Lactobacillus crispatus, L. jensenii, and L. helveticus were enriched in control women; these were the top three species with biomarker significance between the two groups. Eight pathways and four KEGG orthologies of the cervical microbiota of statistical differences were identified between the HR-HPV infection and control women. Collectively, our study described the cervical microbiota and its potential function during HR-HPV infection. Biomarkers of cervical microbiota and the changed bacterial metabolic pathways and metabolites can help clarify the pathogenic mechanism of HR-HPV infection, making them promising targets for clinical treatment and intervention for HR-HPV infection and cervical carcinoma.The relationship between the cervico-vaginal microbiome and high-risk human papillomavirus (HR-HPV) is well observed. However, there is a lack of adequate research regarding the cervical microbiota in HR-HPV infection. Most published research results have used 16S rRNA gene sequencing technology; this technology only focuses on marker sequences, resulting in incomplete gene information acquisition. Metagenomic sequencing technology can effectively compensate for the deficiency of 16S rRNA gene sequencing, thus improving the analysis of microbiota function. Cervical swab samples from 20 females with HR-HPV infection and 20 uninfected (Control) women were analyzed through 16S rRNA gene and metagenomic sequencing. Our results indicated that the composition and function of the cervical microbiota of HR-HPV infection differed notably from that of control women. Compared with control women, Firmicutes was decreased during HR-HPV infection, whereas Actinobacteria was increased. At the genus level, Lactobacillus was enriched in control women, while levels of Gardnerella and Bifidobacterium were lower. At the species level,  Cervical carcinoma is the world’s fourth most common cancer in women, the incidence of which is rising and the trend is getting younger . PreventThe microenvironment of the female reproductive tract has a dynamic balance composed of a large number of microbiotas with self-regulation . Among tPrevious studies regarding microbiota are mostly based on traditional methods of microbial culture; however, most microbiota cannot be isolated and cultured in a laboratory, resulting in limited experimental data, and a prolonged heavy workload . Since tThe core of research regarding reproductive tract microecology is microbiota. Most of the previous studies focused on vaginal microbiota. Studies indicate that the diversity of the cervical microbiome is higher than that of the vagina, and changes of the cervical microbiome in patients with HR-HPV infection are more obvious than those in the vagina . TherefoWe recruited as participants, 40 women of reproductive-age who visited the Department of Gynecology or Physical Examination Center at Guangdong Second Provincial General Hospital and who met the inclusion criteria, from December 2020 to September 2021. Based on the results of HPV DNA, cervical thinprep cytologic tests, and cervical biopsy pathology, the participants were divided into two groups: 20 women infected with HR-HPV (HR-HPV) and 20 uninfected women . All subjects were aged 25–45 y, had engaged in normal sex for one year or more without using hormonal contraception, and were not in their menstrual period, pregnant, or entering menopause. Exclusion criteria were (1) use of antibiotics or vaginal antimicrobials in the past month, and vaginal intercourse or vaginal lavage within the last 3 d; (2) suffering from bacterial vaginosis, candida vaginitis, urinary tract infectious diseases; (3) history of mycoplasma, chlamydia, gonorrhea, syphilis, genital herpes, trichomonas, or other sexually transmitted diseases; (4) previous treatment history of cervical lesions or total hysterectomy; (5) history of systemic diseases such as diabetes, autoimmune disease, and malignant tumors; (6) habits of smoking or drinking; (7) history of HPV vaccination. All subjects volunteered to participate in our research and provided written informed consent. The research was permitted by the ethics committee of the Second Peoples Hospital of Guangdong Province, and the research was carried out based on the principles of the Declaration of Helsinki.Three sterile swab specimens were taken from the cervical canal during a gynecological examination, and stored immediately at −80 °C for DNA extraction. Total genome DNA from specimens was extracted using the Hexadecyltrimethy Ammonium Bromide (CTAB) method. DNA concentration and purity was monitored on 1% agarose gels. Some DNA was destined for 16S rRNA gene sequencing, and the remainder for metagenomic sequencing.® DNA PCR-Free Sample Preparation Kit  following the manufacturer’s recommendations, and index codes were added. Finally, the qualifying libraries were sequenced on an Illumina Novaseq 6000 platform to obtain 250 bp paired-end reads.The V3–V4 region of the bacteria 16S ribosomal RNA gene were amplified by PCR  using primers 341F 5′- CCTAYGGGRBGCASCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′. Sequencing libraries were generated using TruSeqThe raw data was quality-filtered and chimera sequences were removed to obtain high-quality clean data by QIIME . Using t® Ultra DNA Library Prep Kit for Illumina . The quality of the library was assessed by Agilent 2100. All specimen structured libraries were sequenced on an Illumina Novaseq 6000 platform (Illumina). The sequencing strategy was PE150, and the amount of sequencing data for each sample was 6G.Library preparation for metagenomic sequencing was performed using the NEBNextThe raw sequences were cleaned by clearing the sequences which contained low quality bases, and the reads with unknown bases “N”. Then, the sequences were aligned to the human genome database to filter out the reads that may originate from the host using Bowtie v.2.2.4 software . SubsequP < 0.05 was considered to indicate a statistically significant difference.The SPSS v.25.0 software was used to analyze the participants’ general data. Wilcoxon rank-sum test was used for intergroup comparison of the 16SrRNA gene and metagenomic sequencing. Statistical analysis was performed with R software (v.2.15.3). P = 0.318), BMI (P = 0.811), age at first sexual encounter (P = 0.058), or Parity (P = 0.236) between the two groups  and 20 healthy females (“Control” group). There was no obvious difference in age (o groups Table 1.P<0.05).We obtained 3 350 704 raw reads from 40 subjects’ cervical swab samples. After removing barcodes, primers, low-quality bases, short sequences, and chimeras, 2 909 768 effective reads were obtained with an average efficiency rate of 86.63%. A total of 4911 OTUs in Control and 8235 OTUs in HR-HPV were achieved with 97% consistency  effect size (LEfSe) analysis was performed to identify the biomarkers of cervical microbiota between HR-HPV and Control. At the phylum level, Firmicutes was higher in Control, while Actinobacteriota was enriched significantly in HR-HPV. At the genus level, the cervical microbiome of HR-HPV was highly enriched with Gardnerella, Bidfidobacterium, and Atopobium, while Control was enriched with Lactobacillus  approach, were selected for analyzing microbial composition and function, respectively, which solving the problem of less effective data owing to serious host contamination.L. crispatus was the dominant species in Control, accounting for 36.39%. The relative abundance of L. crispatus (17.74%) decreased significantly, while Gardnerella vaginalis increased in HR-HPV  detected, 234 were found to be statistically significant. K01443 with the highest relative abundance was selected for species contribution analysis. With the decreased abundance of ficantly Table 2.In our research, we compared the composition and function of the cervical microbiome in HR-HPV infection and uninfected subjects using 16S rRNA gene and metagenomic sequencing. There were obvious differences in distribution and function of microbiota between the groups, including three species and eight pathways. Further, four KOs within those pathways were identified.L. crispatus decreased in the HR-HPV group, whereas G. vaginalis was increased, even similar to L. crispatus. LEfSe analysis was used to further explore the diagnostic biomarkers associated with HR-HPV. The prediction of biomarkers by the random forest model further validated the results of LEfSe at the genus level, and also highlighted the importance of Staphylococcus and Pseudomonas. L. crispatus, L. jensenii, and L. helveticus were the prominent three species with biomarker significance. Previous cross-sectional and longitudinal research indicates that the vaginal microbiome predominated by L. crispatus and is more common in women without HPV infection or cervical lesions , and other gynecological diseases  to inhibit the activity of cervical cancer cells  is the main mechanism for bacteria to absorb carbohydrates. The abundance of PTS was positively associated with that of tibility . It has tibility . A studytibility , but thiL. crispatus M247 significantly altered vaginal microbiota types and increased HPV conversion rates in women (L. rhamnosus BMX 54) group of 6-months had a much higher HPV conversion rate than a 3-month group, with a statistically significant difference (Following the in-depth research on reproductive tract microbiota in recent years, it is now known that dysbacteriosis contributes to the occurrence and progress of HPV infection, cervical lesions, and even cervical cancer. Therefore, the intervention and treatment of reproductive tract microbiota disorder has been a breakthrough for preventing cervical cancer and removing HPV. Its main purpose is to remove the pathogenic bacteria, and rebuild the balance of reproductive tract microecology to prevent further development of the disease and promote its cure. In an uncontrolled study of 35 HPV-positive women, oral administration of the probiotic in women . In a prfference . The usefference . With thfference . SMMM (Sfference . ClarifiThere were some limitations to this study. Differentiation studies between persistent HR-HPV infection and transient infection were ignored. Subsequently, the illustration of a causal relationship between HPV infection and microbiota by cross-sectional studies alone is insufficient; cohort studies should be performed to identify the potential impact of cervical microbiota biomarkers and altered metabolic pathways.L. crispatus, L. jensenii, and L. helveticus are the predominant three species with biomarker significance between the groups, and they represent potential microbial targets for future treatment. There were some metabolic pathways and metabolites of cervical microbiota that varied significantly; whether these affect the development of HR-HPV infection in women remains unclear. With the rapid development of molecular biology in recent years, the integration of multi-omics techniques such as metagenomics and metabolomics is promising. We plan to further clarify the causal relationship between HPV infection and the cervical microbiota and metabolites through combined metabolomics techniques in the near future.In conclusion, our study is the first in this field to report that combined 16S rRNA gene and metagenomic sequencing can more comprehensively demonstrate the diversity, microbial composition, and function of cervical microbiota between HR-HPV infected and uninfected women. Analysis using metagenomic sequencing can compensate for the lack of 16S rRNA gene sequencing technology. Meanwhile, the results of metagenomic and 16S rRNA gene sequencing gave mutual verification. Our study confirmed that the composition and function of the cervical microbiome in HR-HPV infection differed notably from that of uninfected women. 