Cervical Samples Research Articles

Increased Risk of Infertility in Women Infected with Human Papillomavirus.

Among several causes of infertility, urogenital infections seem to be influencing factors. The effect of bacterial or viral sexually transmitted infections (STIs) on human fertility is not well understood. The aim of this study was to determine the frequency of STIs in cervical samples of infertile and fertile women and study the relationship between these agents and infertility. In this case-control study, cytobrush was used for collecting of cervical sample from each infertile and fertile woman (n=95) who attended Research and Clinical Centers for Infertility in Kerman, Iran. PCR and real-time PCR methods were used to detect the presence of bacterial (genital Ureaplasma species, genital Mycoplasma species, Chlamydia trachomatis (C. trachomatis), and Gardnerella vaginalis) and viral (herpes simplex virus, human papillomavirus (HPV), and Epstein-Barr virus) agents, respectively. Fisher's exact test and the logistic regression with the significance level of ≤5% were used for statistical analyses. In general, 78.94% and 14.73% of specimens were positive for one or more studied microorganisms, respectively. Among studied agents, only the infection with HPV was significantly different between infertile and fertile groups (p=0.005) which may enhance the likelihood of female infertility (OR=5.30, 95% CI:1.47-19.11, p< 0.05). After adjusting for age, irregular menstrual cycle, abnormal vaginal discharge, and ectopic pregnancy, the odds ratio of infertility in HPV-infected women increased (OR=7.02, 95% CI:1.52-32.3, p<0.05). Since HPV infection is asymptomatic, periodic screening of women in reproductive age especially infertile couples is recommended for early diagnosis and prevention of infection progression and cross contamination.

Patterns of synaptic loss in human amyotrophic lateral sclerosis spinal cord: a clinicopathological study

Amyotrophic Lateral Sclerosis (ALS) is mainly characterized by the degeneration of corticospinal neurons and spinal α-motoneurons; vulnerable cells display prominent pTDP-43 inclusions. Evidence gathered from genetics, murine models, and iPSC-derived neurons point to the early involvement of synapses in the disease course and their crucial role in the pathogenic cascade. However, pathology studies, with specimens from large post-mortem cohorts, mapping the pattern of synaptic disturbances over clinical and neuropathological hallmarks of disease progression, are currently not available. Thus, the appearance and progression of synaptic degeneration in human ALS patients are currently not known, preventing a full validation of the murine and in vitro models. Here, we investigated the loss of synaptophysin-positive terminals in cervical, thoracic, and lumbar spinal cord samples from a retrospective cohort of n = 33 ALS patients and n = 8 healthy controls, and we correlated the loss of synapses against clinicodemographic features and neuropathological ALS stage. We found that, although dorsal and intermediate spinal cord laminae do not lose synapses, ALS patients displayed a substantial but variable loss of synapses in the ventral horn of lumbar and cervical spinal cord. The amount of synaptic loss was predicted by disease duration, by the clinical site of onset, and by the loss of α-motoneurons, although not by the fraction of pTDP-43-immunopositive α-motoneurons. Taken together, our findings validate the synaptic pathology observed in other models and suggest that pathogenic pathways unfolding in the spinal microenvironment are critical to the progressive disassembly of local synaptic connectivity.

Reversal of the immunosuppressive tumor microenvironment via platinum-based neoadjuvant chemotherapy in cervical cancer

