Title: Relationship between vaginal microbial dysbiosis, inflammation, and pregnancy outcomes in cervical cerclage. One Sentence Summary

Acknowledgments: We thank all participants of the study and members of Women's Health Research Centre, Imperial College Health NHS Trust.

Funding: Supported by the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre at Imperial College London (Grant Ref P45272) and by the Genesis Research Trust (Grant Ref P51389). DAM is supported by a Career Development Award from the Medical Research Council (MR/L009226/1).

Author contributions L.M.K., D.A.M., C.L., P.T-H., M.S., T.G.T. and P.R.B. conceived and designed the retrospective study. L.M.K., D.A.M., T.G.T and P.R.B. conceived and designed the prospective study. Retrospective data collection and collation was performed by L.M.K., V.T., J.R.C., C.L., F.I-B., Y.F., P.T-H., M.S., T.G.T., and P.R.B. Prospective patient enrollment and sample collection and transvaginal scans were undertaken by L.M.K. and J.R.C. Experiments were performed by L.M.K., Y.S.L. and J.A.K.M. Data analysis was performed by L.M.K., D.A.M., J.R.M., A.S., S.C., E.H., J.K.N and P.R.B. All figures and tables were generated by L.M.K., D.A.M., S.C., and J.R.M. The manuscript was written by L.M.K., D.A.M. and P.R.B, and critically reviewed by all authors.

Competing interests: The authors declare that they have no competing interests

Data and materials availability: Public access to sequence data and accompanying metadata can be obtained at the European Nucleotide Archive’s (ENA) Sequence Read Archive (SRA) (PRJEB11895).
Figures:


Figure 1. Braided suture material for cervical cerclage is associated with worse outcomes.

(A) Retrospective comparison of 10 years of birth outcomes for women receiving a cerclage based on suture material (monofilament, n=344 vs braided, n= 337) revealed higher rates of non-viable births (delivery <24 weeks or intrauterine death) in women receiving a braided cerclage compared to a monofilament alternative (15% vs 5% respectively; P = 0.0001, Fisher’s exact test) and increased rates of preterm birth (24-37 weeks gestation) in women receiving braided cerclage (28% braided vs 17% monofilament; P = 0.0006, Fisher’s exact test) (see Table S3 for details). (B) Ward-linkage analysis of vaginal bacterial genera of cervical vaginal fluid samples (n=197) collected longitudinally before and after insertion of a monofilament (n=24) or braided (n=25) cervical cerclage permitted classification of vaginal bacterial communities into three groups: normal (>90% Lactobacillus abundance), intermediate (30-90% Lactobacillus abundance), or dysbiotic (<30% Lactobacillus abundance). (C) Braided cerclage was associated with a 5-fold increase in microbial dysbiosis within 4 weeks of insertion, which persistent until at least 16 weeks, whereas no change was observed in women receiving a monofilament cerclage (P values = fishers exact before v after cerclage, and 2 way ANOVA: monofilament vs braided at comparable time points).

Figure 2. Bacterial taxonomic groups discriminate between monofilament and braided cerclage. (A) Differentially abundant microbial clades and nodes according to suture material four weeks after insertion were identified using LEfSe analysis and presented as a cladogram. (B) Linear Discriminant Analysis (LDA) was used to estimate the effect size for each differentially abundant species. The vaginal microbiome of patients receiving a monofilament cerclage was enriched with bacilli, whereas those receiving a braided cerclage were comparatively enriched in Bacteroides spp., and Clostridia. (C) Relative abundance bar charts for individual samples highlight maintenance of Lactobacillus genus stability after insertion of monofilament cerclage (P = 0.9; ANOVA), in contrast to the decreased numbers after braided cerclage (P = 0.043; ANOVA) (P values = corrected two-sided Welch’s t-test for monofilament vs. braided). (D) Comparison of total number of bacterial species observed reveals an increase after braided cerclage compared to a monofilament cerclage. (E) Alpha diversity was increased at 16 weeks after braided cerclage insertion compared to monofilament. (*P values = corrected two-sided Welch’s t-test for monofilament vs. braided, ^#P values = ANOVA Bonferroni multiple comparison to before-cerclage samples).