16S rRNA sequencing data for all the samples have been deposited in NCBI with the accession number of PRJNA846153. Metagenomic sequencing data for all the samples have been deposited in NCBI with the accession number of PRJNA847258.The studies involving human participants were reviewed and approved by The ethics committee of Guangdong Second Provincial General Hospital. The patients/participants provided their written informed consent to participate in this study.QZ and BF conceived the study question, and all authors were involved in the study design. BF and QL selected subjects and collected all the samples. BF, QL, ZW and ZO performed the sequencing and data analysis. BF created the first draft of the manuscript. QZ provided guidance throughout the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: In Local Health Unit 7, human papilloma virus (HPV) vaccination campaigns for 12-year-olds have long been implemented by the vaccination services of the Department of Prevention. Due to the pressure of the COVID-19 pandemic on these services, an emergency vaccination campaign was directly managed by primary care pediatricians (PCPs). An initial evaluation of this experience was conducted. Materials and methods: Data on 12-year-olds assisted by PCPs belonging to the 2006 (pre-pandemic) and 2008 (pandemic) birth cohorts were extracted, along with HPV vaccination data. Health district, gender, citizenship, socioeconomic status, and PCPs were evaluated as possible influencing factors in a two-level logistic regression (second level: single PCP). Results: The HPV vaccination gap between males and females increased significantly for the 2008 birth cohort compared to the 2006 birth cohort (11 vs. 4 percentage points). As for PCPs, the vaccination uptake range was 4–71% for the 2008 birth cohort vs. 32–85% for the 2006 cohort. The proportion of variance explained at the second level was overall equal to 9.7% for the 2008 cohort vs. 3.6% for the 2006 cohort. Conclusions: The vaccination campaign carried out during the peak of the COVID-19 pandemic increased the HPV vaccination gaps among Health Districts, genders, and individual PCPs, probably due to a lack of homogeneity in professional practices and attitudes toward HPV vaccination. Catch-up interventions are required in the immediate term, while an equity-lens approach should be taken for reprogramming the vaccination campaign. Greater involvement of schools and families could ensure a more equitable approach and a better uptake. The COVID-19 pandemic has dramatically disrupted the delivery of routine vaccines for children and adolescents. At the beginning of the pandemic, a temporary suspension of vaccination services was enforced, while the fear of contagion limited the attendance at these services. When the COVID-19 vaccines were finally available, a large proportion of the vaccination staff were reallocated to COVID-19 vaccination centers ,3.In Italy, the human papilloma virus (HPV) vaccination, unlike most pediatric immunizations, is not mandatory, resulting in a striking reduction in coverage, with the first HPV dose of 20 percentage points for 12-year-old girls and 16 percentage points for 12-year-old boys .Even before the pandemic, inequalities regarding access to HPV vaccinations for adolescents were described in many countries worldwide. Lower vaccination coverage has been found among ethnic minorities and low-educated or socially disadvantaged population groups; these disparities are less marked when the vaccination is offered for free, as is the case in Italy ,6. AnothHealthcare professionals, particularly general practitioners (GPs) and primary care pediatricians (PCPs), can have a significant influence on the HPV vaccination coverage of their patients . Physici®. The purpose of this study was to assess the impact of this change on the HPV vaccination coverage in terms of the disparities in access by gender, citizenship, and socioeconomic status, and among different PCPs. The COVID-19 emergency seriously tested and reshaped the vaccination services ,15,16,17The study population included birth cohorts from 2006 (pre-pandemic period) and 2008 (pandemic period). Adolescents were linked to their primary care physician on 1 January 2019 (2006 cohort) or 30 April 2022 (2008 cohort) using the population register; those assisted by a GP were not included. Those in the 2006 cohort received personal invitations by mail with the date, time, and place of vaccination from the vaccination services of the Department of Prevention, while those in the 2008 cohort were referred to their respective PCP for vaccination. Each adolescent in the study was characterized by gender , citizenship , and socioeconomic status at the municipal level  . Data re® software was used for statistical analysis. The impact on vaccination coverage disparities by gender, citizenship, and socioeconomic status was measured in terms of the relative risk ratio, using a generalized linear model for the binomial family, considering the risk ratio measured in the 2006 cohort as a reference. A multilevel and multivariate logistic model with random effects was used to take into account the hierarchical structure of the data, with the second level of aggregation being PCPs. The variability among PCPs was measured using the intraclass ρ correlation index, which describes the percentage of variance explained at the second level. To compare the variability among PCPs in the two cohorts under study, the median odds ratio (MOR) was calculated using a generalized structural equation model (GSEM). MOR can be conceptualized as the median increased risk if another PCP with higher HPV coverage was chosen. Therefore, MOR is equal to 1 if there is no coverage difference between PCPs, and the higher the value, the greater the difference . This apA total of 2754 12-year-old patients were enrolled in the 2006 cohort (vaccinated in the pre-pandemic period by the vaccination services of the Prevention Department), and 2208 in the 2008 cohort (vaccinated in the pandemic period by PCPs). As shown in As shown in Foreign citizenship influenced the vaccination coverage significantly and homogeneously in both birth cohorts , but there was no evidence of a differential impact . The municipal index of deprivation of the 52 municipalities of LHU 7 did not show any effect . The range of teen vaccination coverage by individual PCPs showed an increase for the 2008 birth cohort compared to the 2006 cohort  .The multilevel logistic model with mixed effects, adjusted for sex and citizenship, shows that the intraclass ρ correlation coefficient  increased substantially in LHU 7 (12.5% in the 2008 cohort vs. 5.1% in the 2006 cohort) and in Health District A (16.7% vs. 3.8%), while it remained substantially unchanged in HD B (4.4% vs. 3.2%).2 = 5.15, df = 1, p = 0.023), whereas the SARS-CoV-2 infection had the opposite effect . Of note, these two factors have a similar distribution among PCPs; therefore, the intraclass correlation coefficient does not change .In the 2008 cohort, COVID-19 vaccinations within the observation window (July 2021–April 2022) were associated with a significant increase in HPV coverage , whereas it remained almost unchanged in HD B . Overall, in LHU 7, the increase in coverage variability between PCPs was significant .The median odds ratio increased significantly in HD A  and among PCPs. This result is more evident in Health District A, which already showed lower coverage in the 2006 cohort.Other studies have evaluated the impact of the COVID-19 pandemic in terms of the inequality of access to routine vaccinations for children and adolescents who belong to ethnic minorities or live in rural communities or deprived neighborhoods 1.14–17.21. In this study, no disparity in vaccination coverage was observed among non-Italian adolescents, who represent about 7% of the birth cohorts and are distributed uniformly within LHU 7. In addition, no reduction in vaccination coverage was found among adolescents living in disadvantaged municipalities (more detailed neighborhood data are unavailable) [A lower HPV vaccine coverage among male adolescents has long been observed worldwide and is consistent with the delayed recommendation, or lack of recommendation, for HPV vaccinations for boys. The reduction in vaccination coverage in the males from the 2008 cohort compared to the females is an original finding of this study, and may be reasonably linked to the educational attitudes of different PCPs. Vaccinating a 12-year-old boy for HPV may require a conversation with the parents to address objections such as, “My son is too young and not yet sexually active”, or, “HPV infection affects young and adult women”. Parental health literacy is important, but medical counselling can make a difference ,22. FemaThe novelty of this study is the formal assessment of the disparity in HPV vaccination coverage observed among primary care pediatricians, before and after the COVID-19 pandemic. The widening of vaccination coverage disparities among PCPs for the 2008 cohort suggests the presence of different educational attitudes and capabilities, as well as different management methods adopted by PCPs, such as providing vaccination appointments and access times, making it easy to schedule and change an appointment (through an office secretary or self-booking computer-assisted system), and active calling (sending an invitation with the date and time of the appointment).PCPs who work alone cannot delegate to other professionals activities that do not require specific medical skills  . This maHPV vaccination coverage in the 2008 cohort was influenced by the SARS-CoV-2 infection and the COVID-19 vaccination. The former may have reduced vaccination coverage because of the isolation measures that required postponing the appointment. In contrast, the COVID-19 vaccination increased the odds of receiving an HPV vaccination, which could have resulted from a greater propensity toward vaccinations. Interestingly, these factors were distributed homogeneously in the target population and did not significantly influence the large gap observed among PCPs.This local experience shows that careful monitoring of the disparities in HPV vaccination coverage can offer important insights for the evaluation of HPV vaccination campaigns and programs. After several COVID-19 pandemic waves, it is time for resilience and recovery, and this is also true for teen HPV vaccinations. First, it is necessary to reverse the sharp drop in coverage with catch-up campaigns, to avoid an undue burden of HPV infection and disease in the youth population in the medium term . Many strategies have been shown to be effective in improving HPV vaccination coverage among adolescents. For example, social marketing campaigns and personalized invitations and reminders are effective at minimizing socioeconomic variability in the uptake of routine HPV immunizations ,30. ImpoIn the actual Venetian context, many of the proposed interventions are reasonably easier to implement within the framework of the vaccination services of the Department of Prevention than among PCPs. Whatever the health policy indication, the “health equity lens” can provide invaluable help in recognizing and reducing inequalities, and identifying the most effective strategies for reprogramming ,37.


