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

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Abstract

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 L. crispatus and L. iners, whereas for SIL, it was Sneathia spp. and for CC, Fusobacterium spp. We found higher median cervical levels of IL-4 and TGF-β1 mRNA in the CST dominated by Fusobacterium spp. These results suggest that the cervical microbiota may be implicated in cervical cancer pathology. Further cohort studies are needed to validate these findings.

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EMPeror: a tool for visualizing high-throughput microbial community data

Yoshiki Vázquez-Baeza, Meg Pirrung, Antonio González González … (2013)

Background As microbial ecologists take advantage of high-throughput sequencing technologies to describe microbial communities across ever-increasing numbers of samples, new analysis tools are required to relate the distribution of microbes among larger numbers of communities, and to use increasingly rich and standards-compliant metadata to understand the biological factors driving these relationships. In particular, the Earth Microbiome Project drives these needs by profiling the genomic content of tens of thousands of samples across multiple environment types. Findings Features of EMPeror include: ability to visualize gradients and categorical data, visualize different principal coordinates axes, present the data in the form of parallel coordinates, show taxa as well as environmental samples, dynamically adjust the size and transparency of the spheres representing the communities on a per-category basis, dynamically scale the axes according to the fraction of variance each explains, show, hide or recolor points according to arbitrary metadata including that compliant with the MIxS family of standards developed by the Genomic Standards Consortium, display jackknifed-resampled data to assess statistical confidence in clustering, perform coordinate comparisons (useful for procrustes analysis plots), and greatly reduce loading times and overall memory footprint compared with existing approaches. Additionally, ease of sharing, given EMPeror’s small output file size, enables agile collaboration by allowing users to embed these visualizations via emails or web pages without the need for extra plugins. Conclusions Here we present EMPeror, an open source and web browser enabled tool with a versatile command line interface that allows researchers to perform rapid exploratory investigations of 3D visualizations of microbial community data, such as the widely used principal coordinates plots. EMPeror includes a rich set of controllers to modify features as a function of the metadata. By being specifically tailored to the requirements of microbial ecologists, EMPeror thus increases the speed with which insight can be gained from large microbiome datasets.

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The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

Janneke H. H. M. van de Wijgert, Hanneke Borgdorff, Rita Verhelst … (2014)

We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.

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The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR.

M Bartolo, Marc Snijders, Catherina Meijer … (1995)

Sequence analysis of human papillomavirus (HPV) general primer GP5/6 mediated PCR products revealed the presence of short highly conserved sequences adjacent to the 3' ends of both primers. Part of these sequences was used to elongate GP5 and GP6 at their 3' ends to generate the primers GP5+ and GP6+, respectively. Compared with the GP5/6 PCR, GP5+/6+ specific PCR on 22 cloned mucosotropic HPVs revealed an improved HPV detection, reflected by a 10- to 100-fold higher sensitivity and a markedly increased signal to background ratio, especially at the gel level. As determined on purified DNA, the sensitivity of this GP5+/6+ based assay was at the femtogram level for those HPV genotypes which match strongly with the primers (e.g. HPV-16) and at the picogram level for HPV types (e.g. HPV-39 and -51) having four or more mismatches with one or both primers. Application of both methods on 264 cervical scrapes of a cohort of women participating in a prospective follow-up study revealed an increase of total HPV positivity from 39% (GP5/6 PCR) to 43% (GP5+/6+ PCR) of the scrapes. Additional HPV typing by PCR specific for the HPV-6, -11, -16, -18, -31 and -33 revealed that all GP5+/6+ PCR positive cases which were negative by GP5/6 PCR (n = 12) contained HPV types different from these six types. These data indicate that the GP5+/6+ PCR method provides an increased detection level mainly of uncommon, apparently poorly matched HPV types in cervical scrapes and most likely in the enlargement of the spectrum of HPVs detectable by this assay.

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Author and article information

Contributors

Maria Lina Tornesello: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 26 April 2016

Publication date Collection: 2016

Volume: 11

Issue: 4

Electronic Location Identifier: e0153274

Affiliations

[1 ]Dirección de Infecciones Crónicas y Cáncer, Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública (INSP) (Chronic Infectious Diseases and Cancer Division, Center for Research on Infectious Diseases, National Institute of Public Health), Cuernavaca, Morelos, Mexico

[2 ]Private Health Center for Gynecology, Cuernavaca, Morelos, Mexico

[3 ]Centro de Atención para la Salud de la Mujer (CAPASAM) (Center for Women’s Health), Health Services of the State of Morelos, Cuernavaca, Mexico

[4 ]Division of Clinical Research, Instituto Nacional de Cancerología (INCan), SS, Mexico City, Mexico

[5 ]Division of Basic Research, Instituto Nacional de Cancerología (INCan), SS, Mexico City, Mexico

Istituto Nazionale Tumori, ITALY

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: AACh KTP. Performed the experiments: AACh MBR JTS JMB BCC GLE KDR DC. Analyzed the data: AACh KTP JMB BCC AIBG AGC VMM. Contributed reagents/materials/analysis tools: VMM. Wrote the paper: AACh KTP JMB JTS AGC VMM. Participated in the design of the study, the collection of clinical materials, carried out the bioinformatics analysis of the sequenced samples and cytokine expression and the statistical analysis, and drafted the manuscript: AACh KTP. Participated in the processing of samples: MBR. Participated in the process of sequencing of samples: JTS. Participated in designing sequencing strategy and the analysis of the results: JMB. Carried out the bioinformatics analysis: BCC. Participated in the selection of study population, gynecological sampler and patient care: GLE, KDR DC. Participated in the design of the study and the statistical analysis: AIBG. Participated in the analysis of the results and drafted the manuscript: AGC VMM. Contributed with reagents and materials for experiments: VMM. Read and approved the final manuscript: AACh KTP MBR JTS JMB BCC GLE KDR AIBG DC AGC VMM.

[¤a]

Current Address: Consejo Nacional de Ciencia y Tecnología de Mexico (CONACyT), Mexico City, Mexico

[¤b]

Current Address: Unit of Biomedical Research in Cancer, Instituto Nacional de Cancerología (INCan), SS and Biomedical Research Institute, Universidad Nacional Autónoma de México, Mexico City, Mexico

* E-mail: vmarina@ 123456insp.mx

Article

Publisher ID: PONE-D-15-52810

DOI: 10.1371/journal.pone.0153274

PMC ID: 4846060

PubMed ID: 27115350

SO-VID: 2be10ddc-d4ff-4d1a-b71e-32b1181012ad

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 4 December 2015

Date accepted : 25 March 2016