Figure 3. Braided suture induces cytokine release into the cervico-vaginal fluid. (A) Cytokines were detected using a multiplex assay before and 4 weeks after cerclage insertion. An increase in pro-inflammatory cytokines was detected in the cervico-vaginal fluid after braided cerclage (see Table S7 for details). Relative to the concentrations before cerclage, braided suture was associated with an increase in pro-inflammatory cytokines (B) IL-1β, (C) IL-6, (D) IL-8, (E) TNF-α, and (F) MMP-1 but not (G) IL-4 (*P values = Wilcoxon signed rank test for cytokine concentration before and after cerclage). Similar changes were observed when the braided cerclage samples were compared for cytokine concentrations 4 weeks after monofilament cerclage (^#P values = Mann-Whitney for fold change monofilament vs. braided). (H) Mean cytokine profiles grouped by the corresponding microbial classification (normal, intermediate, and dysbiotic) revealed that dysbiosis is associated with increased expression of pro-inflammatory cytokines ICAM-1, IL-1β, IL-6, MMP-1, MCP-1, TNF-α, GM-CSF, and IFN-γ and anti-inflammatory IL-10, but not G-CSF, IL-8, VEGF, RANTES, IL-2 or IL-4. (P value = Mann-Whitney for normal vs dysbiotic).


Figure 4. Braided cerclage induces premature cervical vascularization. (A) Cervical vascularization index (VI), as assessed by transvaginal ultrasound, was greater in patients receiving braided cerclage compared to monofilament cerclage at 4, 8, 12, and 16 weeks after insertion (*P value = Welches corrected t-test for monofilament vs braided, ^#P value = ANOVA, Bonferroni multiple comparison for before vs after cerclage). (B) Linear regression analyses demonstrated a positive correlation between VI and the number of species observed in braided (R²=0.09, P = 0.002) but not monofilament (R²=0.001, P = 0.75) cerclages. (C) A similar relationship was observed between VI and alpha diversity index (braided: R² = 0.14, P = 0.001 and monofilament: R²=0.02, P = 0.14), indicating an interplay between braided suture, increased cervical vascularity, and vaginal microbial dysbiosis.

Tables

Table 1. Patient characteristics for women randomized to receive monofilament and braided cervical cerclages

	Monofilament suture	Braided suture	Total population
n (%)	24/49 (49%)	25/49 (51%)	49/49
Age, Mean ±SD (range) years	32.8 ± 3.0 (27-39)	33.9 ± 3.8 (25-42)	33.5 ± 3.5 (25-42)
BMI, Mean ±SD (range)	24.1 ± 4.2 (18-35)	26 ± 3.6 (21-36)	25.1 ± 4.5 (18-36)
Ethnicity, n (%)
Caucasian	16 (67%)	11 (44%)	27 (55%)
Asian	2 (8%)	7 (28%)	9 (18%)
Black	6 (25%)	7 (28%)	13 (27%)
Parity, n (%)
Para 0	12 (50%)	13 (52%)	25 (51%)
Smoking, n (%)	1 (4%)	2 (8%)	3 (6%)
Cerclage insertion
GA at insertion, mean ±SD w	17^+6 ± 2.8	18^+1 ± 3	17^+0 ± 2.9
CL at insertion, mean ±SD mm	18 ± 5.1	19 ± 4.5	19 ± 5.3
GA at delivery, n (%)
<34^+0w	4/24 (16%)	0/25 (0%)	4 (8%)
34^+1-36^+6 w	2/24 (8%)	8/25 (32%)	10 (20%)
≥37^+0 w	18/24 (75%)	17/25 (68%)	35 (71%)

BMI= body mass index; CL = cervical length (mm); GA = gestational age; w= weeks; SD=standard deviation