Background: Head and neck squamous carcinoma (HNSC) poses a major threat to human life. The role of human papillomavirus (HPV) infection in the initiation and progression of HNSC is becoming more widely accepted. HPV-positive (HPV+) HNSC has shown unique responses to cancer therapies, which may be due to differences in immune cell infiltration. It is critical to determine how the immune responses to HPV in HNSC are regulated. Methods: Transcriptome data of HNSC from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database were analyzed. Then, the CIBERSORT algorithm was used to calculate immune cell infiltration in HNSC. FDCSP expression level was detected by qPCR in the HNSC tissues collected from the Nanfang Hospital. Results: Follicular dendritic cell secreted protein (FDCSP) was highly expressed in HPV+ HNSC, and higher expression of FDSCP was associated with a favorable prognosis. In HPV+ HNSC samples, FDCSP significantly increased the proportion of T follicular helper cells (TFHs). FDCSP expression was also found to be associated with TP53 mutation status in HPV+ HNSC. The function of FDCSP was intimately connected to chemokine pathways, particularly with the C-X-C motif chemokine ligand 13 (CXCL13). We verified that the high expression of FDCSP in HPV+ HNSC and higher FDCSP is closely related to prognosis in HNSC samples we collected by qPCR. Conclusions: Collectively, these findings may provide fresh evidence that FDCSP is a potential chemokine-associated prognostic biomarker in HPV+ HNSC. Head and neck squamous carcinoma (HNSC) is the sixth most common cancer worldwide . HNSC haHuman papillomavirus (HPV) is a tumor-associated virus with unenveloped double-stranded DNA (dsDNA). HPV infection causes nearly all cervical squamous cell carcinomas and approximately 40% of head and neck cancers ,3. In coBecause most previous studies compared HNSC or HPV+ groups to normal groups, studies comparing HPV+ against HPV− cancers are scarce. HPV infection has been shown to increase T-cell infiltration and activate immunological effector cells . In addiFollicular dendritic cell secreted protein  is a tiny, secreted protein that was discovered in follicular dendritic cells (FDCs). The FDCSP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines . FDCSP iOur study performed immunological analyses using HNSC combined data from GEO and TCGA. We analyzed differentially expressed HPV genes and evaluated the correlation and prognosis of FDCSP with immune infiltration in both HPV+ and HPV− HNSC. These findings support the hypothesis that FDCSP is the immune-associated biomarker that regulates HPV-induced HSNC.This study retrieved microarray datasets from the Gene Expression Omnibus (GEO) database. The GSE65858 dataset , based op-value (FDR) < 0.05 were considered the cutoff criterion. The results of DEGs were introduced to the heatmap and the volcano map.The DEGs were determined between HPV+ HNSC and HPV− HNSC samples with the “limma” R package. The |log2-fold change (FC)| > 2.5 and adj. p-value < 0.05.Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted with the DEGs by adopting the “clusterProfiler” package . GO enrihttp://software.broadinstitute.org/gsea/index.jsp, accessed on 1 March 2022) website, according to which the FDCSP expression levels were divided into high expression groups (≥50%) and low expression groups (<50%) and were obtained from the Molecular Signatures Database [http://www.gsea-msigdb.org/gsea/downloads.jsp, accessed on 1 March 2022). We downloaded the c5.go.bp, c5.go.mf, c5go, and KEGG symbol subsets to evaluate related pathways and molecular mechanisms, which were grouped based on gene expression profiles and phenotypes, and set the minimum gene set to 5. With a maximum gene set of 5000 and one thousand resamplings, a p-value of < 0.05 (as needed) and an FDR of < 0.25 (as needed) were considered statistically significant.For gene set enrichment analysis (GSEA), we obtained the GSEA software (version 3.0) from the GSEA   [TIMER2.0  ,20,21,22http://cistrome.org/TIMER/, accessed on 1 March 2022) web server and evaluated the correlation of the FDCSP expression level with the abundance of infiltrating immune cells in HNSC patients using the CIBERSORT_ABS algorithm. The p-values and partial correlation values were calculated by means of purity-adjusted Spearman’s rank correlation test.To determine the association between the FDCSP expression level and immune infiltration according to TCGA tumor data, we used the Tumor Immune Estimation Resource 2.0 (TIMER2.0) , and we integrated the sample mutation data and gene expression data. We also filtered samples for synonymous mutations. A total of 493 HNSC samples with detected mutations were included, of which the plot samples included a total of 387 (78.5%). We used the chi-square test to evaluate the difference in gene mutation frequency in each group of samples. The results of the difference test for all genes are as follows.We downloaded the Simple Nucleotide Variation dataset of level 4 of all TCGA samples processed by MuTect2 software  from GDChttps://string-db.org/, accessed on 1 March 2022), which predicts interactions based on relevant experimental data.PPI prediction analysis for the FDCSP-related proteins was performed using the STRING database version 11.0  is a free online extensible and customizable gene annotation portal tool that supplies a complete resource about gene and protein function. It displays a survey across diverse normal human tissues from the U133plus2 Affymetrix microarray. The values shown are z-scores produced by the barcode function of the R package “frma”. A z-score >5 suggests that the gene is expressed in that tissue.BioGPS  All patients were informed with written consent and the Ethics Committees of Nanfang Hospital approved the collection and use of all clinical specimens (NO: NFEC-2018-027).Total RNA was extracted from the cells using Trizol ). Reverse transcription (RT) and qPCR were performed in accordance with the manufacturer’s instructions . RT-qPCR for each gene was repeated three times. Quantification amounts were normalized to GAPDH levels. Primers are listed in the A total of 6 samples of collected oropharyngeal cancer specimens (3 HPV− and 3 HPV+ determined by qRT-PCR mentioned before) were used. FDCSP  and CXCL13  antibodies were both diluted by 1:500.Seven GEO datasets from various nations were included in the analysis A. Gene eThe combined datasets included 167 HPV+ HNSC samples and 574 HPV− HNSC samples. In total, 52 DEGs with |log2 FC| > 2.5 in the HPV+ HNSC samples that were compared with the HPV− HNSC samples were identified. There were 4 upregulated genes and 5 downregulated genes. The DEGs are presented in the heatmap plot and volcano plot B,C.Based on the better prognosis of HPV+ HNSC, we intended to focus on which genes or proteins inhibited tumor progression, so we focused on genes that were highly expressed in HPV+ HNSC. Outcomes of upregulated genes in DEGs were estimated in TCGA-HNSC by TIMER2.0 , which are dendritic cells (DCs). Because FDCs are characterized by high tissue selectivity, we reasoned that FDCSP would be the same. The FDCSP expression in human normal tissues was then identified using the BioGPS web tool. We obtained the raw data of FDCSP expression and filtered out the expression level >2 cell types in Since the source and function of FDCSP are closely related to the immune system, we attempted to investigate the relationship of FDCSP in HNSC immunity. We estimated stromal, immune, and ESTIMATE scores in GEO cohorts based on gene expression . We knowUsing the TIMER2.0 database, we mapped the immune infiltration of HNSC in both TCGA and GEO cohorts C. Immunep < 0.05 and |R| > 0.2 were filtered out). In HPV− HNSC, FDCSP was positively related to B memory cells (R = 0.287). In HPV+ HSNC, FDCSP was associated with more TFHs (R = 0.333), B memory cells (R = 0.337), CD8+ T cells (R = 0.229), CD4+ memory cells (R = 0.248), and macrophages .To further clarify the effect of FDCSP on immune infiltration in HPV+ HNSCs, we measured the levels of various immune cells using the CIBERSORT score in the GEO cohort. The composition of 21 kinds of immune cells in HNSC GEO cohorts with or without HPV infection is shown in HPV+ HNSC in the GEO cohort was divided into two groups according to the level of FDCSP expression, and the correlation between immune cells was assessed .We further analyzed the prognosis of FDCSP and the degree of infiltration of immune cells in HNSC. From the TIMER2.0 database, we constructed a Cox model of FDCSP and immune cells in HNSC . In HPV−In HNSC, the distribution of genetic mutations was investigated in groups according to high/low FDCSP expression. Significantly mutated gene mutation profiles presented TP53 (82.5%) and CASP8 (11.3%) in HNSCs .p < 0.001). However, there was no significant difference in HPV− HNSC. . We intersected the DEGs in both datasets and obtained 159 genes (112 upregulated genes and 47 downregulated genes) significantly related to FDCSP in HPV+ HNSC .p < 0.05). Gene Oncology (GO) analysis results are listed in Then, we used these 159 DEGs to carry out functional enrichment analysis  to identify FDCSP-related pathways in HPV+ HNSC using the KEGG dataset. The top ten enrichment scores (ES) are shown in In the previous functional enrichment analysis, we found that FDCSP is closely related to the chemokine pathway in HPV+ HNSC. We then intersected the DEGs of FDCSP in HPV+ HSNC and constructed a protein–protein interaction (PPI) network B. We fouIn HNSC tissues collected from Nanfang Hospital (n = 20), there were 17 HPV+ and 3 HPV− samples. We detected mRNA expression of FDCSP and CXCL13. FDCSP was highly expressed in HPV+ and was related to a worse prognosis C,E. In eHPV-induced HNSC tumorigenesis has been demonstrated to exhibit a distinct pathological pattern. Despite poor overall survival in HNSC, HPV+ HNSC showed a better prognosis than HPV− HNSC. Recent studies have shown that improved outcomes in patients with HPV+ HNSC are associated with the high responsiveness of these tumors to chemotherapy, radiotherapy, and targeted therapy ,25. ImmuTo determine which gene contributes to immune dynamics in HPV+ HNSCs, we analyzed seven GEO cohorts and identified DEGs. Four upregulated DEGs were filtered out. Among them, FDCSP was expressed at