BackgroundImmunotherapy favors patients with tumors; however, only 3–26.3% of patients with cervical cancer benefit from single-agent immune checkpoint inhibitors. Combined immunotherapy and chemotherapy has been explored against tumor; however, the combination remains controversial. This study aimed to investigate the tumor immune microenvironment (TIME) and the effects of platinum-based neoadjuvant chemotherapy (NACT) in cervical cancer to identify the clinical value of combining chemotherapy with immunotherapy. MethodsMultiplex immunohistochemistry (IHC) with 11 markers (cluster of differentiation [CD]3, CD8, CD4, CD11c, CD68, forkhead box P3 [Foxp3], programmed cell death 1 [PD-1], programmed cell death 1 ligand 1 [PD-L1], indoleamine 2,3-dioxygenase [IDO], cyclin-dependent kinase inhibitor 2A [p16], and cytokeratin [CK]) was performed to evaluate TIME from 108 matched pre- and post-NACT cervical cancer samples. The mechanism of antitumor immunity triggered by NACT was explored using RNA sequencing (RNA-seq) from four paired samples and subsequently verified in 41 samples using IHC. ResultsThe infiltration rate of the CD8+ T cells in treatment-naive cervical cancer was 0.73%, and those of Foxp3+ regulatory T cells (Tregs) and IDO+ cells were 0.87% and 17.15%, respectively. Moreover, immunoreactive T cells, dendritic cells, and macrophages were more in the stromal than the intratumor region. NACT increased dendritic, CD3+ T, CD8+ T, and CD4+ T cells and decreased Tregs. The aforementioned alterations occurred predominantly in the stromal region and were primarily in responders. Non-responders primarily showed decreased Tregs and no increase in CD8+ T or dendritic cell infiltration. Furthermore, dendritic cells interacted more closely with CD3+ T cells after NACT, an effect primarily observed in responders. RNA-seq data revealed activation of the antigen receptor-mediated signaling pathway and upregulation of major histocompatibility complex (MHC) I and MHC II after chemotherapy, validated using IHC. ConclusionsNACT can reduce Tregs, and when tumor cells are effectively killed, antigen presentation is enhanced, subsequently activating antitumor immunity finitely. Our study provides the molecular characteristics and theoretical basis for the simultaneous or sequential combination of platinum-based NACT and immunotherapy for cervical cancer.

Cervical sample-taking in the primary health service

In Norway, approximately 360000 cervical screening samples were taken in 2020, of which 11000 were registered as inadequate. We therefore wished to investigate doctors' knowledge of cervical sample-taking in the primary health service. An anonymous survey on cervical sample-taking was sent by email to around 4700 members of the Norwegian College of General Practice in September 2021. Of the 1039 doctors who responded to the survey, 820 (79%) reported that they always indicate the reason for taking the sample in the requisition form, and 898 (86%) reported that they avoid taking a sample during menstruation. Only one in three doctors (343) correctly indicated the location of the squamocolumnar junction in postmenopausal women. In response to a question aimed at users of the ThinPrep method, which is particularly sensitive to sampling errors, 426 out of 697 (61%) answered that they either avoid using a lubricant or use a water-based lubricant, while only 35% of the doctors responded that they stop taking the sample if bleeding occurs. The results show that although many doctors have satisfactory knowledge, a continuous focus on cervical sample-taking is essential. Correct sampling and knowledge of anatomical factors in postmenopausal women may be significant for reducing the number of inadequate samples.

Protease-Activated Receptor 1 (PAR1) Expression Contributes to HPV-Associated Oropharyngeal Cancer Prognosis.

