Abstract Microbial-driven spontaneous preterm birth (sPTB) is linked to adverse vaginal microbiome and dysregulated immune responses, yet this knowledge has not translated into predictive tools or therapies. We sampled 186 high-risk pregnant women and assessed 14 complement proteins and the neutrophil immunophenotype at the cervicovaginal interface to expand potential targets. Alterations in classical and alternative complement pathway components were associated with bacterial community composition and preceded cervical shortening and sPTB. At 12 +0 –16 +6 weeks, women with Community State Type (CST) IV had significantly higher concentrations of C1q, C4, C4b, Factor B, D, C3b/iC3b, and Factor H, I than those with CST I. Women who later developed a short cervix and delivered preterm showed lower L. crispatus abundance and elevated complement activation. Neutrophils were the dominant local immune cell and exhibited enhanced activation relative to peripheral neutrophils, with altered expression of CD11b, CD62L, CD63 and CD66b. Cervical neutrophil CD63, CD66b, and CD88 differed between CST IV and CST I, though not by pregnancy outcome. Neutrophil abundance correlated with cytokines, complement proteins, and MMPs, suggesting roles in inflammation and tissue remodelling. These findings highlight a microbiota-driven complement–neutrophil axis present before cervical remodelling and sPTB, identifying potential complement-based predictors and therapeutic targets.

Placental production of steroids, like progesterone, drives increasing circulating concentrations during pregnancy. It is postulated that reduced cervicovaginal progesterone contributes to cervical shortening and increased preterm birth (PTB) risk. Although vaginal progesterone is recommended for PTB prevention, its effectiveness remains uncertain. While serum and urinary progesterone are routinely measured in pregnancy, little is known about cervicovaginal fluid (CVF) progesterone concentrations. To address this, we adapted existing mass spectrometry-based assays to quantify 13 steroid hormones in CVF collected between 12-24 weeks gestation from uncomplicated term pregnancies (n = 14) and those with adverse outcomes (n = 11). Only progesterone, androstenedione, and cortisol were detected above detection limits. In uncomplicated term pregnancies, mean concentrations of these steroids were 6.61 ± 8.13, 4.16 ± 2.98 and 0.34 ± 0.21 ng/g of CVF, respectively. Cervicovaginal progesterone was weakly, positively associated with gestational age (r2= 0.15, p-value= 0.02) which was also observed in paired samples collected at 12 and 22 weeks (n = 5, paired Wilcoxon, p-value = 0.03). No differences in CVF steroid levels were observed between women who subsequently delivered preterm (n = 9) or at term (n = 15). Our study provides a robust method for quantifying CVF steroids and suggests that second-trimester PTB risk is not associated with cervicovaginal progesterone concentrations.

The mechanisms by which vaginal microbiota shape spontaneous preterm birth (sPTB) risk remain poorly defined. Using electronic clinical records data from 74,913 maternities in conjunction with metaxanomic (n = 596) and immune profiling (n = 314) data, we show that the B blood group phenotype associates with increased risk of sPTB and adverse vaginal microbiota composition. The O blood group associates with sPTB in women who have a combination of a previous history of sPTB, an adverse vaginal microbial composition and pro-inflammatory cervicovaginal milieu. In contrast, women of blood group A have a higher prevalence of vaginal Lactobacillus crispatus, a lower risk of sPTB, with sPTB cases showing no association with vaginal microbiota composition or inflammation. We found that cervicovaginal fluid contains ABH(O) glycans and shows variable binding to key vaginal bacteria. This indicates that cervicovaginal ABH(O) glycans influence microbiota-host interactions implicated in sPTB risk, suggesting a novel target for sPTB prediction and prevention.

