Cervical Microbiome and Cytokine Profile at Various Stages of Cervical Cancer: A Pilot Study

<div><p>Cervical cancer (CC) is caused by high-risk human papillomavirus persistence due to the immunosuppressive tumor microenvironment mediated by cytokines. Vaginal microbiota determines the presence of certain cytokines locally. We assessed the association between cervical microbiota diversity and the histopathological diagnosis of each stage of CC, and we evaluated mRNA cervical expression levels of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ across the histopathological diagnosis and specific bacterial clusters. We determined the cervical microbiota by high throughput sequencing of 16S rDNA amplicons and classified it in community state types (CST). Mean difference analyses between alpha-diversity and histopathological diagnosis were carried out, as well as a β-diversity analysis within the histological diagnosis. Cervical cytokine mRNA expression was analyzed across the CSTs and the histopathological diagnoses. We found a significant difference in microbiota's diversity in NCL-HPV negative women vs those with squamous intraepithelial lesions (SIL) and CC(p = 0.006, p = 0.036).When β-diversity was evaluated, the CC samples showed the highest variation within groups (p<0.0006) and the largest distance compared to NCL-HPV negative ones (p<0.00001). The predominant bacteria in women with normal cytology were <i>L</i>. <i>crispatus</i> and <i>L</i>. <i>iners</i>, whereas for SIL, it was <i>Sneathia spp</i>. and for CC, <i>Fusobacterium spp</i>. We found higher median cervical levels of IL-4 and TGF-β1 mRNA in the CST dominated by <i>Fusobacterium spp</i>. These results suggest that the cervical microbiota may be implicated in cervical cancer pathology. Further cohort studies are needed to validate these findings.</p></div

Analysis of reproductive/sexual lifestyle-related risk factors in the study population (cervical lesion and cervical cancer patients).

<p>Analysis of reproductive/sexual lifestyle-related risk factors in the study population (cervical lesion and cervical cancer patients).</p

Cervical cytokine mRNA levels normalized to GAPDH, in NCL versus SIL and in NCL versus CC.

<p>(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).</p

Community composition of cervical samples at the species level as determined by massively parallel sequencing on the 454 platform.

<p>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 <i>Pseudomonas oleovorans</i>; cyan–CST II dominated by <i>L</i>. <i>iners</i>; orange–CST I dominated by <i>L</i>. <i>crispatus</i>; green–CST IV dominated by <i>Sneathia</i>; blue–CST V dominated by <i>G</i>. <i>vaginalis</i>; yellow–CST VIII dominated by <i>Fusobacterium spp</i>.; red–CST VI dominated by <i>S</i>. <i>agalactiae</i>, and purple–CST VII dominated by <i>Fusobacterium necrophorum</i>. 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.</p

Distribution of samples in each community state type (CST).

<p>Distribution of samples in each community state type (CST).</p

Community compositions according to histopathological diagnosis groups.

<p>Bar chart of relative abundance of species per group.</p

Estimated mean difference of bit units (Shannon diversity index) in cervix between NCL regardless of HPV status and SIL or CC.

<p>Estimated mean difference of bit units (Shannon diversity index) in cervix between NCL regardless of HPV status and SIL or CC.</p

Comparison of microbiota composition between different studies and the present study.

<p>Comparison of microbiota composition between different studies and the present study.</p

Mean difference analysis between the Shannon diversity index or the phylogenetic diversity whole tree and the histopathological diagnosis.

<p>Mean difference analysis between the Shannon diversity index or the phylogenetic diversity whole tree and the histopathological diagnosis.</p

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.

<p>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.</p