Temporal composition of the cervicovaginal microbiome associates with hrHPV infection outcomes in a longitudinal study.

BACKGROUND: Persistent infections with high-risk human papillomavirus (hrHPV) can cause cervical squamous intraepithelial lesions (SIL) that may progress to cancer. The cervicovaginal microbiome (CVM) correlates with SIL, but the temporal composition of the CVM after hrHPV infections has not been fully clarified. METHODS: To determine the association between the CVM composition and infection outcome, we applied high-resolution microbiome profiling using the circular probe-based RNA sequencing technology on a longitudinal cohort of cervical smears obtained from 141 hrHPV DNA-positive women with normal cytology at first visit, of whom 51 were diagnosed by cytology with SIL six months later. RESULTS: Here we show that women with a microbial community characterized by low diversity and high Lactobacillus crispatus abundance at both visits exhibit low risk to SIL development, while women with a microbial community characterized by high diversity and Lactobacillus depletion at first visit have a higher risk of developing SIL. At the level of individual species, we observed that a high abundance for Gardnerella vaginalis and Atopobium vaginae at both visits associate with SIL outcomes. These species together with Dialister micraerophilus showed a moderate discriminatory power for hrHPV infection progression. CONCLUSIONS: Our results suggest that the CVM can potentially be used as a biomarker for cervical disease and SIL development after hrHPV infection diagnosis with implications on cervical cancer prevention strategies and treatment of SIL.

Longitudinal analysis on the ecological dynamics of the cervicovaginal microbiome in hrHPV infection.

The cervicovaginal microbiome (CVM) is a dynamic continuous microenvironment that can be clustered in microbial community state types (CSTs) and is associated with women's cervical health. Lactobacillus-depleted communities particularly associate with an increased susceptibility for persistence of high-risk human papillomavirus (hrHPV) infections and progression of disease, but the long-term ecological dynamics of CSTs after hrHPV infection diagnosis remain poorly understood. To determine such dynamics, we examined the CVM of our longitudinal cohort of 141 women diagnosed with hrHPV infection at baseline with collected cervical smears at two timepoints six-months apart. Here we describe that the long-term microbiome dissimilarity has a positive correlation with microbial diversity at both visits and that women with high abundance and dominance for Lactobacillus iners at baseline exhibit more similar microbiome composition at second visit than women with Lactobacillus-depleted communities at baseline. We further show that the species Lactobacillus acidophilus and Megasphaera genomosp type 1 associate with CST changes between both visits. Lastly, we also observe that Gardnerella vaginalis is associated with the stability of Lactobacillus-depleted communities while L. iners is associated with the instability of Megasphaera genomosp type 1-dominated communities. Our data suggest dynamic patterns of cervicovaginal CSTs during hrHPV infection, which could be potentially used to develop microbiome-based therapies against infection progression towards disease.

In-depth insights into cervicovaginal microbial communities and hrHPV infections using high-resolution microbiome profiling.

The cervicovaginal microbiome (CVM) correlates with women's cervical health, and variations in its composition are associated with high-risk human papillomavirus (hrHPV) infection outcomes. Cervicovaginal microbes have been grouped into five community state types (CSTs) based on microbial community composition and abundance. However, studying the impact of CSTs in health and disease is challenging because the current sequencing technologies have limited confident discrimination between closely related and yet functionally different bacterial species. Circular probe-based RNA sequencing (ciRNAseq) achieves high-resolution microbiome profiling and therefore provides in-depth and unambiguous knowledge about the composition of the CVM. Based on ciRNAseq profiling of a large cohort of cervical smears (n = 541), we here define subgroups of CSTs I, III, and IV based on intra-CST differences with respect to abundances of Lactobacillus acidophilus (CSTs I-A vs. I-B and CSTs III-A vs. III-B), Lactobacillus iners (CSTs I-A vs. I-B and CSTs III-A vs. III-B), and Megasphaera genomosp type 1 (CSTs IV-A vs. IV-B). Our results further support the existence of subgroups of CST IV-C that are dominant for non-Lactobacillus species and have intermediate microbial diversity. We also show that CST V is associated with uninfected conditions, and CST IV-A associates with hrHPV-induced cervical disease. In conclusion, we characterized new subdivisions of cervicovaginal CSTs, which may further advance our understanding of women's cervical health and hrHPV-related progression to disease.