Human papillomavirus (HPV)-associated oropharyngeal cancer occasionally has a poor prognosis, making prognostic risk stratification crucial. Protease-activated receptor-1 (PAR1) is involved in carcinogenesis, and its expression is regulated by alpha-arrestin domain-containing protein 3 (ARRDC3). It is also involved in the tumor microenvironment. We sought to evaluate the predictive ability of PAR1, ARRDC3, and tumor-infiltrating lymphocyte (TIL) scores in patients with oropharyngeal, hypopharyngeal, and uterine cervical cancers, serving as comparators for HPV-associated oropharyngeal cancer. Immunohistochemical analysis of p16, ARRDC3, and PAR1 expression was performed on 79 oropharyngeal, 44 hypopharyngeal, and 42 uterine cervical cancer samples. The TIL scores were assessed and classified into the following groups based on invasion: low: 0-10%, medium: 20-40%, and high: > 50%. For prognostic analysis, the three groups were evaluated by dividing them into low, medium, and high categories, or alternatively into two groups using the median value as the cutoff. p16 was expressed in 44 (56%) oropharyngeal, 8 (18%) hypopharyngeal, and all uterine cervical cancer samples. ARRDC3 was detected in 39 (49%) oropharyngeal, 25 (57%) hypopharyngeal, and 23 (55%) uterine cervical cancer samples. PAR1 was expressed in 45 (57%) oropharyngeal, 22 (50%) hypopharyngeal, and 22 (50%) uterine cervical cancer samples. Patients diagnosed with p16-positive oropharyngeal cancer had a substantially improved prognosis compared to those diagnosed with p16-negative cancer. The PAR1-negative cases had a considerably improved prognosis compared to the positive cases (disease-specific survival [DSS] and -negative cases (disease-free survival [DFS]). Multivariate analysis revealed that ARRDC3-positive cases had an appreciably better DSS prognosis than patients with p16-negative oropharyngeal cancers. PAR1-positive patients among patients with p16-positive oropharyngeal cancer had a poor prognosis. With respect to DFS, patients with PAR1-positive and p16-negative oropharyngeal cancer had a 35-fold higher recurrence rate than those with PAR1-negative and p16-negative oropharyngeal cancer. Our results suggest that PAR1 expression affects the prognosis and recurrence rate of HPV-associated oropharyngeal cancer.

MiR-214-5p increases the radiosensitivity of cervical cancer by targeting ROCK1 expression.

The tolerance of cervical cancer to radiotherapy is a major factor affecting treatment outcomes. The miR-214-5p is involved in the regulation of biological processes such as tumor proliferation and metastasis. The aim of the study was to explore the role of miR-214-5p and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) in cervical cancer and their response to radiotherapy in cervical cancer patients. Fifty-three cervical cancer tissue samples were collected to analyze the level of miR-214-5p in patients with different responses to radiotherapy. Cervical cancer cell lines with radiation resistance were selected to explore the role of miR-214-5p in radiosensitivity. The wound healing, transwell migration, clone formation assay, and in vivo analysis were utilized to evaluate the effect of miR-214-5p on the radiation sensitivity of cervical cancer cells. Patients with poor radiotherapy responses demonstrated low levels of miR-214-5p. The upregulation of miR-214-5p decreased migration and invasion ability of radiotherapy-resistant cells. The bioinformatic analysis showed that ROCK1 is a candidate target gene of miR-214-5p, and this was confirmed with dual luciferase reporter assay showing that miR-214-5p directly interacts with the 3'untranslated region (3'UTR) of ROCK1. Decreased ROCK1 improved the radiosensitivity of cervical cancer in vitro and in vivo, and the overexpression of ROCK1 decreased the radiosensitivity effect of miR-214-5p in cervical cancer cells. The miR-214-5p can regulate the radiation sensitivity of cervical cancer cells by targeting the mRNA of ROCK1 and regulating its expression.

Two Enhances the Cisplatin Sensitivity of Cervical Cancer Cells via Suppression of c-MET Expression.

The drug resistance of chemotherapeutic agents leads to unsatisfactory survival rates for cervical cancer (CC) patients. We aimed to explore the effect of FOXP2 on the sensitivity of CC cells to cisplatin (DDP) and its mechanism in Changde, China in 2018. A Total of 6 cervical cancer tissue samples including 3 patients with cisplatin sensitivity and 3 patients with cisplatin resistance, who received DDP-based treatment, were obtained from Changde First People's Hospital, Changde City during 2021, and FOXP2 level was detected by Western blot. The expression levels of FOXP2 and c-MET (hepatocyte growth factor receptor, c-MET) in cells were determined by q-PCR and Western blot analysis. The cell survival, apoptosis, and clone formation were analyzed by flow cytometry, MTT assay, or clone formation assay. Dual-luciferase reporter assays and Chromatin immunoprecipitation were applied to verify the regulation between FOXP2 and c-MET. FOXP2 was downregulated in cisplatin-resistant cervical cancer tissues and cells compared with control. FOXP2 overexpression in SiHa/DDP cells inhibited cell proliferation and promoted cell apoptosis, whereas down-regulation of FOXP2 in SiHa cells had the opposite result. FOXP2 enhanced chemosensitive to DDP in CC cells. FOXP2 is negatively correlated with c-MET expression level in SiHa and SiHa/DDP cells. Mechanistically, FOXP2 binds to the promoter region of c-MET to regulate its expression in CC cells negatively. Overexpression of c-MET can attenuate the enhancement of DDP-induced apoptosis caused by FOXP2 overexpression. This is a novel study on the role of FOXP2 in promoting the DDP sensitivity of CC cells by inhibiting c-MET. The FOXP2/c-MET signaling axis uncovered in the present study may be a novel therapeutic target for the DDP therapy resistance of CC.

