Cervical microbiome is altered in cervical intraepithelial neoplasia after loop electrosurgical excision procedure in china

Abstract

Although human papillomavirus (HPV) infection is a major cause leading to the development of cervical intraepithelial neoplasia (CIN), the relationship between genital microbiome and HPV persistence/clearance is not well established. Loop electrosurgical excision procedure (LEEP) is one of standard treatments of CIN 2/3 globally, yet little is known about how the LEEP influence genital microbiota. We conducted a prospective study of 26 patients with CIN2/3 who underwent analysis of cervical microbiome before and after 3 months of LEEP treatment. Cervical swabs were collected, and microbiomes were analyzed by 16S ribosomal RNA gene sequencing. A decrease of cervical microbial diversity was observed after 3 months of LEEP treatment. Notably, a significant shift from community type of a Prevotella-containing and lack of a consistent dominant species to lactobacillus iners dominated microbiome correlated with LEEP. Particularly, Leptotrichia and clostridium were further decreased after LEEP treatment (P = 0.049 and P = 0.002, respectively). Our results suggest that the cervical microbiome is altered after LEEP treatment in patients with CIN2/3. Further studies with larger sample sizes are needed to validate these findings.

Figure 1

16S rRNA sequence analysis of cervical swabs reveals four distinct bacterial community structures. (A) Heatmap of bacterial taxa identified by 16S rRAN V3-4 sequencing of cervical swabs collected from 26 patients with or without LEEP procedure. Cervicotypes (CTs) were determined based on the dominant species: non-iners Lactobacillus (i.e., higher percentage of Lactobacillus crispatus) (CT1), Lactobacillus iners (CT2), Gardnerella (CT3), and mixed microbial community containing Prevotella (CT4). (B) Principal component analysis (PCA) plot constructed from 52 samples. The first two principal components (PC1 and PC2) can explain 71.08% of the data variance. Different colors denote 4 distinct Cts. P = 0.001 for CTs.

Figure 2

Cervical microbiome richness and diversity indices associated with LEEP status. The number of species observed decreased in patients with LEEP (A). Diversity, as assessed by Inverse Simpson (B) and non-parametric Shannon (C) indices followed the same pattern. Kruskall-Wallis test (Dunn’s post hoc).

Figure 3

Principal coordinates analysis (PCoA) and genus level relative abundance for 26 samples from the patients before LEEP. (A) Bray-Curtis dissimilarity PCoA was used to generate ordination of beta-diversity in two dimensions. Principal coordinates 1 and 2 (PC1 and PC2) explain 40.33% and 14.78% of the variance in Bray-Curtis dissimilarity respectively (x and y axes). Samples are colored according to the menopause status. (B) Relative abundance was shown for the top fifteen genus.

Figure 4

Bacterial taxonomic groups discriminate between before and following LEEP procedure. (A) Differentially abundant microbial clades and nodes according to LEEP surgery were identified using LEfSe analysis and presented as a cladogram. (B) Histogram of the LDA scores was used to features differentially abundant between LEEP and no LEEP state. The cervical microbiome of patients following LEEP surgery was enriched with Bacilli and Firmicutes, whereas those before LEEP were comparatively enriched Actinobacteria and Bifidobacteriales. Relative abundance bar charts for individual samples highlight decreased numbers of Leptotrichia amnion (C) and Clostridium sensu strict (D) after LEEP (Welchi’s t-test).