Assessing the Cervicovaginal Microbiota in the Context of hrHPV Infections: Temporal Dynamics and Therapeutic Strategies.

Cervical cancer is the third leading cause of female cancers globally, resulting in more than 300,000 deaths every year. The majority of all cervical cancers are caused by persistent infections with high-risk human papillomaviruses (hrHPV) that can progress to cancer via a series of premalignant lesions. Most women, however, clear this infection within a year, concomitant with disease regression. Both hrHPV clearance and disease regression have been associated with the composition of the cervicovaginal microenvironment, which is defined by the host immune system and the cervicovaginal microbiome (CVM). A healthy microbiome is generally characterized by a high abundance of Lactobacillus species, and a change in the composition may cause bacterial vaginosis (BV), which is associated with an increased susceptibility to persistent hrHPV infections and disease. In this review, the composition of the CVM is discussed, with emphasis on the possible causes that drive changes in the cervicovaginal microbiota in relation to hrHPV infections, disease progression, and disease regression. The literature search focused on the composition of the CVM and its correlation with hrHPV infections and neoplastic lesions as well as the current efforts to adjust the microbiome against adverse viral outcomes.

Targeted RNA next generation sequencing analysis of cervical smears can predict the presence of hrHPV-induced cervical lesions.

BACKGROUND: Because most cervical cancers are caused by high-risk human papillomaviruses (hrHPVs), cervical cancer prevention programs increasingly employ hrHPV testing as a primary test. The high sensitivity of HPV tests is accompanied by low specificity, resulting in high rates of overdiagnosis and overtreatment. Targeted circular probe-based RNA next generation sequencing (ciRNAseq) allows for the quantitative detection of RNAs of interest with high sequencing depth. Here, we examined the potential of ciRNAseq-testing on cervical scrapes to identify hrHPV-positive women at risk of having or developing high-grade cervical intraepithelial neoplasia (CIN). METHODS: We performed ciRNAseq on 610 cervical scrapes from the Dutch cervical cancer screening program to detect gene expression from 15 hrHPV genotypes and from 429 human genes. Differentially expressed hrHPV- and host genes in scrapes from women with outcome "no CIN" or "CIN2+" were identified and a model was built to distinguish these groups. RESULTS: Apart from increasing percentages of hrHPV oncogene expression from "no CIN" to high-grade cytology/histology, we identified genes involved in cell cycle regulation, tyrosine kinase signaling pathways, immune suppression, and DNA repair being expressed at significantly higher levels in scrapes with high-grade cytology and histology. Machine learning using random forest on all the expression data resulted in a model that detected 'no CIN' versus CIN2+ in an independent data set with sensitivity and specificity of respectively 85 ± 8% and 72 ± 13%. CONCLUSIONS: CiRNAseq on exfoliated cells in cervical scrapes measures hrHPV-(onco)gene expression and host gene expression in one single assay and in the process identifies HPV genotype. By combining these data and applying machine learning protocols, the risk of CIN can be calculated. Because ciRNAseq can be performed in high-throughput, making it cost-effective, it can be a promising screening technology to stratify women at risk of CIN2+. Further increasing specificity by model improvement in larger cohorts is warranted.

Novel high-resolution targeted sequencing of the cervicovaginal microbiome.

BACKGROUND: 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. 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. RESULTS: We 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. CONCLUSIONS: CiRNAseq 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.

Cervical cancer risk profiling: molecular biomarkers predicting the outcome of hrHPV infection.

INTRODUCTION: Cervical cancer affects half a million women worldwide annually. Given the association between high-risk human papillomavirus (hrHPV) infection and carcinogenesis, hrHPV DNA testing became an essential diagnostic tool. However, hrHPV alone does not cause the disease, and, most importantly, many cervical lesions regress to normal in a year because of the host immune system. Hence, the low specificity of hrHPV DNA tests and their inability to predict the outcome of infections have triggered a further search for biomarkers. AREAS COVERED: We evaluated the latest viral and cellular biomarkers validated for clinical use as primary screening or triage for cervical cancer and assessed their promise for prevention as well as potential use in the future. The literature search focused on effective biomarkers for different stages of the disease, aiming to determine their significance in predicting the outcome of hrHPV infections. EXPERT OPINION: Biomarkers such as p16/Ki-67, hrHPV genotyping, hrHPV transcriptional status, and methylation patterns have demonstrated promising results. Their eventual implementation in the screening programs may support the prompt diagnosis of hrHPV infection and its progression to cancer. These biomarkers will help in making clinical management decisions on time, thus, saving the lives of hrHPV-infected women, particularly in developing countries.