Rational design of genotyping nanodevice for HPV subtype distinction

There are more than 200 subtypes of human papillomavirus (HPV), and high-risk HPVs are a leading cause of cervical cancer. Identifying the genotypes of HPV is significant for clinical diagnosis and cancer control. Herein, we used programmable and modified DNA as the backbone to construct fluorescent genotyping nanodevice for HPV subtype distinction. In our strategy, the dye-labeled single-stranded recognize-DNA (R-DNA) was hybridized with Black Hole Quencher (BHQ) labeled single-stranded link-DNA (L-DNA) to form three functionalized DNA (RL-DNA). Through the extension of polycytosine (poly-C) in L-DNA, three RL-DNAs can be more firmly adsorbed on graphene oxide to construct reliable genotyping nanodevice. The genotyping nanodevice had low background noise since the dual energy transfer, including Förster resonance energy transfer (FRET) from dye to BHQ and the resonance energy transfer (RET) from dye to graphene oxide. Meanwhile, the programmability of DNA allows the proposed strategy to simultaneously and selectively distinguish several HPV subtypes in solution using DNA labeled with different dyes. To demonstrate clinical potential, we show multiplexed assay of HPV subtypes in cervical scrapes, and it has been successfully applied in HPV-DNA analysis in cervical scrapes samples. The genotyping nanodevice could be developed for simultaneous and multiplex analysis of several oligonucleotides in a homogeneous solution by adjusting the recognition sequence, demonstrating its potential application in the rapid screening of multiple biomarkers.

Genotype profiles of high-risk human papillomavirus in women of reproductive age: A community-based study.

This research is a preliminary study to observe the high-risk HPV infection profile among asymptomatic women, as a basis for developing Indonesian-specific reagents and implementing a national vaccination program. The research subjects were female employees/families of BUMN (state-owned enterprises) who underwent annual routine medical check-up. The research sample was a cervical swab which was examined using the Cobas® 6800 platform for HPV identification and INNO Lipa (Fujirebio) kit for identification of the genotype. Out of 858 cervical swab samples collected in this study, 31 samples were excluded as they had invalid results from the initial examination, so the remaining 827 samples continued this study protocol. Of those samples, 69 (8%) samples gave positive results, giving an overall HPV prevalence of 8%. Among HPV-positive samples, HPV 52 was the most common genotype (N = 15, 21.7%) found as both single-infection and multiple infections. The median age of subjects was 38 years. There were nine samples (13%) with multiple infections of two or more genotypes and seven samples (10.1%) with no specific genotype identified. HPV prevalence was 8%, with HPV 52 being the most common high-risk type, making it a necessity to develop a diagnostic kit and vaccine for national vaccination program that is specific for Indonesian population which includes this genotype.