significantly higher levels in HPV+ HNSC and was associated with a favorable prognosis in HPV+ HNSC. FDCSP is specifically highly expressed in the gingival epithelium, tonsils, and tonsil epithelium. FDCSP is a unique secreted protein by follicular dendritic cells (FDCs) that has received little attention and research. FDCs contribute to the pathogenesis of a variety of immune-related diseases, including HIV/AIDS, prion diseases, chronic inflammation, and autoimmune diseases ,27,28. HWe screened out FDCSP using HPV+ versus HPV− in multiple GEO datasets and validated them in the TCGA database. Additionally, increased FDCSP expression indicates a better prognosis in HNSC. We further calculated the immune score based on the GEO cohort’s gene expression matrix and performed correlation analysis with FDCSP. FDCSP was significantly associated with the Immune Score and ESTIMATE Score in HPV+ HNSC. The CIBERSORT algorithm is an algorithm that calculates the scores of 21 immune cells in the bulk expression matrix based on the model obtained by single-cell sequencing . We furtLower FDCSP levels with lower TFH infiltration had a significantly worse prognosis in the TCGA HPV− HNSC group than those with higher TFH infiltration. In TCGA HPV+ HNSC, higher FDCSP levels with higher TFH infiltration have a significantly better prognosis than those with lower TFHs. These findings suggest that FDCSP works in tandem with TFHs in HPV+ HNSC. Follicular T helper cells, as the name implies, are a subset of activated T cells that specialize in providing T-cell assistance to corresponding B cells in germinal centers (GCs). GCs are specialized microenvironments formed after infection where activated B cells can mutate their B-cell receptors to undergo affinity maturation . TFHs arIt is now clear that the p53 status of cancer cells has a significant impact on the immune response . HNSCs hIn HPV+ HNSC, aberrantly expressed genes were enriched in immune processes, including the chemokine signaling pathway, T-cell activation, and cytokine signaling pathway . To furtIn normal lymphoid tissue, FDC networks can regulate recirculating resting B cells and divert antigen-activated B cells to clonal expansion to generate germinal centers (GCs) . FDCs seWe have demonstrated for the first time that FDCSP is required for the immune infiltration of HNSC. Increased FDCSP was significantly associated with a better prognosis, especially in HPV+ HNSC. Due to their close relationship with CXCL13, FDCs appear to play an important role in modifying the immune environment, particularly THFs, during the HPV+ HNSC tumor immunity process. Additional single-cell sequencing and experiments are necessary to decipher the dynamic role of FDCs in the immune response of HPV-induced tumors as well as the causal relationship between FDCs and CXCL13.


P = 0.11), β-HPVs , and γ-HPVs infections  was not associated with the HNSCC development. Our data did not suggest an HPV-related etiology for HNSCC pathogenesis. Nonetheless, this study provides novel insights into the diversity of β-, and γ-HPVs in different HNSCC anatomical subsites.Human papillomavirus (HPV) causes a subset of head and neck squamous cell carcinoma (HNSCC). Knowledge of determinants of α-, β-, and γ-HPVs types in the oral cavity is required for a better understanding of HNSCC development. Oral rinse samples of 498 HNSCC cases and 242 controls from the IROPICAN study—a large multicenter case-control study in Iran—were screened for 21 α-HPV, 46 β-HPVs, and 52 γ-HPVs using bead-based HPV genotyping assays. α-HPVs were detected only in 1.2% of the patients and 2.9% of the controls from which HPV16 was the most prevalent type among participants. β-HPVs were detected in 43.8% of the patients and 38.6% of the controls where the lip and oral cavity (45.5%) had the highest positivity. Values for γ-HPV prevalence in patients and controls were 26.1% and 24.7%, respectively. The highest percentage of γ-HPV positivity was found in the larynx (30.4%). Concerning the β genus, HPV23 and HPV38 were the most prevalent types among the patients and controls, respectively. For the γ genus, SD2 in cases and HPV134 in controls were the most prevalent types. Overall, detection of α-HPVs  is responsible for a subset of neck squamous cell carcinoma (HNSCC), but knowledge of the prevalence of and risk factors for oral HPV infection, especially cutaneous types in Iran, remains unknown. In a large retrospective study, the authors used a sensitive assay for the detection of α-, β-, and γ-HPVs in oral rinse samples of HNSCC and matched controls. They find that the α-HPV contribution to HNSCC in Iran is lower than global prevalence. High-risk α-HPVs or cutaneous β- and γ-HPVs were not associated with the HNSCC development. Besides, this study provides novel insights into the diversity of β- and γ-HPVs in different HNSCC anatomical subsites. Head and neck squamous cell carcinoma (HNSCC) is a debilitating disease that involves multiple anatomical subsites that comprise the lip and oral cavity, pharynx, and larynx . Annuall2–A broad spectrum of mucosal and cutaneous HPV types has been isolated from oral cavity samples –9. Certa–13–There is a discrepancy in the HPV-attributable fraction of HNSCC among Iranian patients , 21–24. 21–A total of 498 HNSCC cases, of which 198 lip and oral cavity , 44 pharynx , 219 larynx (ICD-O: C32), and 37 other subsites of the head and neck , and 242 controls from the IROPICAN study were included in the study. All β-globin negative samples were excluded from the final analysis . Charactn = 4; 0.8%) was the most prevalent α-HPV type among the HNSCC patients, and controls .Six out of 492 (1.2%) cases and seven out of 239 (2.9%) controls were positive for any α-HPVs. Characteristics of the 13 α-HPV-positive participants are reported in Table S1. Nine types of 21 types  were detected in the participants (Table S1). HPV16 (n = 216) among the patients and 38.6% (n = 93) among the controls. The lip and oral cavity showed the highest prevalence of β-HPV DNA , followed by larynx , pharynx , and other anatomical subsites   .HPVs from β-HPV-2, -1, -3, -5, and -4 species were the most abundant in patients and controls .The most frequent β-HPV types detected in HNSCC patients were HPV23 (8.9%), HPV38 (7.9%), HPV24 (6.1%), HPV5 (6.1%), and HPV49 (6.0%), while the most frequent β-HPV types in controls were HPV38 (9.1%), HPV23 (8.3%), HPV124 (7.9%), HPV145 (5.8%), and HPV111 (5.4%). Multiple β-HPV infections were found in 23.5% (116 out of 216 β-HPV-positive samples) and 21.6% (52 out of 93 β-HPV-positive samples) of the patients and controls, respectively .n = 128) and 24.9% (n = 59), respectively. The larynx subsite contained most of the γ-HPV positive samples , followed by the lip and oral cavity , other subsites , and pharynx  (n = 50) of the patients and 9.2% (n = 22) of the controls  . For γ-H, 16.3%) . The moscontrols .P = 0.041) and HPV168 (P = 0.048) , β-HPV , and γ-HPV . There was no significant difference in the frequency of individual α, β, and γ types between the cases and controls except for HPV134 (= 0.048) . We obse= 0.048) .Recent progress has highlighted the importance of HPV infections in the HNSCC pathogenesis from which high-risk mucosal HPV types are considered causal factors, especially in the oropharyngeal cancer development , 25–27. 25–28–In this study, a small fraction of HNSCC was positive for α-HPV DNA, lower than the previous estimates from Iran that mainly involved oral cavity and larynx fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples , 31. FurPrevious findings are in favor of an increased risk of oropharyngeal cancers with the HPV infection , 33. HowWe also provided additional data regarding the β- and γ-HPV prevalence in the HNSCC cases and matched controls. According to our results, 43.8% of the patients and 38.6% of the controls were β-HPV positive, in a fair agreement with a report of 41.8% positivity for any β-HPV in the samples from the oral region of the head and neck cancer patients and controls , 34. In In a nested case-control study by Agalliu et al., 62.1% of the HNSCC patients were positive for any β-HPV (mainly any β1 and β2). Any β-HPV positive cases had an increased risk of HNSCC incidence by two times more than the controls; the same association occurred for the types β1-HPV5 and β2-HPV38. In addition, the β1-HPV5 type increased the risk of oropharyngeal, oral cavity, and laryngeal cancers . In the The higher prevalence of β1- and -2 HPV species in our findings are inconsistent with the previous reports , 10. AccSimilar to β genus, we did not find any association between the γ-HPV positivity and HNSCC risk. In this study, γ7-HPV134 and γ8-HPV168 were found to be statistically significantly associated with the HNSCC risk. The possible role of these cutaneous HPV types in the development of head and neck cancer needs further evaluation. The γ-HPV genus is a ubiquitous and continuously growing HPV genus found in both cutaneous and mucosal sites , 40. InfWe are aware that a number of limitations may have influenced our results. Firstly, patients with tumors in oral, oropharyngeal, and larynx/pharynx sites may not have done appropriate specimen collection containing the epithelial cells; this may result in a small amount of HPV containing mucus and debris from the exfoliated cells. Secondly, an extremely rare number of oropharyngeal cancer patients in the current study caused inefficient power to detect HPV infection. It is noteworthy that in general, less than 1% of head and neck cancers in Iran are oropharyngeal ones. Hence, we suggest a pooled analysis of oropharyngeal cancers from the WHO Eastern Mediterranean Regional Office (EMRO) region or the International Head and Neck Cancer Epidemiology (INHANCE) consortium.In conclusion, our data were not suggestive of an HPV-related etiology in the HNSCC patients in Iran. However, this study provides novel insights into the diversity of β-, and γ-HPVs in different HNSCC anatomical subsites. To better understand the natural history and transmission dynamics of β- and γ-HPV in patients versus controls in the development of HNSCC, studying the HPV status in the anogenital and anal specimens is highly recommended.Oral rinse specimens were originated from the Iran Opium and Cancer (IROPICAN) study, which was conducted in 10 