Does the gut microbiota play a role in changing the local immune response to an adverse vaginal microbiota? We show that in those with an adverse vaginal microbiota, a healthy gut can protect you and dampen inflammation at the local cervicovaginal level. There is growing evidence to implicate the role of the vaginal microbiota in reproductive health. We have recently shown that an adverse vaginal microbiota accompanied by a heightened inflammatory response is linked to euploid miscarriage. However, it is unclear what modulates this immune response as some women with suboptimal vaginal microbial composition go on to deliver term pregnancies. This was a prospective observational cohort study based at Queen Charlotte’s and Chelsea Hospital, Early Pregnancy Unit, London between July 2017 and February 2019. We analysed the vaginal and rectal microbiota in 47 miscarriage patients (29 euploid and 18 aneuploid, determined following cytogenetic analysis) and 49 viable pregnancies. Here we investigate the role of the rectal microbiota in relation to miscarriage and the local cervical vaginal immune response. We used 16S rRNA gene based metataxonomics to interrogate the vaginal and rectal microbiota in 29 euploid and 18 aneuploid miscarriages and 49 viable pregnancies. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid. There was no difference in the richness or diversity of the rectal microbiota in the different pregnancy outcomes. In patients with an adverse vaginal microbiota, there was a significantly higher Firmicutes/Bacteroidetes ratio (a marker of gut dysbiosis) in euploid miscarriage compared to aneuploid miscarriage (P=0.02). Finegoldia magna, Prevotella bivia, Dialister invisus and Peptoniphilus gorbachii were enriched in euploid miscarriage whereas relative abundance of Clostridium XIVa and Bacteroidetes was higher in aneuploid miscarriage. In the whole cohort, anti-inflammatory cervical vaginal cytokines were significantly higher in patients with a healthy gut microbiota (low F/B ratio). In those with a healthy vaginal microbiota, rectal dysbiosis was not associated with cervicovaginal cytokines. However, in patients with an adverse vaginal microbiota a low F/B ratio was associated with significantly increased concentrations of anti-inflammatory cervicovaginal cytokines (IL4 and IL10) compared to patients with a high F/B ratio (P=0.01 and 0.04 respectively). This study provides initial insights into the potential role the gut microbiota has to play in miscarriage. The next steps would involve longitudinal preconception profiling of the patient cohort alongside stool cultures and detailed diet and lifestyle questionnaires to further advance our knowledge on the topic. We postulate there may be crosstalk between the vaginal microbiota and gut dysbiosis in euploid miscarriages with an effect of gut dysbiosis on inflammatory responses. This provides a rationale for healthy eating to maintain gut health, and using live biotherapeutics orally and vaginally to improve reproductive health and pregnancy outcomes. No

Several studies have associated seminal microbiota abnormalities with male infertility but have yielded differing results owing to their limited sizes or depths of analyses. The semen microbiota during recurrent pregnancy loss (RPL) has not been investigated. Comprehensively assessing the seminal microbiota in men with reproductive disorders could elucidate its potential role in clinical management. We used semen analysis, terminal-deoxynucleotidyl-transferase-mediated-deoxyuridine-triphosphate-nick-end-labelling, Comet DNA fragmentation, luminol reactive oxidative species (ROS) chemiluminescence, and metataxonomic profiling of semen microbiota by 16S rRNA amplicon sequencing in this prospective, cross-sectional study to investigate composition and bacterial load of seminal bacterial genera and species, semen parameters, ROS, and sperm DNA fragmentation in men with reproductive disorders and proven fathers. 223 men were enrolled, including healthy men with proven paternity (n=63), the male partners in a couple encountering RPL (n=46), men with male factor infertility (n=58), and the male partners of couples with unexplained infertility (n=56). Rates of high sperm DNA fragmentation, elevated ROS, and oligospermia were more prevalent in the study group compared with control. In all groups, semen microbiota clustered into three major genera-dominant groups (1, Streptococcus; 2, Prevotella; 3, Lactobacillus and Gardnerella); no species clusters were identified. Group 2 had the highest microbial richness (p<0.001), alpha-diversity (p<0.001), and bacterial load (p<0.0001). Overall bacterial composition or load has not been found to associate with semen analysis, ROS, or DNA fragmentation. Whilst global perturbation of the seminal microbiota is not associated with male reproductive disorders, men with unidentified seminal Flavobacterium are more likely to have abnormal seminal analysis. Future studies may elucidate if Flavobacterium reduction has therapeutic potential.

Lactobacillus displacement from the vaginal microbiome associates with adverse health outcomes and is linked to increased risk of preterm birth. Glycans mediate bacterial adhesion events involved in colonisation and infection. Using customised glycan microarrays, we establish glycan interaction profiles of vaginal bacteria implicated in reproductive health. Glycan binding signatures of the opportunistic pathogens Escherichia coli, Fusobacterium nucleatum and Streptococcus agalactiae to oligomannose N-glycans, galactose-terminating glycans and hyaluronic acid, respectively are highly distinct from Lactobacillus commensals. Binding to sulphated glycosaminoglycans by vaginal bacteria is pH dependent, as is binding to neutral and sialic acid-terminating glycans by F. nucleatum. Adhesion of Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis, S. agalactiae and F. nucleatum to vaginal epithelial cells is partially mediated by chondroitin sulphate. S. agalactiae binding to chondroitin sulphate C oligosaccharides is inhibited by L. crispatus. This study highlights glycans as mediators of vaginal bacterial binding events involved in reproductive health and disease.