The linkage of NF-κB signaling pathway-associated long non-coding RNAs with tumor microenvironment and prognosis in cervical cancer

BackgroundNF-κB signaling pathway participate closely in regulating inflammation and immune response in many cancers. Long non-coding RNAs (lncRNAs) associated with NF-κB signaling have not been characterized in cervical cancer. This study revealed the linkage between tumor microenvironment and NF-κB signaling-associated lncRNAs in cervical cancer.Materials and methodsThe expression profiles of cervical cancer samples from The Cancer Genome Atlas (TCGA) database were downloaded. NF-κB signaling-associated lncRNAs were screened as a basis to perform molecular subtyping. Immune cell infiltration was assessed by ESTIMATE, Microenvironment Cell Populations (MCP)-counter and single sample gene set enrichment analysis (ssGSEA). The key NF-κB signaling-associated lncRNAs were identified by univariate analysis, least absolute shrinkage and selection operator, and stepAIC.ResultsThree molecular subtypes or clusters (cluster 3, cluster 2, and cluster 1) were categorized based on 27 prognostic NF-κB signaling-associated lncRNAs. Cluster 2 had the worst prognosis, highest immune infiltration, as well as the highest expression of most of immune checkpoints. Three clusters showed different sensitivities to immunotherapy and chemotherapy. Six key NF-κB signaling-associated lncRNAs were screened to establish a six-lncRNA risk model for predicting cervical cancer prognosis.ConclusionsNF-κB signaling-associated lncRNAs played an important role in regulating immune microenvironment. The subtyping based on NF-κB signaling-associated lncRNAs may assist in the selection of optimal treatments. The six key NF-κB signaling-associated lncRNAs could act as prognostic biomarkers in prognostic prediction for cervical cancer.

Transcriptional activity of the long control region in human papillomavirus type 33 intratype variants

BackgroundHigh-risk human papillomaviruses (HPVs) are responsible for the development of cervical and other anogenital cancers. Intratype sequence variants of certain high-risk HPV types (e.g. 16, 18 and 31) are thought to have different oncogenic potential, partly due to nucleotide sequence variation in the viral long control region (LCR). The LCR has an important role in the regulation of viral replication and transcription. The purpose of this study was to explore sequence variation in the LCR of HPV 33 intratype variants in Hungary and to see whether there are differences in the transcriptional activities of the variants.MethodsThe complete HPV 33 LCR was amplified from HPV 33 positive cervical samples. After sequencing the LCR variants, multiple sequence alignment and phylogenetic analyses were carried out. Representative HPV 33 LCR sequence variants were selected for cloning and functional analysis. After transient transfection of HeLa cells, luciferase reporter assays were used to analyse the transcriptional activities of different LCR variants.ResultsAltogether 10 different variants were identified by sequence analysis of the HPV 33 LCR. The results of phylogenetic analysis showed that 3 variants belonged to sublineage A1, while the other 7 variants clustered with sublineage A2. Variants belonging to sublineage A2 had significantly lower transcriptional activities than variants belonging to sublineage A1. Within sublineage A2, the two variants analysed had significantly different transcriptional activities, which was shown to be caused by the A7879G variation.ConclusionsNucleotide variation in the HPV 33 LCR can result in altered transcriptional activity of the intratype variants. Our results can help to understand the correlation between LCR polymorphism and the oncogenic potential of HPV 33 variants.

Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA

CRISPR-Cas12a RNA-guided complexes have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, they are limited by the complexity of the operation, risk of carry-over contamination, and degradation of CRISPR RNA (crRNA). In this study, a Cas12a-based single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA)-mediated one-step diagnostic method (CasDOS) was established to overcome these drawbacks. mD-crRNA consisted of wild-type crRNA (Wt-crRNA) with ssDNA extensions at the 3′ and 5′ ends. Compared to Wt-crRNA, mD-crRNA exhibited a 100–1000-fold increase in sensitivity in the one-step assay, reducing the cis-cleavage activity of Cas12a to avoid excessive cleavage of the target DNA in the early stages of the reaction, leading to increased amplification and accumulation of the target amplicons, and improved the speed and intensity of the generated fluorescence signal. The detectability of CasDOS was 16.6 aM for the constructed plasmids of Streptococcus agalactiae (GBS), human papillomavirus type 16 (HPV16), and type 18 (HPV18). In clinical trials, CasDOS achieved 100% accuracy in identifying the known genotypes of the five HPV DNA samples. Moreover, CasDOS showed complete concordance with the qPCR results for GBS detection in ten vaginal or cervical swab samples, with a turnaround time from sampling to results within 30 min. In addition, mD-crRNA remained stable after Ribonuclease R treatment, suggesting that it might be more suitable as a raw material for the CRISPR detection kit. In conclusion, we have developed a universal, rapid, and highly sensitive one-step CRISPR detection assay.

