Certain types of human papillomavirus (HPV) induce long-lasting infections that cause cervical cancer. This study evaluated the prevalence of HPV infections and the distribution of their genotypes among clinic patients and healthy women in Beijing, China.Cervical specimens were collected from 12,100 patients and 1176 subjects who underwent physical examinations at Dongzhimen Hospital, Beijing University of Chinese Medicine, between March 2016 and September 2020. HPV genotyping was performed using commercial kits designed to detect 15 high-risk and 2 low-risk HPV genotypes.There was a higher overall prevalence of HPV among the clinic patients (21.0%) than among the healthy women (11.9%). The most common HPV genotypes among the patients were: HPV-52 (5.4%), HPV-16 (3.4%), HPV-58 (3.2%), HPV-51 (2.6%), HPV-39 (2.0%), HPV-56 (2.0%), and HPV-66 (2.0%). Among the healthy women: HPV-52 (3.0%), HPV-51 (1.8%), HPV-58 (1.6%), HPV-66 (1.5%), HPV-16 (1.2%), HPV-56 (1.2%), and HPV-18 (1.1%). Multiple HPVs were detected in 29.1% of the gynecological outpatients and in 23.6% of the women receiving physical examinations. The most common pairs of HPV types detected were HPV-52 and HPV-16 in the clinic patients, and HPV-52 and HPV-56 in the healthy women. Age-specific HPV positivity and peak prevalence were observed among the individuals in both groups for women aged less than 25 years and those between 61 and 65 years of age.Our results provide current estimates of HPV prevalence and genotypes in the Beijing region. The epidemiological characteristics observed also provide a reference for the development of cervical cancer screening strategies and selection of HPV vaccine antigen targets for this region. A comparison of these HPV prevalence data with those from other regions suggests that regional vaccines may be an important direction for future research. Cervical cancer (CC) is a highly preventable disease, yet remains the fourth most frequently diagnosed cancer, and fourth leading cause of cancer-related mortality, among women worldwide . In 2020Persistent infection with human papillomavirus (HPV) is the main etiologic factor for CC . There aOver the last few decades, trends in CC incidence and mortality have been observed to vary in different countries . SimilarFor this cross-sectional and retrospective study, a total of 13,276 patients were enrolled from Dongzhimen Hospital Beijing University of Chinese Medicine  between March 2016 and September 2020. These patients visited the hospital for various reasons, including: leukorrhagia, menstrual disorders, pelvic inflammation, vaginitis, cervicitis, undiagnosed abdominal pain, genital warts, cervical intraepithelial neoplasia, and for physical examination. This study was approved by the Ethics Committee of the Dongzhimen Hospital Beijing University of Chinese Medicine (Grant No.: 2022DZMEC-094-01).All of the subjects were asked to refrain from sexual activity and avoid washing their genital area for 48 h prior to sample collection. Cervical samples were obtained by using a cervical brush and then were placed in a preservative buffer solution. The samples were stored at 2–8 °C for no more than 7 days. DNA was isolated using a nucleic acid extraction reagent  in compliance with the manufacturer’s instructions.4 copies/mL and 1 × 103 copies/mL, respectively. To quantify the amount of HPV in the samples and to avoid false-negative test results, internal quality control was performed alongside the samples. Quality controls were included in all of the experiments, including DNA amplification and genotyping, with positive and negative controls included in the PCR assays.For HPV detection and genotyping, two commercial HPV Genotyping Kits  were used to detect fifteen high risk (HR)-HPV types  and a combination of two low-risk (LR)-HPV types (6/11) with use of TaqMan real-time fluorescent quantitative polymerase chain reaction (PCR) using a Slan-96P Real-time PCR System assay . Samples were reported to be HPV positive if any of the 17 HPV DNA types were detected. In brief, each 4-μL sample of DNA was mixed with 35.6 μL of PCR Mix and 0.4 μL DNA Taq polymerase. Amplification of HR-HPV types included the following steps: 94 °C for 2 min, followed by 40 amplification cycles . Single-point fluorescence was detected at 62 °C. To amplify LR-HPV types, the following steps were programmed: 37 °C for 2 min, 94 °C for 2 min, and a total of 40 amplification cycles . Single-point fluorescence was detected at 60 °C. The detection limits of the assays were 1 × 10P values less than 0.05 were considered statistically significant. The test criterion was α = 0.05 and was used throughout the study. If one or more of the expected numbers was ≤ 5, or the P value was close to 0.05, Fisher’s exact test was applied.All of the statistical analyses performed for this study used SPSS 21.0 for Windows. Prevalence of HPV infection, genotype distribution, and both single and multiple HPV infections were analyzed individually. Prevalence of HPV infections