes in women with obesity. We sought to describe metabolic-obesity phenotypes of women using novel staging tools and investigate relationships with inflammation. METHODS: In a cross-sectional study, we collected fasting blood samples from sixty-four females with body mass index (BMI) ≥28 kg/m2. Participants were classified as metabolically healthy or metabolically unhealthy obesity (MUO) using the cardiometabolic disease staging system (CMDS) and Edmonton obesity staging system (EOSS). Data were analyzed using independent sample t tests, Pearson's correlations, and multiple logistic regression. RESULTS: Mean (SD) age was 40.2 (9.3) years with median (IQR) BMI 31.8 (30.3-35.7) kg/m2. The prevalence of MUO was 46.9% and 81.3% using CMDS and EOSS criteria, respectively. Women with raised CMDS scores had higher C3 (1.34 [0.20] vs. 1.18 [0.15], p = 0.001) and C-reactive protein (CRP) (2.89 [1.31-7.61] vs. 1.39 [0.74-3.60], p = 0.034). C3 correlated with insulin (r = 0.52), hemoglobin A1c (r = 0.37), and C-peptide (r = 0.58), all p < 0.05. C3 above the median (>1.23 g/L) increased odds of raised CMDS score, when controlled for age, BMI, ethnicity, and smoking (OR = 6.56, 95% CI: 1.63, 26.47, p = 0.008). CONCLUSION: The prevalence of MUO was lower using CMDS than EOSS. C3 and CRP may be useful clinical biomarkers of risk or treatment targets in women with obesity.

Changes in Serum N-Glycome for Risk Drinkers: A Comparison with Standard Markers for Alcohol Abuse in Men and Women.

Background and aim: Glycomic alterations serve as biomarker tools for different diseases. The present study aims to evaluate the diagnostic capability of serum N-glycosylation to identify alcohol risk drinking in comparison with standard markers. Methods: We included 1516 adult individuals (age range 18-91 years; 55.3% women), randomly selected from a general population. A total of 143 (21.0%) men and 50 (5.9%) women were classified as risk drinkers after quantification of daily alcohol consumption and the Alcohol Use Disorders Identification Test (AUDIT). Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) was used for the quantification of 46 serum N-glycan peaks. Serum gamma-glutamyltransferase (GGT), carbohydrate-deficient transferrin (CDT), and red blood cell mean corpuscular volume (MCV) were measured by standard clinical laboratory methods. Results: Variations in serum N-glycome associated risk drinking were more prominent in men compared to women. A unique combination of N-glycan peaks selected by the selbal algorithm shows good discrimination between risk-drinkers and non-risk drinkers for men and women. Receiver operating characteristics (ROC) curves show accuracy for the diagnosis of risk drinking, which is comparable to that of the golden standards, GGT, MCV and CDT markers for men and women. Additionally, the inclusion of N-glycan peaks improves the diagnostic accuracy of the standard markers, although it remains relatively low, due to low sensitivity. For men, the area under the ROC curve using N-glycome data is 0.75, 0.76, and 0.77 when combined with GGT, MCV, and CDT, respectively. In women, the areas were 0.76, 0.73, and 0.73, respectively. Conclusion: Risk drinking is associated with significant variations in the serum N-glycome, which highlights its potential diagnostic utility.

Enhanced Immunomodulatory Effect of Intravenous Immunoglobulin by Fc Galactosylation and Nonfucosylation.

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC).　Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis highlighted the low-dose (G2)2 glycoform of IVIG (0.1 g/kg) as an effective immunomodulatory agent as the 10-fold higher dose of native IVIG. These preliminary results suggest that the anti-inflammatory activity of IVIG is in part mediated via activating FcγR blockade by galactosylated, nonfucosylated IgG and that such nonfucosylated IgG glycoforms bound to FcγRs on immune cells play immunomodulatory roles in health and disease. This study provides insights into improved therapeutic strategies for autoimmune/inflammatory diseases using glycoengineered IVIG and recombinant Fc.

Identification and characterization of O-linked glycans in cervical mucus as biomarkers of sperm transport: A novel sheep model.

Cervical mucus plays an important role in female fertility, since it allows the entry of motile and morphological normal sperm while preventing the ascent of pathogens from the vagina. The function of cervical mucus is critically linked to its rheological properties that are in turn dictated by O-glycosylated proteins, called mucins. We aimed to characterize the O-glycan composition in the cervical mucus of six European ewe breeds with known differences in pregnancy rates following cervical/vaginal artificial insemination with frozen-thawed semen, which are due to reported differences in cervical sperm transport. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway (n = 28-30 ewes/breed). We identified 124 O-glycans, from which 51 were the major glycans with core 2 and fucosylated glycans as the most common structures. The use of exogenous hormones for synchronization did not affect the O-glycan composition in both high-fertility ewe breeds, but it did in the other four ewe breeds. There was a higher abundance of the sulfated glycan (Galß1-3[SO3-GlcNAcß1-6]GalNAc), fucosylated glycan (GlcNAcß1-3(Fucα1-2Galß1-3)GalNAc) and core 4 glycan (GlcNAcß1-3[GlcNAcß1-6]GalNAc) in the low-fertility Suffolk breed compared with NWS (high fertility). In addition, core 4 glycans were negatively correlated with mucus viscosity. This novel study has identified O-glycans that are important for cervical sperm transport and could have applications across a range of species including human.