Testing for Human Papillomaviruses in Urine, Blood, and Oral Specimens: an Update for the Laboratory.

Twelve high-risk alpha human papillomavirus (HPV) genotypes cause approximately 690,000 cancer cases annually, with cervical and oropharyngeal cancer being the two most prominent types. HPV testing is performed in laboratory settings for various applications of a clinical, epidemiological, and research nature using a range of clinical specimens collected by clinicians or by individuals (self-collected specimens). Here, we reflect on the importance and justification of using the right test for the right application and provide practical updates for laboratories either participating in or anticipating involvement in HPV testing in three specimen types, namely, urine, blood, and oral specimens, which are considered "alternative" specimens by many. In addition to clinician-collected cervical samples and self-collected cervicovaginal samples, first-void urine is emerging as a credible specimen for HPV-based cervical cancer screening, triage of HPV screen-positive women, monitoring HPV vaccine impact, and HPV testing in groups for which a less invasive sample is preferred. Detection of cell-free DNA (including HPV DNA) in blood has great promise for the early detection of HPV-attributable oropharyngeal cancer (HPV-AOC) and potentially other HPV-driven cancers and as an adjunct prognostic marker in long-term tumor surveillance, including treatment response. The moderate sensitivity of HPV testing in oral rinses or swabs at HPV-AOC diagnosis prevents its use in HPV-AOC secondary prevention but represents a promising prognostic tool in HPV-AOC tertiary prevention, where the HPV persistence in oral rinses throughout treatment may predict early HPV-AOC recurrences and/or the development of secondary HPV-AOC. The increasing sophistication of specific collection devices designed for alternative samples and the enhanced precision of novel molecular technologies are likely to support the evolution of this field and catalyze potential translation into routine practice.

Evaluation of the Roche MagNA Pure 96 nucleic acid extraction platform for the SPF10 line probe assay system.

To evaluate the MagNA Pure 96 (MP96) nucleic acid extraction system as an alternative for cervical specimen processing for human papillomavirus (HPV) genotyping detection by reverse hybridization line probe assay (LiPA-25), compared to the well-established extraction system MagNA Pure LC 2.0 (MPLC). A total of 200 cervical samples preserved in ThinPrepCyt® solution were extracted by MP96 and MPLC, respectively, and then purified nucleic acids were amplified using the SPF10 PCR primer set. Amplification products were subjected to SPF10-DNA enzyme immunoassay (DEIA) and LiPA-25. The concordance between different extraction methods in this study was reflected in the comparison of the results of the DEIA and LiPA-25. Agreement of HPV-positive results (DEIA) was 97.5% (Kappa=0.932). Pair-wise analyses of either HPV grouping (any HPV genotypes, any high-risk HPV genotypes, any low-risk HPV genotypes, any nonavalent vaccine-targeted HPV, and any non-vaccine-targeted oncogenic HPV genotypes) identified >95% agreement (all Kappa>0.900). For the two extraction methods, there was no statistical difference (chi-square test: P=0.690) for single versus multiple genotypes, and concordant, compatible, and discordant genotypes were observed in 87.0%, 9.5%, and 3.5% of 200 samples, respectively. HPV genotyping results of the MPLC system and the MP96 system showed a high degree of concordance. Combined with the advantages of high-throughput and anti-contamination of MP96, the MP96 extraction system could be more suitable for the testing of samples in future studies.