with single- versus multiple-HPV genotypes in different groups were compared using the Chi-square test. Binomial distribution analysis was used to calculate 95% confidence interval (95%CI). Factors associated with HPV prevalence were evaluated and reported as odds ratios (ORs) and 95% CI. Two-sided A total of 13,276 subjects were included in this study with a median age of 39 years (range 18–85). Among these patients, 12,100 experienced various gynecopathy conditions, including: leukorrhagia, menstrual disorders, pelvic inflammation, vaginitis, cervicitis, undiagnosed abdominal pain, genital warts, or cervical intraepithelial neoplasia. In addition, 1176 healthy women were included who underwent physical examinations.P < 0.05) between the prevalences of: HPV-52 , HPV-16 , HPV-58 , HPV-39 , HPV-6/11 , HPV-68 , and HPV-82 . The prevalence of certain HPV types  was higher among patients than among the controls, although these differences were not statistically significant. The pooled OR for the patients with mixed gynecopathy associated with the presence of any HPV type was 1.961 (95%CI 1.635–2.352).A total of 2675 subjects  were positive for HPV DNA. According to group, the prevalence of HPV DNA was 21.0% (95%CI 20.2–21.7) among the clinic patients and 11.9% (95%CI 10.0–13.9) among the healthy controls . The 26–30 years age group showed a marked decrease in HPV prevalence for both groups  that was followed by a gradual decrease in prevalence in both groups. For the patient group, this included a decrease to 17.8% in the 46–50 years group, an increase to 20.4% in the 61–65 years group, and then a decrease to 15.0% in the ≥ 66 years group. Meanwhile, in the healthy women group, a gradual decrease led to a low point at 5.1% for the 56–60 years group, followed by a second smaller peak in HPV prevalence (14.3%) in the 61–65 years age group, and then 2.7% for the ≥ 66 years group.To explore whether the prevalence of HPV exhibits an age-dependent trend in our cohort, the cases were stratified into ten groups according to age Fig. . In bothThis cross-sectional study reports the prevalence of HPV infections and genotype distributions among two groups of women, clinic patients and healthy women, between 2016 and 2020 in Beijing, China. These epidemiological data support the availability of HPV vaccines in this region. Previous studies have reported that the prevalence of HPV varies considerably in China, from 6.2 to 50.64% , 15. It Some multicenter studies have shown that geographical variations contribute to the prevalence and distribution of HPV in mainland China , 21. ForCompared with studies based on general populations, the prevalence of HPV among clinic-based patients varies greatly . For exaConsistent with the results of previous studies , 19, we It remains unclear whether competitive or cooperative interactions exist among co-infecting HPV genotypes. Infection by multiple HPV types has been associated with a greater risk of CC than infection by a single HPV type . An assoBased on the results of the current study, it is observed that cervical HPV infections exhibit a bimodal distribution with age. A similar overall trend was observed for age-specific HPV positivity and peak prevalence among individuals aged less than 25 years and aged 61–65 years among gynecological patients and healthy females. The first peak corresponds with age at first sexual intercourse. Number of births is also recognized as a risk factor. In contrast, incidence of cervical HPV infections has previously been reported to have similar persistence rates independent of age , 26. A pCC can be effectively controlled with organized screening and vaccination programs. HPV causes CC in three necessary steps: acquisition, persistence linked to development of precancer, and invasion . VaccinaRegional variations in the distribution of certain HPV types should be taken into account when creating vaccines that are tailored to different geographic regions . The 9vHThere were limitations associated with the present study. First, due to the retrospective design of this study which relied on a medical record system, each observed clinical diagnosis could not be accurately stratified due to its complexity and non-uniformity. Thus, a prospective cohort study is needed to evaluate the relationship between HPV genotype and disease type, especially cervical intraepithelial neoplasia. Second, according to the cross-sectional design of this study, observed associations cannot be interpreted as temporally linked with infection . Third, In conclusion, the present results characterize the epidemiological characteristics of HPV infections among patients and the general population in Beijing. The HPV genotypes detected differ from the current 9-valent vaccine HPV types associated with this area. It has been hypothesized that HPV types 51, 56, 53, and 39 may be better adapted to individuals of Chinese ancestry. In the future, the development of HPV vaccines according to region may be an effective strategy.