Background:Pregnancy of unknown location (PUL) is classified if an early pregnancy is not visualised on transvaginal ultrasonography (TVUS). Biomarkers currently used to triage PUL outcomes have varying accuracy. Delayed or missed diagnosis of ectopic pregnancies (EP) continue to cause significant morbidity and mortality. We investigated whether maternal plasma microRNAs (miRNAs) can predict and differentiate high-risk EP from viable (VIUP) or non-viable (NVIUP) intrauterine pregnancies. Methods:Plasma was collected from women with PUL/EP (n = 120), mostly between four to eight weeks' gestation, where outcomes of EP (n = 39), VIUP (n = 58) and NVIUP (miscarriage, n = 23) were determined using TVUS. Nanostring nCounter miRNA assay was used to examine the expression of ∼800 miRNAs in 22 women. Differentially expressed miRNAs were validated using RT-qPCR in 98 women. Results:Nanostring nCounter miRNA assay identified 19 miRNAs which were expressed significantly higher in EP/NVIUP compared with VIUP. Two miRNAs were validated in a second, separate validation cohort using RT-qPCR: hsa-miR-21-5p in EP was 2.8-fold higher than in VIUP (p = 0.03, ROC AUC = 0.64), and hsa-miR-411-5p had 0.2-fold decreased expression (p = 0.02, ROC AUC = 0.66). Combining the divergent miRNAs as a ratio improved discrimination of EP from VIUP (p < 0.001, ROC AUC = 0.74). Conclusion:Plasma miRNAs are differentially expressed in EP and VIUP and are detectable as early as four gestational weeks. Exploring miRNA targets may further understanding of EP pathophysiology, offering the potential to use miRNA as predictive and diagnostic markers in early pregnancy.

To understand the relationship between endometrial CD138 expression and the reproductive tract microbiota. CD138+ staining of endometrium is associated with general delay in endometrial maturation but not the composition of the microbiota. Chronic endometritis (CE) is a purported cause of recurrent miscarriage (RM). This asymptomatic inflammation of the endometrium has been associated with RM, with several studies demonstrating a higher prevalence than the general population. CE has previous been associated with a reduction in Lactobacilli species and a dysbiotic endometrial microbiota. CD138 is a widely used plasma cell marker utilised to aid the diagnosis of CE. However, CD138, a transmembrane heparan sulfate proteoglycan, is expressed by other cells in human endometrium. Its expression has previously been shown to vary across the menstrual cycle. The implications of this remain under-explored. Translational cohort study embedded within a randomised controlled trial with a subset of 103 samples derived from a trial population of 737 women. Women aged ≥18 to < 42 yrs, with a history of two or more first trimester consecutive miscarriages were recruited from specialist Recurrent Pregnancy Loss (RPL) clinics. A timed endometrial biopsy alongside endometrial microbial sampling was performed following ovulation (10±4 days). Additional samples were obtained from the Tommy’s National Reproductive Health Biobank. Samples underwent CD138 immunostaining, histological and molecular dating analysis, immune profiling, cytokine analysis and bacterial metataxonomic profiling with 16S ribosomal RNA (rRNA) sequencing analysis. Non-immune expression of CD138 was demonstrated across all endometrial cell populations. Stromal expression was very high (>200 CD138-positive stromal cells/10mm2) in 26 out of 27 proliferative endometrial samples. While CD138 immunoreactivity in the stroma declines markedly following ovulation (Mann Whitney U-Test; p < 0.005), gene expression analysis demonstrated persistently elevated SDC1 (CD138) transcript levels in a subset of non-immune stromal cells. Immunohistochemistry revealed three patterns of CD138 immunoreactivity: punctate staining, diffuse staining, and a mixed pattern. When compared to CD138 negative samples, conspicuous diffuse staining in the stromal compartment was associated with significantly earlier histological dating (p < 0.01) and lower molecular dating ratios (p < 0.01). Sequencing of paired vaginal and ectocervical swabs and endometrial Tao brush samples collected from 114 patients demonstrated tightly interconnected microbial ecosystems across the low reproductive tract. There was no association between the pattern or severity of CD138 immunoreactivity in luteal phase endometrium and vaginal, ectocervical or endometrial community state types (p < 0.05). Analysis of supernatants of vaginal and ectocervical swabs and Tao Brush revealed an inverse correlation between the pattern and severity of stromal CD138 immunoreactivity in endometrial stroma and secreted levels of TNF-α, CXCL/BRAK and VEGF (q-value < 0.05). This study relied on self-reported ovulation testing using commercially available urine LH tests. In addition, multiple statistical testing was utilised across differing data types. The consistency of the results seen, however, adds credence to the findings. This study challenges the assumptions underlying the reliability of CD138 IHC to detect plasma cells infiltration. It suggests the possibility of a confounding relationship of delayed endometrial maturation within the literature on CE to date. CD138-based CE testing and treatment should not therefore be performed outside of a research context. Yes