Human papillomavirus spectrum of HPV-infected women in Nigeria: an analysis by next-generation sequencing and type-specific PCR

BackgroundHuman papillomavirus (HPV) infection and cervical cancer are leading health problems and causes of death in many parts of the world. There are ~ 200 HPV types that can infect humans. This study aims to understand the spectrum of HPV infections in Nigerian women with normal or abnormal cytology.MethodsWe screened cervical samples from 90 women with possible HPV infections collected in two regional hospitals in Nigeria. The first screening was done using next-generation DNA sequencing (NGS), identifying multiple HPV types in many samples. Thereafter, type-specific PCR analysis was used to verify the NGS-identified HPV types in each sample.ResultsNGS analysis of the 90 samples from the Nigerian cohort identified 44 HPV types. The type-specific PCR confirmed 25 HPV types out of the 44 HPV types detected by NGS, and ~ 10 of these types were the most prevalent. The top five prevalent types found in the Nigerian cohort were HPV71 (17%), HPV82 (15%), HPV16 (16%), HPV6 (10%), and HPV20 (7%). Among the PCR-confirmed HPV types, we found 40.98% high-risk HPV types, 27.22% low-risk HPV types, and 31.15% undetermined HPV types. Among these 25 HPV types in Nigeria, only six were included in the current nine-valent HPV vaccine. We also observed strikingly high multiple HPV infections in most patients, with as many as nine HPV types in a few single samples.ConclusionsOur NGS-PCR approach of HPV typing in the Nigerian cohort samples unveiled all possible HPV types currently circulating in Nigerian people. We confirmed 25 HPV types using NGS and PCR, with many samples infected with multiple HPV types. However, only six of these types are part of the nine-valent HPV vaccines indicating the need to develop region-specific selective vaccines.

Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples.

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.

Distribution of human papillomavirus prevalence, genotype, and PAP test results in women in the southeast of Turkey.

The aim of this study was to investigate the prevalence, genotype, and distribution of cervical human papillomavirus (HPV) in women living in southeast Turkey. A total of 13,300 cervical smear materials were scanned and 899 cases found to be HPV-positive were included in the study. Cases were divided into seven groups according to age (under 19 years of age, 20-24, 25-29, 30-39, 40-49, 50-59, and over 60 years old) and six groups according to HPV types (HPV type 16, HPV type 18, HPV type 16-18 association, HPV type 16-high risk (HR) association, HPV Type 18-HR association, and HPV HR [31,33,35,39,45, 51,52,56,58,59,66, and 68]). SurePath liquid-based cytology preparations were evaluated, and HPV tests were performed using real-time-polymerase chain reaction. A total of 6.7% of cervical smear samples were positive for HPV DNA. The mean age of these cases was 41 years (range 15-78 years). All HPV types showed the highest rate of positivity in the 30-39 age groups. Regarding the distribution of HPV types, most of the cases were in the HPV HR group (66%). The most common atypia category detected in cytological examination was "Atypical squamous cells of undetermined significance" (ASC-US) (27%). It was determined that the prevalence of HPV in the southeast of Turkey is lower than the world average, the most common HPV type in our region is HPV-HR, and HPV peaks at older ages compared to what has been reported for other regions of the world.

Sequence analysis of exons 30 and 31 of LAMA3 gene variants and its association with human papillomavirus infection predisposition: no evidence was found.

Human papillomavirus (HPV) is associated with cervical cancer. For the infection to occur, most HPV types depend on interactions with heparan sulfate proteoglycans (HSPGs); however, non-HSPGs receptors are also involved. Laminin 332 is a crucial component of the epidermis's base membrane. It has shown interactions with HPV that suggest its function as a transient viral receptor in the extracellular matrix (ECM). We provide new information about Laminin 332 and HPV by identifying LAMA3 gene allelic variants from exons 30 and 31 and their distribution among women with and without HPV infection. We included 192 cervical cancer scrape samples from two groups of patients, 96 samples from patients with a low-grade squamous intraepithelial lesion (LSIL) and 96 samples from HPV-negative samples without LSIL. Identification of the HPV type was performed using an LCD-Array kit. Exons 30 and 31 of LAMA3 were amplified by PCR and analyzed by Sanger's sequencing. We identified a wide range of HPV types. The most frequent low-risk (lrHPV) HPV types were 6, 42, 44, and 90. For high-risk (hrHPV) HPV were 16, 31, 56, and 66. Only the genetic variant rs1131521 was identified in both groups. However, no significant association was observed between rs1131521 and the study groups. A single silent polymorphism was identified in both groups with similar frequency, whereas no mutations related to increased epithelial friability were identified.