Micro-Heterogeneity of Antibody Molecules.

Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly efficacious biopharmaceuticals for a wide range of clinical indications. Full-length IgG mAbs are large proteins that are subject to multiple posttranslational modifications (PTMs) during biosynthesis, purification, or storage, resulting in micro-heterogeneity. The production of recombinant mAbs in nonhuman cell lines may result in loss of structural fidelity and the generation of variants having altered stability, biological activities, and/or immunogenic potential. Additionally, even fully human therapeutic mAbs are of unique specificity, by design, and, consequently, of unique structure; therefore, structural elements may be recognized as non-self by individuals within an outbred human population to provoke an anti-therapeutic/anti-drug antibody (ATA/ADA) response. Consequently, regulatory authorities require that the structure of a potential mAb drug product is comprehensively characterized employing state-of-the-art orthogonal analytical technologies; the PTM profile may define a set of critical quality attributes (CQAs) for the drug product that must be maintained, employing quality by design parameters, throughout the lifetime of the drug. Glycosylation of IgG-Fc, at Asn297 on each heavy chain, is an established CQA since its presence and fine structure can have a profound impact on efficacy and safety. The glycoform profile of serum-derived IgG is highly heterogeneous while mAbs produced in mammalian cells in vitro is less heterogeneous and can be "orchestrated" depending on the cell line employed and the culture conditions adopted. Thus, the gross structure and PTM profile of a given mAb, established for the drug substance gaining regulatory approval, have to be maintained for the lifespan of the drug. This review outlines our current understanding of common PTMs detected in mAbs and endogenous IgG and the relationship between a variant's structural attribute and its impact on clinical performance.

Importance and Monitoring of Therapeutic Immunoglobulin G Glycosylation.

The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a profound impact on the safety and efficacy of therapeutic IgG monoclonal antibodies (mAbs). Fc glycosylation of a mAb is an established critical quality attribute (CQA), and its oligosaccharide profile is required to be thoroughly characterized by state-of-the-art analytical methods. The Fc oligosaccharides are highly heterogeneous, and the differentially glycosylated species (glycoforms) of IgG express unique biological activities. Glycoengineering is a promising approach for the production of selected mAb glycoforms with improved effector functions, and non- and low-fucosylated mAbs exhibiting enhanced antibody-dependent cellular cytotoxicity activity have been approved or are under clinical evaluation for treatment of cancers, autoimmune/chronic inflammatory diseases, and infection. Recently, the chemoenzymatic glycoengineering method that allows for the transfer of structurally defined oligosaccharides to Asn-linked GlcNAc residues with glycosynthase has been developed for remodeling of IgG-Fc oligosaccharides with high efficiency and flexibility. Additionally, various glycoengineering methods have been developed that utilize the Fc oligosaccharides of IgG as reaction handles to conjugate cytotoxic agents by "click chemistry", providing new routes to the design of antibody-drug conjugates (ADCs) with tightly controlled drug-antibody ratios (DARs) and homogeneity. This review focuses on current understanding of the biological relevance of individual IgG glycoforms and advances in the development of next-generation antibody therapeutics with improved efficacy and safety through glycoengineering.

Novel diagnostic options for endometriosis - Based on the glycome and microbiome.

Background: Endometriosis is a chronic gynaecological disease whose aetiology is still unknown. Despite its prevalence among women of reproductive age, the pathology of the disease has not yet been elucidated and only symptomatic treatment is available. Endometriosis has high latency and diagnostic methods are both limited and invasive. Aim of review: The aim of this review is to summarise minimally invasive or non-invasive diagnostic methods for endometriosis and their diagnostic efficiencies. Furthermore, we discuss the identification and diagnostic potential of novel disease biomarkers of microbial or glycan origin. Key scientific concepts of review: Great efforts have been made to develop minimally invasive or non-invasive diagnostic methods in endometriosis. The problem with most potential biomarker candidates is that they have high accuracy only in cases of severe disease. Therefore, it is necessary to examine other potential biomarkers more closely. Associations between gastrointestinal and genital tract microbial health and endometriosis have been identified. For instance, irritable bowel syndrome is more common in women with endometriosis, and hormonal imbalance has a negative impact on the microbiome of both the genital tract and the gastrointestinal system. Further interrogation of these associations may have potential diagnostic significance and may identify novel therapeutic avenues. Glycomics may also be a potent source of biomarkers of endometriosis, with a number of glyco-biomarkers already approved by the FDA. Endometriosis-associated microbial and glycomic profiles may represent viable targets for development of innovative diagnostics in this debilitating disease.