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

Statistical analysis

Assessment of differences in outcomes of viability and preterm birth between cerclage suture material groups (braided versus monofilament) was performed using the Fisher exact test for categorical variables and Mann-Whitney for continuous variables. We used a linear mixed-effects model incorporating suture material group, maternal age, parity, previous preterm birth, and hospital location as fixed effects and ethnicity (Asian, Black, or Caucasian) as a random effect to compare braided versus monofilament suture material for the two primary outcomes (viability and preterm birth). The contributions of fixed-effects terms (p-value and F statistics) were calculated using the analysis of variance (ANOVA) with Satterthwaite approximation for degrees of freedom.

Examination of statistical differences between vaginal microbiota were performed at bacterial genera and species levels using the Statistical Analysis of Metagenomic Profiles (STAMP) software package (71). Ward linkage hierarchical clustering analysis (HCA) of bacterial genera was performed using a clustering density threshold of 0.75. Samples were classified according to the percentage of Lactobacillus spp. reads as a proportion of the total number of reads per sample into the following groups: normal (>90% Lactobacillus spp.), intermediate (30-90% Lactobacillus spp.), or dysbiotic (<30% Lactobacillus spp.). Bacterial species data were classified into community state types (CSTs) as described by Ravel et al (25): CST I (L. crispatus), CST II (L. gasseri), CST III (L. iners), CST IV (mixed bacterial species), and CST V (L. jensenii). To identify potential associations between suture material and differing degrees of dysbiosis, an alternative classification of the species data was performed, as described by Borgdorff et al (26) into communities characterized by healthy Lactobacillus spp. dominance, L. iners, or moderate or severe dysbiosis.

The effects of suture material and time from cerclage insertion on bacterial genera, number of species observed and alpha diversity were assessed using One-way ANOVA, Kruskal-Wallis, and Dunn’s multiple comparisons where appropriate.

Linear discriminant analysis (LDA) effect size (LEfSe) method (27) characterized differentially abundant taxonomic features of the two suture materials before and 4 weeks after cerclage insertion. An alpha value of 0.01 was used for factorial Kruskal-Wallis test between classes, and a threshold of 3.0 was used for logarithmic LDA score for discriminative features.

The Wilcoxon signed rank test compared cytokine analyte concentrations before and 4 weeks after cerclage insertion. The Mann-Whitney test was used to test for differences among suture material types. Analyte expression was classified according to the corresponding microenvironments (Fig. 1C), and the Mann-Whitney test compared cytokine expression in the presence of a normal or dysbiotic microbiome.

Cervical vascularization was compared according to suture material from the time of cerclage insertion and as a function of the corresponding bacterial classification, using Kruskal-Wallis and ANOVA multiple comparison analyses where appropriate. We used linear regression to assess for correlations between cervical vascularity, the number of observed species, and Shannon index of alpha diversity, according to suture material.

Supplementary information

Figure S1. Linear mixed effects modeling of retrospective outcome data.

Figure S2. Gestation at birth as a function of suture material used in the prospectively recruited cohort.

Figure S3. Ward hierarchical clustering analysis of species sequence data.

Figure S4. Longitudinal assessment of vaginal bacterial community structure following suture insertion.

Figure S5. In vitro adherence assay of suture material with E. coli or L. jensenii.

Figure S6. Longitudinal comparison of bacterial genera increased following braided suture insertion.

Figure S7. Quantitative PCR assessment of A. vaginae and G. vaginalis at 4 weeks after cerclage

Figure S8. V1-V3 hypervariable region sequence alignment against major vaginal Lactobacillus species

Table S1. Patient demographics for retrospective study cohort.

Table S2. Contributing confounder analysis for non-viable pregnancy and preterm birth <37 weeks.

Table S3. Bacterial genus classification according to time from cerclage

Table S4. Bacterial species classification into community state types according to time from cerclage.

Table S5. Species classification into normal, L. iners dominant, intermediate and severe dysbiosis according to time from cerclage.

Table S6. Mean bacterial counts of Atopobium vaginae and Gardnerella vaginalis before and 4 weeks after cerclage insertion, as assessed by quantitative PCR.

Table S7. Mean fold change in analyte expression detectable in cervico-vaginal fluid before and after cerclage insertion.

Table S8. DNA identity for the V1-V3 region used in the analysis for the 4 main lactobacilli in CST I, II, III, and V.

References and notes
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