Comparison of the Clinical Accuracy of Xpert HPV Assay on Vaginal Self-Samples and Cervical Clinician-Taken Samples within the VALHUDES Framework

The accuracy of high-risk human papillomavirus testing with the Xpert HPV assay on vaginal self-samples was compared with clinician-taken samples within the VALHUDES framework. We recruited 523 women in five Belgian colposcopy clinics, of whom 483 (median age, 40 years; interquartile range, 31 to 49 years) were included in the main analysis (226 collected with Evalyn Brush and 257 collected with Qvintip). Cervical samples were collected with Cervex-Brush. Colposcopy and histology outcomes were considered as the reference standard. The Xpert HPV assay had similar accuracy for cervical intraepithelial neoplasia ≥2 on self-collected versus clinician-collected samples [relative sensitivity,0.96 (95% CI, 0.91-1.02); and relative specificity,0.96 (95% CI, 0.89-1.04)]. The relative accuracy slightly differed by vaginal collection device [sensitivity ratios of 0.98 (95% CI, 0.90-1.06) and 0.94 (95% CI, 0.87-1.02) for Evalyn and Qvintip, respectively; specificity ratios of 1.06 (95% CI, 0.95-1.19) and 0.88 (95% CI, 0.80-0.98) for Evalyn and Qvintip, respectively]. No difference in CT values was observed between vaginal and cervical samples. In conclusion, the sensitivity of Xpert HPV assay for cervical intraepithelial neoplasia ≥2 on vaginal self-samples was similar to that of cervical specimens. The clinical specificity was lower than on clinician-collected samples when self-samples were taken with Qvintip.

Lymph node metastasis-related gene signature shows good performance in predicting prognosis and immune infiltration in cervical cancer.

This study aimed to construct a lymph node metastasis-related gene signature to predict prognosis and immune infiltration in patients with cervical cancer. Clinical and RNA sequencing data of 193 patients with cervical cancer, which were divided into lymph node metastasis (N1) and non-lymph node metastasis (N0) groups, were acquired from TCGA. Differentially expressed genes (DEGs) between the N1 and N0 groups were detected, and protein-protein interaction combined with LASSO analysis was conducted to further screen lymph node metastasis-related genes. Univariate and multivariate Cox regression analyses were performed to establish a predictive signature. The genetic features, potential biological behavior, and immune infiltration characteristics of the predictive signature were explored. Furthermore, the sensitivity of patients to chemotherapy drugs was estimated based on the predictive signature and the expression of TEKT2 and RPGR was investigated in the cervical cancer tissue samples. A total of 271 lymph node metastasis-related DEGs, including 100 upregulated and 171 downregulated genes, were identified. Two genes, TEKT2 and RPGR, were associated with lymph node metastasis and prognosis in cervical cancer, and were used to construct a lymph node metastasis-related predictive signature. Based on the predictive signature, patients with cervical cancer were divided into high- and low-risk groups. The high-risk group, characterized by a higher tumor mutation burden and somatic mutation rate, indicated a poor overall survival. The activation of immune infiltration and increased expression of checkpoint genes were observed in the high-risk group, indicating that they might benefit from immunotherapy. Cytarabine, FH535, and procaspase-activating compound-1 were estimated as reasonable chemotherapy options for patients in the high-risk group, whereas two taxanes and five tyrosine kinase inhibitors, including etoposide and vinorelbine, had therapeutic significance for patients in the low-risk group. The expression of TEKT2 and RPGR was significantly downregulated in cervical cancer tissues, especially in metastatic lymph node tissues. The lymph node metastasis-related predictive signature based on TEKT2 and RPGR showed good performance in predicting the survival outcomes of patients with cervical cancer. The risk score of the predictive signature was related to genetic variation and immune infiltration, which could guide immunotherapy and chemotherapy strategies.