different provinces of Iran among HNSCC cases and sex- and age-matched controls during April 2016 and April 2019. Detailed information on the study method and design has been described elsewhere . BrieflyPatients and controls were instructed to rinse their mouth vigorously with 10 mL of mouthwash  for 60 s. The patients were asked to do this before surgery to restrict the presence of blood cells in the oral rinse, then spit it into RNase/DNase-free 50 mL conical centrifuge tubes. The samples were then centrifuged for 10 min, and the oral cell pellets were resuspended in 1 mL of remaining saline solution and stored at −80°C.Oral rinse samples were shipped to the International Agency for Research on Cancer  for DNA extraction and HPV DNA genotyping. Samples were centrifuged at 4,000 g for 5 min for pelleting the oral cell and washed with phosphate-buffered saline. Nucleic acid extraction was performed using the EZ1 DNA Tissue kit. Briefly, cell pellets were then resuspended in 180 μL of G2 buffer  and 20 μL of proteinase K (Qiagen) and incubated overnight at 56°C under agitation at 750 rpm in a Thermomixer Comfort instrument . DNA was then extracted using the EZ1 instrument (Qiagen), according to the manufacturer’s instructions, eluted in a final volume of 50 μL, and stored at 20°C until analysis. The samples were then tested for the presence of α-, β-, and γ-HPV types using multiplex bead-based assays that combine multiplex PCRs and Luminex technology , as described elsewhere , 45.The multiplex type-specific PCR method uses specific primers for the detection of 21 high-risk α-HPV types ; 46 β-HPVs from β-species 1 , β-species 2 , β-species 3 , β-species 4 (92), and β-species 5 (96 and 150); and 52 γ-HPVs from γ-species 1 , γ-species 2 (48 and 200), γ-species 3 (50), γ-species 4 (156), γ-species 5 , γ-species 6 , γ-species 7 , γ-species 8 , γ-species 9 (116 and 129), γ-species 10 , γ-species 11 , γ-species 12 , γ-species 13 (128), γ-species 14 (131), γ-species 15 (179), γ-species 18 (156), γ-species 19 , γ-species 20 (163), γ-species 21 (167), γ-species 22 (172), γ-species 23 (175), γ-species 24 (178 and 197), γ-species 25 (184), and γ-species 27 (201), and SD2 . Frequencies and percentages were calculated for categorical variables. We used a logistic regression model and adjusted it for the study centers, age, sex, place of residence, tobacco use, opium use, and socioeconomic status to estimate the association between genotypes of α-, β-, and γ-HPVs and risk of HNSCC. We chose the covariates of models if the type I error was less than P = 0.2 . MoreoveThe data set used and analyzed during study are available from the corresponding authors on reasonable request.


Axillary reverse mapping (ARM) is a novel intraoperative technique developed in recent years. This study was aimed at determining the effect of combined use of fluorescence dye and methylene blue, as well as its feasibility of ARM in patients with breast cancers who undergo modified radical mastectomy.  From January 2016 to June 2017, 46 patients with primary breast cancer at stage I-IV  who received modified radical mastectomy were enrolled in this study. The exclusion criteria included preoperative radiotherapy/chemotherapy or bilateral disease. Patients were divided into 2 groups: methylene blue group  and methylene blue+indocyanine green (ICG) group . ARM was performed before surgery. P = 0.032). There was a statistical difference of visualization rate between clinical groups of N0-N1 and N3-N4 . Although there was no significant difference found in the total drainage volume and arm perimeter of patients between the two groups (P > 0.05), the harden diameter of the injection site in methylene blue+ICG group was significantly longer than that in methylene blue group (P < 0.05). Among the 44 patients with different molecular profiling of breast cancer, there was no significant difference of visualization rate of ARM nodes in luminal A group , luminal B group , HER2 group , and basal-like group .  The overall visualization rate of ARM nodes was 80.4% (37/46). The visualization rate was significantly increased in methylene blue+ICG group  than that in methylene blue group  ( ARM using methylene blue+ICG presented greater identification rate than that with methylene blue alone, especially in patients with more invasive breast cancer. Our finding offers support for the improvement of ARM in future breast cancer management. Although new markers for oncogene expression were identified and showed promise in guiding treatment strategies for breast cancer, the condition of the axillary lymph nodes affects treatment and remains one of the most important prognostic factors of outcome . CurrentAxillary reverse mapping (ARM) is a technique described initially by Thompson et al.  and Nos From January 2016 to June 2017, 46 patients who underwent modified radical mastectomy were enrolled in this study. They were diagnosed as primary breast cancer at clinical stage I-IV  in our hospital. Those with preoperative radiotherapy/chemotherapy or bilateral disease were excluded. Ethical approval for this study was provided by the local ethical committee of our hospital. The median age of the study cohort was 50 years old (range 34-77). Informed consent was obtained from each patient prior to surgery.Patients were divided into 2 groups. The control group (methylene blue group) consisted of patients receiving ARM using methylene blue alone . The experiment group (methylene blue+ICG group) received the combination of ICG and methylene blue . In the methylene blue group, 2.0 ml of methylene blue  was subcutaneously injected into the upper inner arm. Then, the injection site was massaged, and the arm was raised for 10 min until the blue lymphatic vessels were observed in the upper extremity. Similar procedure was used in the methylene blue+ICG group for injection of 2.0 ml methylene blue plus 5 mg ICG . The clinical characteristics of the study cohort were summarized in The axillary reverse mapping (ARM) technique is developed for identifying and preserving lymphatic drainage from the arm during ALND. ARM was performed before the surgery. Before ALND, 2.0 ml of methylene blue with or without 5 mg of ICG in 2 ml of normal saline (NS) was subcutaneously injected into the ipsilateral upper inner arm of experiment or control group, respectively . The injThe primary ALND was indicated based on enlarged axillary lymph nodes by imaging assessment. ARM node labeled with ICG was observed by a fluorescence imaging camera , while methylene blue was viewed with the naked eyes Figures  and 3. IAll patients were diagnosed with the evaluation of postoperative TNM stage based on the revised TNM staging system adopted by NCCN Clinical Practice Guidelines in Oncology: Breast Cancer (Version 1.2016).Molecular profiling of breast cancer was performed for each patient and identified as luminal A, luminal B, basal-like, and overexpressed HER2 type and the estrogen/progesterone receptor (ER/PR) status as well as HER2/neu status.T test analysis were used to estimate intergroup differences. A P value < 0.05 was considered to be statistically different.Statistical analysis was conducted by using SPSS, version 17.0. The chi-square and P = 0.032). Moreover, among the 24 patients in the methylene blue+ICG group, the diagnosis result on lymph node showed that 20 (83.31%) cases belonged to clinical N0-N1 and 4 (16.67%) cases to clinical N3-N4, while in the methylene blue group, 16 (76.19%) belonged to clinical N0-N1 and 5 (23.81%) to clinical N3-N4. The differences of clinical N0-N1 stage and N3-N4 stage between methylene blue group and methylene blue+ICG group presented statistically significant .The overall successful dyeing rate of ARM nodes was 80.4% (36/46). 22 patients received dyeing of methylene blue alone, while 24 patients were given treatment of methylene blue+ICG. Visualization rate was significantly higher in the methylene blue+ICG  group than that in the methylene blue alone group  (P > 0.05)  .P > 0.05). The difference of limb perimeter between the patients in the group of methylene blue+ICG was to some extent higher than that in methylene blue group from day 1 to day 6. At days 7 and 8, perimeter difference of the two groups tended to be consistent   .In the methylene blue group, the number of positive axillary lymph node dissections was 11 , and in the methylene blue+ ICG group, the number was 11 , indicating that the positive rate of lymph node in methylene blue+ICG group was slightly lower than that of methylene blue group .The concept underlying the ARM technique assumes that lymphatics draining the upper extremity are not involved in the metastasis of the primary breast tumor , 6. HoweBased on the previous finding of the presence of metastasis in the ARM nodes, our data indicated 17.3% of the metastatic rate in the ARM nodes, which is similar to that of the earlier results of 13% by Kumar et al.  and 14% The initial studies on ARM in 2007 marked a blue dye applied in mapping axillary upper extremity lymph nodes and lymphatics. In accordance with the initial reports, blue dye has been generally used to delineate the axillary upper extremity lymph drainage. The result of visualization rates in the axilla of ARM lymph nodes during ALND ranged from 39% to 90% while of ARM lymphatics from 47% to 86% , 11. Altt = 2.431, P = 0.19 < 0.05). These findings suggested that methylene blue+ICG could improve identification rate of ARM node, especially for patients with more invasive breast cancer.In our study, the identification rate of the ARM node was 80.4%, which is similar to 61% by Thompson et al. . Our datAlthough there were no statistical differences in the total drainage volume and perimeter difference between ICG+methylene blue group and methylene blue alone group, the harden diameter of the injection site was significantly increased in methylene blue+ICG group than that in methylene blue alone group. These results indicate the feasibility of ARM using fluorescence+blue dye over blue dye alone in the Chinese breast cancers patients who received modified radical mastectomy.ARM using methylene blue+ICG presents greater identification rate of ARM node than that with methylene blue alone, which provides a potential tool for the surgical therapy towards patients with invasive breast cancer.


