Table 1.
Analysis of reproductive/sexual lifestyle-related risk factors in the study population (cervical lesion and cervical cancer patients).
Fig 1.
Community composition of cervical samples at the species level as determined by massively parallel sequencing on the 454 platform.
Unsupervised heatmap of the relative abundance of microbial taxa found in the cervical microbial communities of 29 subjects, based on the Bray Curtis dissimilarity metric. The species present in relative abundance of 1% in at least one sample are listed on the X axis. The first bar on the left side represents the treatment as follows: red–HPV-negative without lesion; blue–HPV-positive without lesion; orange–squamous intraepithelial cervical lesion; green–cervical cancer. CST are depicted in the second left barside; pink–CST III, dominated by Pseudomonas oleovorans; cyan–CST II dominated by L. iners; orange–CST I dominated by L. crispatus; green–CST IV dominated by Sneathia; blue–CST V dominated by G. vaginalis; yellow–CST VIII dominated by Fusobacterium spp.; red–CST VI dominated by S. agalactiae, and purple–CST VII dominated by Fusobacterium necrophorum. Sample names appear on the right side of the graph. The cladograms at the top of the species names indicate the approximate evolutionary relationships between the species. CST: Community State Type. Dx: Histopathological diagnosis.
Fig 2.
Community compositions according to histopathological diagnosis groups.
Bar chart of relative abundance of species per group.
Table 2.
Distribution of samples in each community state type (CST).
Fig 3.
Comparison of the Shannon diversity index and the PD whole tree logistic according to the histopathological diagnosis group.
The boxplots show the distribution of the H’ (bit units) and PD values across all the samples.
Table 3.
Mean difference analysis between the Shannon diversity index or the phylogenetic diversity whole tree and the histopathological diagnosis.
Table 4.
Association analysis between histopathological diagnosis and alpha diversity indexes.
Table 5.
Estimated mean difference of bit units (Shannon diversity index) in cervix between NCL regardless of HPV status and SIL or CC.
Fig 4.
Beta-diversity of microbial communities by histopathological diagnosis.
A. PCoA profile of the histopathological diagnosis displayed with weighted UniFrac distances. Each figure represents one sample colored according to its histopathological diagnosis. Red circles represent HPV-negative NCL samples; blue squares represent HPV-positive NCL samples; orange triangles represent SIL and green triangles represent CC. A1. Principal component (PC)-1 accounted for 33.44% of the variation in the composition of the microbiota due to the presence of Sneathia spp. and Fusobacterium spp. A2. PC-2 accounted for 26.85% of the variation inthe composition of the microbiota due to the presence of Bifidobacteria spp. and Pseudomonas spp. A3. PC-3 accounted for 10.38% of the variation in the composition of the microbiota due to the presence of Lactobacillus spp. and Streptococcus spp. B. B1. Variation of weighted UniFrac distances within each histological diagnosis group. B2. Variation of weighted UniFrac distances compared with -HPV-negativeNCL.
Fig 5.
Cervical cytokine mRNA levels normalized to GAPDH, in NCL versus SIL and in NCL versus CC.
(A) IL-4, (B) IL-6, (C) IL-10, (D) TNFα, (E) IFN-γ, (F) TGF-β1. * p value for the Mann-Whitney test (p = 0.04).
Fig 6.
Cervical cytokine mRNA levels normalized to GAPDH per community state type (CST).
(A) IL-4, (B) IL-6, (C) IL-10, (D) TNFα, (E) IFN-γ, (F) TGF-β1. *p value for Kruskal Wallis test (p≤0.05).
Fig 7.
INF-γ, IL-4, TGF-β1 and IL-10 expression level per sample in community state type CST I, CST IV and CSTVIII and microbiome composition.
Cervical expression level of INF-γ (A), TGF-β1 (B), IL-4 (C) and IL-10 (D) normalized with GAPDH gen. (E) Microbiome composition in relative abundance.
Table 6.
Comparison of microbiota composition between different studies and the present study.
Fig 8.
Suggested mechanism of microbiome changes during immunosuppression development.
The cervical epithelium is represented in each stage of CC as departing from a normal epithelium (left) and its longitudinal change when a HR-HPV infects it and it progresses to SIL and CC. Microbiome composition and diversity is depicted according to the main bacteria per stage. Lactobacillus are represented as purple rectangles, Pseudomonas oleovorans as red rectangles, Fusobacterium and Sneathia as shaped rods, Streptococcus agalactieas purple circles, and HPV as blue circles. After infection takes place, the microbiome changes and its diversity increases. HPV proteins E2, E6 and E7 enhance IL-10 expression and macrophages type 2 presence. The latter is also enhanced by TGFβ-1, which is in turn stimulated by the microbiota present. The diversity of the microbiota increases, through its toxins (FadA from Fusobacterium spp.), which disturb tight junctions and promote a metastasis similar to colon carcinoma.