RNA-based high-risk HPV genotyping and identification of high-risk HPV transcriptional activity in cervical tissues.

Nearly all cervical cancers are initiated by a persistent infection with one of the high-risk human papillomaviruses (high-risk HPV). High-risk HPV DNA testing is highly sensitive but cannot distinguish between active, productive infections and dormant infections or merely deposited virus. A solution for this shortcoming may be the detection of transcriptional activity of viral oncogenes instead of mere presence of high-risk HPVs. In this study, fresh-frozen cervical tissues (n = 22) were subjected to high-risk HPV DNA detection using the line probe assay and to targeted RNA next-generation sequencing using single-molecule molecular inversion probes. Targeted RNA sequencing was applied for (1) RNA-based genotyping of high-risk HPV, giving information on specific HPV-subtype (2) discrimination of E2, E6, and E7 transcripts and (3) discovery of possible non-HPV cancer biomarkers. Data were analyzed using computational biology. Targeted RNA sequencing enabled reliable genotyping of high-risk HPV subtypes and allowed quantitative detection of E2, E6, and E7 viral gene expression, thereby discriminating cervical lesions from normal cervical tissues. Moreover, targeted RNA sequencing identified possible cervical cancer biomarkers other than high-risk HPV. Interestingly, targeted RNA sequencing also provided high-quality transcription profiles from cervical scrape samples, even after 1 week of dry storage or storage in Preservcyt fixative. This proof of concept study shows that targeted RNA sequencing can be used for high-risk HPV genotyping and simultaneous detection of high-risk HPV gene activity. Future studies are warranted to investigate the potential of targeted RNA sequencing for risk assessment for the development of cervical lesions, based on molecular analysis of cervical scrapes.

Gene expression profile of a selection of Polycomb Group genes during zebrafish embryonic and germ line development.

Polycomb Group (PcG) genes are transcriptional repressors that are described to be important during development and differentiation. There is significant interest in PcGs proteins because of their role in stem cell biology and tumorigenesis. In this study we characterize the expression of a selection of PcG genes in the adult germline of zebrafish and during embryogenesis. In adults, expression of selected PcG genes is found to be enriched in germ line over somatic tissues. Therefore, the germ line of adult zebrafish was analyzed for the expression pattern of a selection of PcG genes by whole mount in situ hybridization. We detected presence of the tested PcG gene transcripts at early stages of both oogenesis and spermatogenesis. This enriched expression for early stages of gametogenesis is also observed in developing gonads at 4 and 5 weeks post fertilization. Additionally, zebrafish embryos were used to study the spatio-temporal expression patterns of a selection of PcG genes during development. The PcG genes that we tested are maternally loaded and ubiquitously expressed at early developmental stages, except of ezh1. The expression of the PcG genes that were assessed becomes enriched anteriorly and is more defined during tissue specification. The data shown here is an important resource for functional PcG gene studies in vivo.

PQN-75 is expressed in the pharyngeal gland cells of Caenorhabditiselegans and is dispensable for germline development.

In Caenorhabditis elegans, five pharyngeal gland cells reside in the terminal bulb of the pharynx and extend anterior processes to five contact points in the pharyngeal lumen. Pharyngeal gland cells secrete mucin-like proteins thought to facilitate digestion, hatching, molting and assembly of the surface coat of the cuticle, but supporting evidence has been sparse. Here we show pharyngeal gland cell expression of PQN-75, a unique protein containing an N-terminal signal peptide, nucleoporin (Nup)-like phenylalanine/glycine (FG) repeats, and an extensive polyproline repeat domain with similarities to human basic salivary proline-rich pre-protein PRB2. Imaging of C-terminal tagged PQN-75 shows localization throughout pharyngeal gland cell processes but not the pharyngeal lumen; instead, aggregates of PQN-75 are occasionally found throughout the pharynx, suggesting secretion from pharyngeal gland cells into the surrounding pharyngeal muscle. PQN-75 does not affect fertility and brood size in C. elegans but confers some degree of stress resistance and thermotolerance through unknown mechanisms.

A Forward Genetic Screen for Suppressors of Somatic P Granules in Caenorhabditis elegans.

In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.