Among POIA, younger individuals (vs. older), single individuals (vs. married), those with high English proficiency (vs. low English proficiency), those with health insurance and those born in the USA (vs. those born outside the USA) were more likely to obtain HPV vaccination . Public health measures promoting HPV vaccination among POIA immigrants may substantially improve vaccination rates among this population.Approximately one-quarter of annual global cervical cancer deaths occur in India, possibly due to cultural norms promoting vaccine hesitancy. We sought to determine whether people of Indian ancestry (POIA) in the USA exhibit disproportionately lower human papilloma virus (HPV) vaccination rates than the rest of the US population. We utilised the 2018 National Health Interview Survey to compare HPV vaccine initiation and completion rates between POIA and the general US population and determined factors correlating with HPV vaccine uptake among POIA. Compared to other racial groups, POIA had a significantly lower rate of HPV vaccination (8.18%  POIA had a significantly lower rate of HPV vaccine initiation than other races , although the duration of US residence did not significantly affect vaccine uptake in the POIA population (P = 0.5017). While proficiency in the English language was a strong predictor of HPV vaccine uptake among POIA (P = 0.0286) and all respondents who got at least one dose of the HPV vaccine were very proficient in English, education level did not affect HPV vaccine initiation rates (P = 0.2310). Similarly, while having health insurance was a key factor in receipt of the HPV vaccine (P = 0.0204), and all who claimed to have received at least one dose were insured, level of family income did not play role in HPV vaccine initiation among POIA (P = 0.9072). On multivariable analysis, being born in the USA and having insurance were independent predictors of HPV vaccine uptake among POIA  [et al., evaluating data from the 2015 NHIS, found that Asian women had significantly lower odds of completing the HPV vaccination series when compared to White women [P = 0.7658), although a difference is seen in initiation rates (P = 0.0091), echoing our findings among POIA. These data suggest that racial disparities in HPV vaccination rates that may have existed in the past may have dissipated in recent years.Regarding HPV vaccine series completion rates, we found that POIA between the ages of 18 and 64 who initiate HPV vaccination are equally likely to complete the three-dose series compared to other major racial groups in the USA. These data echo findings from the 2018 National Immunization Teen Survey. This work found that Asian American teenagers (aged 13–17) completed the HPV vaccine series at roughly the same rate as White, Black and Hispanic teenagers (53.1% ctively) . Intereste women . While tOur data should be viewed in light of its inherent limitations. As a survey study, it is limited by the questions fielded and is subject to recall bias. Furthermore, we recognise that in 2016, guidelines changed to allow for two doses of HPV instead of three, and our study defined completion of the series using the older definition which would have applied to the majority of people in our cohort. We recognise, however, there was some variability in the guidelines and there was no way for our study to control for this.Despite these limitations, our study has a number of strengths. Given the design of the NHIS which is representative of the entire US population, this dataset gives us key insights into factors affecting HPV vaccination rates among POIA. Importantly, while our data demonstrate that POIA are less likely to receive an HPV vaccine than other racial groups, they are just as likely to complete their vaccination schedule once started. As such, targeted public health measures increasing awareness and acceptance of HPV vaccination among POIA may be particularly effective in increasing immunity to HPV among this community. Furthermore, our finding that those born in the USA were more likely to get vaccinated provides hope that, with acculturation and growing acceptance of the vaccine among those born here, HPV vaccination will be higher among POIA in the USA than it is in their ancestral homeland. Among those POIA who continue to migrate to the USA, recognition of disparities in HPV vaccine uptake by clinicians is crucial to increasing HPV vaccination rates within this population. Given recent policy changes allowing for HPV vaccine administration in both men and women up to 45 years old, clinicians should enquire about and discuss HPV vaccination with their migrant POIA patients to promote shared decision making and increased HPV vaccine uptake in this population. Finally, our data highlight insurance as being a key factor that can enhance HPV vaccination rates, suggesting that making HPV vaccines available regardless of insurance status may be a key public health measure to improve vaccination rates and ultimately reduce preventable malignancies associated with HPV – not just for POIA, but for the population as a whole.


