Abstract
BackgroundThe cervicovaginal microbiome (CVM) plays a significant role in women’s cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM.ResultsWe introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes’ abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women.ConclusionsCiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions.
Highlights
The cervicovaginal microbiome (CVM) plays a significant role in women’s cervical health and disease
Using CiRNAseq, we show that hrHPVpositive women with high-grade cervical intraepithelial neoplasia acquire a characteristic community state types (CST) IV microbiome as observed by earlier 16S Ribosomal RNA (rRNA)-seq studies
An obvious question is how the CVM behaves in high-risk human papillomavirus (hrHPV)-positive women with no cervical lesions, low-grade cervical lesions, high-grade cervical lesions, and cancer, and such studies are on the way
Summary
The cervicovaginal microbiome (CVM) plays a significant role in women’s cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Recent efforts have focused on the host immune response and the cervicovaginal microbiome (CVM) [2,3,4] The latter has a significant role in women’s cervical health and disease [5]. Recent investigations have determined that these microbiome alterations occur at the genus level and at the species level, suggesting that specific microbial species and CST are associated with progressive or regressive behavior of cervical lesions and could act as biomarkers for the disease [16,17,18]. Studying the CVM and elucidating its function currently relies mostly on short length 16S rRNA gene sequencing (16S rRNA-seq), which struggles to distinguish microbes at this taxonomic rank [19,20,21,22]
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