Ureaplasma, Chlamydia, and Gardnerella) in the lower part of the female genital system has a positive correlation with the frequency of HPV infection and, consequently, a possible influence on faster progression to cervical dysplasia caused by HPV. We consider that inclusion of screening for sexually transmitted infections as monitoring in women with HPV infection could help to find women at risk of cervical cancer progression.We conducted a prospective study with the aim of determining HPV prevalence and type distribution in the general female population of Southern Croatia (SDC), and to detect the presence of other microorganisms in the lower part of the female reproductive system and their possible influence on the frequency of HPV infection. Data were collected during routine check-up exams. All participants were examined by a gynecologist, and cervico-vaginal scrapings/swabs were collected, for cytological (Pap smear) and microbiological  analysis. Informed consent was obtained from all participants with accompanying questionnaire. A total of 1050 asymptomatic women living in SDC participated in the study during a one-year period, and 107 of them (10.2%) had HR-HPV infection. We found that the presence of some bacteria ( Cervical cancer (CC) is the fourth cause of cancer in women worldwide with 604,127 new cases  and the fourth leading cause of cancer death in women with 341,231 deaths  in 2020 . GenitalThe presence of HPV is necessary for CC developing but not sufficient cause of cervical cancer. The development of HPV-induced cancer is associated with some co-factors, such as other sexually transmitted infections (STIs) and even the presence of a high diversity in the cervico-vaginal microbiota (CVM) ,7.There are only a few studies about the prevalence and distribution of HPV types in Croatia, based on laboratory test results, related to cervical samples of selected women, referred by their gynecologists to laboratory for HPV DNA testing, with either a normal or abnormal Pap smear ,9.The aims of the present study were to determine the prevalence of HPV and HPV distribution of HPV-16 and -18 and 12 other HR HPV genotypes in the general female population of Southern Croatia , to detect the presence of other microorganisms in the lower part of the female reproductive system  and their possible influence on the frequency of HPV infection, as well as to determine the association between HPV infection and abnormal Pap test results.We performed a prospective study to determine HPV prevalence and type distribution of outpatient women in the SDC. Data were collected during routine check-up exams by a group of gynecologists across the county, from four gynecological practices of different parts of the county, that were unaffiliated with the study. Selection bias was eliminated because the gynecologists sent samples of all women  who came for regular gynecological examination on specific predetermined days over a period of one year. Exclusion criteria were pregnancy, lower genital tract dysplasia, or malignancy and if they suffered from more severe diseases .The study involved a total of 1050 women living in SDC, during a one-year period. All participants underwent an examination performed by a gynecologist, and cervico-vaginal scrapings/swabs were collected for cytological (Pap smear) and microbiological  analysis . A strucLactobacillus spp. as dominant bacterium. On the other hand, disruption of vaginal homeostasis we marked as CVM dysbiosis, which meant some other bacterial growth in pure culture or in the predominant number ). Detection of genital Mycoplasma hominis and Ureaplasma urealyticum were performed using the commercially available Mycoview kit  with a cut-off value of 104 CCU/mL. Chlamydia trachomatis (CT) and HR-HPV were detected in cervical exfoliated cells using a real-time FDA approved PCR assay  based on concurrent individual genotyping for HPV-16 and HPV-18 and the pooled detection of 12 other HR-HPV types. The cytology was reported using the Bethesda system, either as atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesion (LSIL), or high grade squamous intraepithelial lesion (HSIL) [Bacterial cultivations were performed from the material obtained from cervical swabs using standard laboratory procedures. Normal healthy CVM was defined as vaginal homeostasis with The study has been approved by the Ethics Committee of the Teaching Institute for Public Health of Split and Dalmatia County, TIPH SDC, within which the work was performed . All parts of the study were performed in accordance with the Declaration of Helsinki.2) test was performed. Statistical analysis was performed using the statistical package SPSS 17.0 . A p-value < 0.05 was considered statistically significant.In this study, all variables were qualitative. The age variable was defined for two age groups, namely for women aged 29 years or less and for women aged 30 years or more . The obtained results were stored in the MS Excel database. Structural pie charts and tables were used to display the selected variables. To display the structure according to the modalities of the selected variables, in addition to nominal values, relative numbers in percentages are also used. The Kolmogorov–Smirnov test was used to check the data distribution. For the statistical processing of qualitative variables that had a normal distribution, the Chi-square , 304 (29%) of them were ≤ 29 years old and 746 (71%) were ≥30 years old , in 317 out of 1050 women (30.2%). In our study, HR-HPV infection was proved in cervical samples of 107 (10.2%) women. Although the majority of tested women, i.e., 98% of them (1029), had a negative Chlamydia trachomatis (CT) test on regular check-up, the primary pathogenic bacterium CT was detected in 21 (2%) cases. Bacterial growth is a normal finding in a cervical swab, as it is often contaminated with CVM when the swab is taken. These bacteria of CVM are in balance in the mixed physiological flora of the lower parts of the genital system (mainly with Lactobacillus sp.) and do not cause infections. In our study, 869 (82.8%) women had such a normal flora, and we marked them in Streptococcus agalactiae, Escherichia coli, Gardnerella vaginalis) in the predominant number or in pure culture, half of which accounted for Gardnerella vaginalis (GV), in 90 samples.According to the data in Out of the total number of 1050 asymptomatic women on regular gynecological examination, 40 of them (3.8%) had an abnormal PAP result (including LSIL and HSIL), while 1010 of them (96.2%) had a normal cytological finding .p = 0.3898).No statistically significant dependence was found between the age of women and Pap test results. For women aged ≤29 years, 14 of 304 (4.6%) had an abnormal cytological result, while 26 (3.5%) of 746 women in the group aged ≥30 years had an abnormal result of the Pap smear  had HR-HPV infection, while the rest of 943 (89.8%) women were negative for HR-HPV .p < 0.001)  .We found that the presence of some bacteria  in the lower part of the female reproductive system have positive correlation with frequency of HPV infection.Ureaplasma, there were a significantly higher proportion of women with a positive HPV test, i.e., in 19.2% samples, while among Uu-negative women, only 6.3% had an HPV infection (p < 0.001). Out of the 1029 women who were negative for CT test, only 9.7% was found to have a positive HPV test, while 33% of women who were CT positive had an active HPV infection at the same time. A statistically significant association between a positive result for CT and HPV infection was established (p < 0.001) (It is clear from the data in < 0.001) .p = 0.218598).In our study, there were 181 women with detected CVM dysbiosis . HPV wasGardnerella vaginalis (GV) was isolated in half of the women with CVM dysbiosis, i.e., in 49.7% (90 of 181 women with the disorder). We analyzed separately the connection between GV and HPV. According to the data in p < 0.05).However, we noticed that the bacterium p < 0.001) . In the group of HPV negative women, only 2.5% had an abnormal Pap smear. This represents a statistically significant difference (< 0.001) .N = 1050) who attended a regular gynecological visit, HPV-16 was detected in 38 (3.6%), HPV-18 in 7 (0.7%), while other HR-HPV genotypes were determined in 89 (8.5%) tested women. In this research, 107 (10.2%) women were HPV positive, and among positive samples, the following genotype distribution was detected: HPV-16 in 35.5% and HPV-18 in 6.5%, while the other HPV types were established in 83%  had proven HPV infection. Genotype HPV-16 was detected in 3.6% women and HPV-18 in 0.7%, while other HR-HPV genotypes were determined in 8.5% tested women. Our present results are different from those found in other studies in Croatia which looked at HPV prevalence, because they differ according to the class of observed female population. To our knowledge, this is the first population-based study in Croatia that analyzed the prevalence of HPV infection and genotype distribution in asymptomatic women from the general population.The percentage of HPV-positive women in the present study is in agreement with the data published in the meta-analysis of a systematic literature review conducted by de Sanjose et al., who showed that, among women with regular cytology worldwide, 10.4% was HPV positive . The majDespite being the most common STI and the causal agent of cervical cancer, it is still not clear why the majority of HPV infections resolve without consequences, while only a small proportion of them persist, causing intraepithelial lesions and cervical cancer . CurrentUreaplasma urealyticum, Chlamydia trachomatis, and Gardnerella vaginalis showed a significant association with HPV infection.Of all the bacteria detected in genital