Introduction: Early prediction of pregnancies destined to miscarry will allow couples to prepare for this common but often unexpected eventuality, and clinicians to allocate finite resources. We aimed to develop a prediction model combining clinical, demographic, and sonographic data as a clinical tool to aid counselling about first trimester pregnancy outcome. Material and methods: This is a prospective, observational cohort study conducted at Queen Charlotte's and Chelsea Hospital, UK from March 2014 to May 2019. Women with confirmed intrauterine pregnancies between 5 weeks and their dating scan (11-14 weeks) were recruited. Participants attended serial ultrasound scans in the first trimester and at each visit recorded symptoms of vaginal bleeding, pelvic pain, nausea and vomiting using validated scoring tools. Pregnancies were followed up until the dating scan (11-14 weeks). Univariate and multivariate analyses were performed to predict first trimester viability. A model was developed with multivariable logistic regression, variables limited by feature selection, and bootstrapping with multiple imputation was used for internal validation. Results: 1403 women were recruited and after exclusions, data were available for 1105. 160 women (14.5 %) experienced first trimester miscarriage and 945 women (85.5 %) had viable pregnancies at 11-14 weeks' gestation. The average gestational age at the initial visit (calculated from the menstrual dates) was 7 + 1 weeks (+/-12.2 days). A multivariable logistic regression model was developed to predict first trimester viability and included the variables: mean gestational sac diameter, presence of fetal heart pulsations, difference in gestational age from last menstrual period and from mean sac diameter on ultrasonography, current folic acid usage and maternal age. The model demonstrated good performance (optimism-corrected area under curve (AUC) 0.84, 95 % CI 0.81-0.87; optimism-corrected calibration slope 0.969). Conclusion: We have developed and internally validated a model to predict first trimester viability with good accuracy prior to the 11-14 week dating scan, which now needs to be externally validated prior to clinical use.

INTRODUCTION:Preterm delivery (PTD) is the leading cause of death in children under 5 years of age. Cervical shortening detected by ultrasound can be used to predict PTD, but prediction is not perfect, and complementary diagnostic markers are needed. Recently, specific plasma microribonucleic acid (miRNAs) detected in early second trimester were shown to be associated with spontaneous PTD in high-risk women with a singleton pregnancy. The aim of this study was to explore to what extent these miRNAs are associated with spontaneous PTD and cervical length in a general population. MATERIAL AND METHODS:This study is a nested case-control study within the CERVIX study. The CERVIX study evaluated the ability of cervical length screening with transvaginal ultrasound to identify women at risk of PTD. In the present study, women who delivered spontaneously <34 weeks (n = 61) were compared with a control group of women who delivered at full term (39 + 0 to 40 + 6 gestational weeks, n = 205). Archived serum samples were analyzed with RT-qPCR for miRNA expression levels of let-7a-5p, miR-150-5p, miR-15b-5p, miR-185-5p, miR-191-5p, miR-19b-3p, miR-23a-3p, miR-374a-5p, and miR-93-5p. The mean relative expression was compared between the groups. Sub-analyses were performed for women delivering <32, <30, and <28 weeks vs the full-term group. RESULTS:The analyzed miRNAs were not significantly differentially expressed in women delivering <34 weeks compared to those delivering at full term. MiR-191-5p and miR-93-5p were significantly overexpressed in women who delivered <32 weeks, and further increase in fold change was observed with decreasing gestational age at delivery. The level of miR-15b-5p was significantly higher in women delivering at <30 weeks compared to those delivering at full term. CONCLUSIONS:Our study shows that overexpression of miR-93-5p, miR-15b-5p, and miR-191-5p in serum at early gestation is associated with spontaneous PTD in a general population. Further research is needed to evaluate the potential of these miRNAs as future biomarkers for spontaneous PTD, as well as their pathophysiological role in spontaneous PTD.