swabs in the present study, after statistical analyses, we found that only Ureaplasma urealyticum (≥104 CCU/mL). Among Uu-positive cases, there were a significantly higher proportion of women with a positive HPV test, i.e., in 19.2% of the samples, while among Uu-negative cases, only 6.3% of women had HPV infection. Our results are in agreement with previous research work from Greece, which showed that 25.4% of Uu-positive women simultaneously had an HPV infection. Some other authors have also found a relationship between Uu and HPV infection. Their results suggest that Uu infection increases the risk of HPV infection, which may have a synergistic effect on the development of HPV-related cervical dysplasia [Ureaplasma can be considered colonization or infection. A cut-off value of more than 104 CCU/mL Uu in a sample is an additional criterion to distinguish colonization from infection, and Kim et al. also indicated that only a Uu with ≥ 104 CCU/mL was significantly associated with HPV infection. They also found this relationship in asymptomatic women and, based on this, they recommended HR-HPV testing for women with Uu infection, regardless of symptom status because of a possible synergistic effect between Ureaplasma and HPV in cervical dysplasia caused by HPV [In the present study, 30.2% participants had positive cervical swab for ysplasia ,16,17. IChlamydia trachomatis (CT) are asymptomatic, they can be carriers of CT infection, which can promote HR-HPV infection. In our study, only 2% women were CT positive and among them, 33% had HPV infection at the same time, while only 9.7% of HPV positive women was CT negative. Our results are in agreement with Lv et al., who revealed about four-fold higher risk of HR-HPV infection in CT-positive women compared with CT-negative women. They suggested that CT infection may damage the mucosal barrier and could increase cervical susceptibility to HPV and indicated the necessity of screening and treatment for CT in HR-HPV-positive women [Although the majority of individuals infected with ve women . Franchive women .Lactobacillus spp. They produce lactic acid and hydrogen peroxide, and in that way, they maintain low pH and increase the viscosity of cervical mucus, and thus, protect the cervical epithelial barrier [The cervico-vaginal microbiota (CVM) has a symbiotic relationship with their host and plays a significant role in the health and disease of the female reproductive tract. It is the first line of defense against infections and appears to play a key role in the acquisition, persistence, and clearance of HPV infection. The heterogeneity of the cervico-vaginal microbiota can vary according to biological and environmental factors. Women with a specific vaginal microbiota composition may be more likely to acquire HPV or to show a faster progression to dysplasia ,7,19,20. barrier ,21.Lactobacillus population, and an increase in CVM diversity, which is mainly composed of bacteria associated with BV, as well as often being associated with strict anaerobic species, including Gardnerella vaginalis (GV) [The researchers noted that the development of HPV-induced cancer is associated with a disruption of CVM homeostasis, which is involved in the control of viral persistence and is, therefore, a prognostic marker for disease. A state of vaginal dysbiosis or microbial imbalance indicates changes in the composition of CVM, with a decrease in mucus production, a decrease in the amount of lis (GV) ,7,22. Solis (GV) . Abdalahlis (GV) . The reslis (GV) . Gillet lis (GV) .In our present study, in 17.2% of tested women, CVM dysbiosis was detected, while 82.8% showed a normal genital microbiota. No statistically significant association between CVM imbalance and a positive HPV test result was found . However, we noticed that the bacterium GV was isolated in half of the women with CVM dysbiosis, i.e., in 90 (49.7%). According to our results, it is clear that among 90 women who had a positive cervical swab for GV, a significantly higher proportion of those had an HPV infection at the same time, i.e., in 18.9%, while only 9.4% of women who were GV negative had an HPV infection. This result is in agreement with other authors who found an association between GV presence and HPV infection, as well as a highly positive correlation with precancerous progression. It is suggested that monitoring the presence of GV and the CVM diversity could help find women at risk for progression to cervical cancer .In his paper, Mandeli emphasized the importance of simultaneous diagnosis of sexually transmitted infections in the genital tract as a key for determining unfavorable clinical outcomes of cervical cancer . In the Gardnerella vaginalis, can influence the susceptibility to HR-HPV infection . Furthermore, we found that bacteria Ureaplasma urealyticum and Chlamydia trachomatis had a very high positive association with HR-HPV infection, and consequently, a possible synergistic effect in the development of cervical dysplasia caused by HPV. Therefore, we believe that it is necessary to raise the level of care for women’s health by introducing a screening program for both HPV and other STIs, which should be available to women worldwide. We did not perform testing for Mycoplasma genitalium (MG) at the time of conducting our study, but nowadays, MG is recognized as important as CT infection, and screening for MG should be included in future research.In our study, on more than 1000 participants of general population, we proved that CVM dysbiosis, mostly caused with Screening for STIs other than HPV could be very good indicator for monitoring women with HPV infection to prevent progression to cancer. We consider that this work represents our contribution to new knowledge about the prevalence and distribution of HPV genotypes in Croatia and the importance of screening, especially in the context that Croatia still does not have a nation-wide screening program for cervical cancer prevention, although this is being prepared.Ureaplasma, Chlamydia, and Gardnerella) in the lower part of the female genital system affect the frequency of HPV infection and, consequently, can affect the faster progression to cervical dysplasia. We consider that including screening for sexually transmitted infections as monitoring in women with HPV infection could help identify women at risk for progression to cervical cancer.The data from this population-based study on the prevalence and distribution of HPV genotypes in general female population of Southern Croatia (SDC), who underwent routine gynecological visits, will be valuable for better organization of HPV-based cervical cancer screening and vaccination program in this region and in Croatia. In this study, we found that the presence of some bacteria (


HPV has been detected in approximately 50% of invasive penile cancers but with a large span between 24 and 89%, most likely due to different types of tumors and various methods for HPV analysis. Most studies of HPV in penile cancer have been performed using paraffin-embedded tissue, argued to be at risk for contaminated HPV analysis. Viral activity of HPV, by the use of HPV mRNA expression is well studied in cervical cancer, but seldom studied in penile cancer.The aim was to determine prevalence of HPV types in fresh tissue of penile cancers compared to non-malignant age-matched penile controls. Additional aims were to analyze the viral expression and copy numbers of HPV16-positive tumors and 10 mm adjacent to the tumor.Fresh tissue from penile cancer cases was biopsied inside the tumor and 10 mm outside the tumor. Controls were males circumcised for non-malignant reasons, biopsied at surgery.PCR and Luminex assays were used for identification of HPV types. HPV16-positive samples were investigated for copy numbers and expression of HPV16-mRNA.n = 135) and age-matched controls (n = 105), HPV was detected in 38.5% (52/135) and 11.4% (12/105), respectively (p < 0.001), adjusted odds ratio 12.8 . High-risk HPV types were found in 35.6% (48/135) of tumors and 4.8% (5/105) of controls (p < 0.001). Among tumors and controls, HPV16 was present in 27.4% (37/135) and 1% (1/105), respectively (p < 0.001). Among HPV16-positive penile cancers, mean HPV16 viral copy/cell was 74.4 (range 0.00003–725.4) in the tumor and 1.6 (range 0.001–14.4) 10 mm adjacent from the tumor. HPV16-mRNA analysis of the tumors and 10 mm adjacent from the tumors demonstrated viral activity in 86.5% (32/37) and 21.7% (5/23), respectively.Among tumors  than among age-matched non-malignant penile samples (11.4%). HPV16 predominates (27.4%) in penile tumors. HPV16 expression was more common in penile cancer than in adjacent healthy tissue, strongly suggesting an etiological role for HPV16 in the development of penile cancer.The online version contains supplementary material available at 10.1186/s12885-022-10324-w. Penile cancer is a rare cancer worldwide, with an incidence of 0.45–1.7/100000 in Europe . HistoloTwo major pathways of malignant transformation to penile cancer have been described; one HPV-driven and the other derived from inflammatory skin diseases such as lichen sclerosus (LS) and lichen planus (LP) . Other rHPV has been detected in approximately 50% of invasive penile cancers but with a large span between 24 and 89%, most likely due to different types of tumors and various methods for HPV analysis –13. HPV Viral activity of HPV, by the use of HPV mRNA expression is well studied in cervical cancer , 20, butTo the best of our knowledge no study has been published analyzing HPV among biopsied fresh tissue from both penile cancer and from age-matched penile non-malignant samples.The primary aim of this study was to determine the prevalence of different HPV types in fresh tissue of invasive penile cancer and 10 mm adjacent to the tumor, and to compare that to corresponding HPV prevalence in fresh biopsies from age-matched non-malignant penile controls. A secondary aim was to conduct a blinded review of subtype of penile cancer and correlate penile cancer subtype to the prevalence of HPV. Furthermore, we aimed to determine copy numbers, status of integration and viral activity among HPV16-positive samples inside the tumor and 10 mm adjacent to the tumor.The study was approved by the ethical board in Lund with diary number 2015/907. Inclusion criteria were men over 18 years, having penile cancer surgery at Skane University Hospital (SUH) in Sweden between 22nd of June 2015 and 10th of August 2021. SUH is one of two national referral centers for penile cancer. Excised tumor tissues were transported fresh to the Department of Pathology, SUH and a 3 mm single-use punch biopsy was taken from the tumor and submerged in 1 mL RNAlater (Ambion). Another 3 mm single-use punch biopsy was taken 10 mm outside the macroscopic margins of the tumor and submerged in 1 mL RNAlater (Ambion).Controls comprised age-matched men over 18 years, circumcised for non-malignant reasons at urological departments in Skane. At surgery, a biopsy of approximately 5 mm was taken from excised penile skin, and submerged in 1 mL RNAlater (Ambion) as described .The biopsies were transported to the Department of Microbiology where they were transferred to 1 mL GITS-solution  and 1% mercaptoethanol) and incubated at room temperature overnight. Then DNA was extracted with the Total NA-kit  using MagNA Pure LC (200 μL input and 100 μL output). Sample adequacy was assessed by testing 5 μL of the sample for the human beta globin gene with a real-time PCR . SimultaThe integration of HPV-DNA into the human genome most frequently disrupts the E2 gene . As a suHistopathological diagnosis and classification of the subtype of penile cancer was retrospectively reviewed by two experienced pathologists subspecialized in uropathology and participating in the national multidisciplinary team conference of penile cancer in Sweden. A recent study showed that pathologists who have experience of penile cancer diagnostics have a good concordance in identifying HPV-related and non-HPV-related histological subtypes of penile cancer .The glass slides were retrieved from the pathology archive, and one representative slide of the tumor was chosen for each case in hematoxylin-eosin (H&E) stain. Assessment of histological subtype and histological grade was carried out using glass slides (106 cases) and high-resolution digital slides 40 cases). The slides were scanned using NanoZoomer S360 (S60 for large histologic sections) by Hamamatsu, and Sectra IDS7 software. The pathologists were blinded to the results of the HPV detection by PCR in the tumor material to avoid any bias in tumor assessment. The histological subtypes were determined according to the WHO criteria in “Classification of tumors of the urinary system” and the International Society of Urological Pathology recommendations (2016) 0 cases. . For claCircumcised skin was subjected to routine histopathological examination at the Department of Pathology at SUH. All specimens were stained with haematoxylin and eosin. All diagnoses were performed by the expert team of six uro-pathologists belonging to the Swedish national Penile Cancer Center in Malmö.All cases and controls completed a questionnaire, regarding medical history, medication, smoking habits, number of sexual partners and former symptoms and procedures performed on the penis . Having previously had a punch biopsy taken for histopathologic evaluation was more common among the penile cancer cases (20.5%) than the controls (2%), (p < 0.001). Previous surgery on the penis was more common among the penile cancer cases (31.5%) than among the controls (11.9%), (p = 0.001). Having had a diagnosis of penile cancer before was only seen in the penile cancer cases (11.6%) and not among the controls 0%.The study included 135 men with penile cancer, with a median age of 72 years (interquartile range 66–79 years)  (Table HPV was detected in 38.5% 52/135) of penile cancers and in 11.4% (12/105) of the controls  of the samples. HR HPV types were detected in 25.8% (34/132) of the samples, with HPV16 present in 17.4% (23/132). Only LR HPV types were seen in 4.5% (6/132) of the samples. All cases of HPV occurrence adjacent to the tumor also had at least one HPV type identical to that in the tumor, except for one case where HPV6 was adjacent to the tumor but HPV-negative in the tumor. In two samples 10 mm adjacent to the tumor, the HPV analysis was not adequate, due to no detectable human DNA in the sample. In one sample 10 mm adjacent to the tumor, the analysis was not performed.In all HPV16-positive cases, the E2/E7 ratio showed HPV16 to be episomal in 59.5% (22/37) of the tumors and in 69.6% (16/23) adjacent to the tumors. HPV was integrated in 27% (10/37) and 13% (3/23) in the tumor and adjacent to the tumor, respectively. Mixed forms of HPV16 were seen in 13.5% (5/37) in the tumor and in 4.3% (1/23) adjacent to the tumor. A weak E7 signal only was seen in one case 10 mm adjacent to the tumor. Viral activity shown by HPV16 mRNA expression was detected in 86.5% (32/37) of the tumors, with a mean mRNA expression of 15.1 (median 8.9) HPV16 mRNA copies/HPV16 DNA copy. Adjacent to the tumor, 21.7% (5/23) of the samples showed viral activity, with a mean mRNA expression of 73.1 (median 7.4) HPV16 mRNA copies/HPV16 DNA copy. In the HPV16-positive tumors the mean viral copy/cell was 74.4  and in the HPV16-positive adjacent tissue it was 1.6  (Supplementary p < 0.001). The subtype of penile cancer where HPV was second most common was in warty subtypes, where HPV was seen in 88.9% (8/9) compared to in 34.9% (44/126) of other subtypes (p = 0.002). Warty-basaloid subtypes of penile cancer were HPV-positive in 85.7% (18/21) compared to 29.8% (34/114) in other subtypes (p < 0.001) with an increased adjusted OR for HPV of 9.0 . In the usual subtype of penile cancer, HPV was found in 13.6% (11/81) (p < 0.001). Decreased risk for HPV was seen in the usual subtype of penile cancer with an adjusted OR of 0.03 .Classification of subtypes of penile cancer showed that the usual type was seen in 60% 81/135) of cases  and LP in 10.5% (11/105). Lichenoid dermatitis was seen in 7.6% (8/105). In LS, HPV was seen in 11.1% (7/63) and in 37.5% (3/8) of lichenoid dermatitis, but HPV was not found in LP  than in non-malignant controls (11.4%), and that HR HPV was seven times more common in invasive penile cancer (35.6%) than in non-malignant controls (4.8%). HPV16 was the predominant HPV type.Only a few other studies have searched for the presence of HPV in fresh penile tissue. Martins et al. found HPV in 89% (49/55) of penile cancer cases, which was substantially higher than our HPV prevalence (38.5%) among invasive penile cancer cases . In compIn our study, HPV16 was found to be the predominant HPV type, in agreement with other studies of HPV in penile cancer .p = 0.0573). Furthermore, our finding of a higher prevalence of HPV16 viral activity in penile cancer cases (86.5%), than in samples taken adjacent to the tumor (21.7%), suggests that HPV is more active in the tumor and probably has a role in the malignant transformation process.In the present study we analyzed HPV also outside of the tumor in order find out possible differences between tumors and adjacent healthy tissue. Interestingly, HPV was relatively frequently detected (30.3%) in the tissue adjacent to the tumors. However, there was a tendency of a higher proportion of HPV16 positive tumors compared to that of the adjacent tissue . Next-generation sequencing appears to yield a higher HPV integration rate than the E2/E7 ratio method used in our study, which is affected by limitations such as decreased detection of HPV integration when a large excess (at least 10-fold) of episomal HPV16 is simultaneously present [The observed mean HPV16 viral load (74.4 virus copies per cell) in the tumor was similar to that of Heideman et al., who found a mean HPV16 load of 72 copies per cell . Other s present .In our study, the HPV prevalence was highest in basaloid subtypes of penile cancer (100%), followed by warty subtypes (88.9%) and warty-basaloid subtypes (85.7%). This is in alignment, but slightly higher than former studies where a meta-analysis showed the pooled prevalence of HPV to be 84% in basaloid penile cancers, 58.7% in warty subtypes and 75.7% in warty-basaloid subtypes .The strengths of our study were that HPV was analyzed in fresh biopsies both in and adjacent to the tumor, and that biopsied age-matched controls were used. Another strength was that not only the presence of HPV16 DNA was studied; also the viral activity was investigated in HPV16-positive samples, both in and adjacent to the tumor. A further strength is that histological subtypes of penile tumors were reviewed by two experienced uropathologists, blinded to each other.One limitation was that compared to the controls, who completed the questionnaire at the hospital before the treatment, there was a relatively high proportion of missing questionnaires Table  from theKnowledge of HPV as a strong risk factor (adjusted OR 12.8) for invasive penile cancer is important in the work of preventing penile cancer. The nine-valent HPV vaccine targets the HPV types HPV6, 11, 16, 18, 31, 33, 45, 52 and 58, covering the majority of HR HPV types found here, which leads to the conclusion that vaccination of boys is important for preventing development of penile cancer. Knowledge about viral activity in penile cancer tumors, further indicates the important role of HR HPV in the malignant transformation.Additional file 1.Additional file 2.Additional file 3.