Lactobacillus spp. and a higher pH. In cervical dysplasia progression, a strong association is found with new bacteria, and with the deregulation of pathways and hyperexpression of cytokines leading to chronic inflammation. Conclusions: in HPV progression, there is a strong correlation between potential biomarkers, such as Sneathia and Delftia found in community state types IV and II, and chronic inflammation with cytokine overexpression. Better analysis of these factors could be of use in the prevention of the progression of the disease and, eventually, in new therapeutic strategies.Background: in recent years, many studies were carried out to explore the role of vaginal microbiota in HPV infections and cervical intraepithelial neoplasia (CIN) progression. The aim of this study was to conduct a review of the literature to analyze the interaction between the vaginal microbiota, the CIN, and the immunological response. Methods: we performed a literature search, considering papers published between November 2015 and September 2021. Results: despite significant evidence suggesting a role of vaginal microbiota in the pathogenesis of HPV-related lesions, some studies still struggle to demonstrate this correlation. However, the vaginal microbiota of HPV-positive women shows an increased diversity, combined with a reduced relative abundance of  Human papillomavirus (HPV) infection is the most frequent sexually transmitted disease, and is the main significant cause of the development of cervical precancerous lesions (dysplasia) and cervical cancer ,2. It isThere are two peaks of prevalence of the infection: one between 15 and 26 years old, and another one around 45 years old. It is demonstrated that the incidence decreases with age, while persistence increases ,8.Known factors associated with a longer persistence of HPV infection and progression to cervical cancer are: a high number of sexual partners, early initiation of sexual activity, the concomitant presence of sexually transmitted infections, impaired immunity, genetic background, use of oral contraceptives, and smoking ,11,12,13HPV has a particular tropism for the transitional zone of the cervix, which lays between the paved epithelium of the esocervix and the cylindrical epithelium of the endocervix. This zone represents, from a biological standpoint, a vulnerable area because it permits easier access to the basal layer that is a target of some oncogenic agents, such as HPV . The mosRecent publications show that immune status is significantly correlated with viral infection, as well as the vaginal microbiota that is involved in the modulation of the immune system of the female lower genital tract ,18. FurtLactobacillus-dominated (LD), with four predominant species: Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii [Gardnerella, Atopobium, Mobiluncus, Prevotella, Streptococcus, Staphylococcus, Ureaplasma, and Megasphaera) and commensal microorganisms, such as the opportunistic fungus, Candida albicans [Concerning women in their reproductive age, the vaginal normal microbiota is jensenii ,20. Thesalbicans .Ethnicity, genetic disposition, lifestyle and diet, hygiene status, infections, antibiotic use, sexual activity, and estrogen are known to be important risk factors influencing the proper functionality and characteristics of the vaginal microbiome ,23.Lactobacillus spp. and the predominance of microaerophilic bacteria  [Lactobacillus-dominant (NLD) microbiota of BV and a higher risk of HPV acquisition and its decreased clearance, leading to the development of precancerous dysplasia and progression to cervical cancer [Lactobacillus spp. with higher rates of dysbiotic BV in patients with precancerous and cancerous lesions is reported [Bacterial vaginosis (BV) represents a dysbiosis of the vaginal microenvironment and microbiome biofilms, correlating with the reduction in ophilus) ,25,26,27l cancer ,30,31,32reported ,30,31.The aim of this review is to analyze the interaction between HPV-related cervical intraepithelial neoplasia, vaginal microbiota, and immunological response.Lactobacillus non-dominant vaginal microbiota (VMB) and HPV infection; in particular, four of the papers analyze the vaginal community state types (CSTs) and their changes associated with HPV infection. In five studies, microbes correlated with changes in VMB are detected, and in two papers microbe markers of HPV infection/LSIL /HSIL /ICC  are identified. Three studies also describe the importance of the increase in vaginal pH in changing VMB, leading to a greater susceptibility to HPV infection. One study attempts to characterize local immune signatures in patients at various stages of cervical carcinogenesis by measuring the levels of some cytokines, assessing that patients with invasive carcinoma, but not precancerous neoplasm, display increased levels of genital immune mediators, leading to chronic inflammation.We found 11 records from the bibliographic search. After excluding three works that were not pertinent with the aim of the research, we accurately examined the other eight papers. We then performed the additional selection, accordingly to the PRISMA diagram, which provided us with a set of six papers. Of the six works under consideration, all investigate the interaction between a Techniques used for HPV detection are commercially available DNA tests, such as the Roche Linear Array HPV Genotyping test, Hybribio HPV typing kit, and Digene Hybrid Capture DNA test, or through an assay of polymerase chain reaction (PCR) using specific primers (Illumina MiSeq).For the study of vaginal microbiota, the initial assessment includes diagnostic methods such as microscopic evaluation, Gram stain test, and microbiological cultures. In some cases, DNA is extracted through DNA isolating kit.Among the samples, five out of six are from the vagina, and two of six from the cervix. Only in one case is a double sample taken, one from the vagina and one from the cervix, and the sample from the cervix is diluted. All the other samples are not diluted.Further characteristics of the studies analyzed are summarized in Lactobacillus-dominant microbiota, when compared to Hispanic and Black women [The structure of vaginal microbiota (VMB) is controlled by numerous factors. The most intrinsic factor is ethnicity, which is significantly associated with modifications in vaginal composition; Caucasian and Asian women display a greater prevalence of ck women . These dck women . Female ck women ,39, actick women . Other eck women  and receck women .Ravel and colleagues  sought tFive major groups of microbial communities were retrieved from the analysis, in accordance with previous data in the literature on microbial biofilm specimens of vaginal diversity . The fivLactobacillus, which constitute >50% of all sequences obtained, represent the most important part of vaginal communities (73%). Lactobacillus crispatus represents the main agent of vaginal community I (26.2% of the samples), while Lactobacillus gasseri represents the main agent of group II (6.3%), and Lactobacillus iners of group III (34.1%), whereas group V (5.3%) is dominated by Lactobacillus jensenii. The remaining communities, observed in 27% of the samples, constitute a large heterogeneous group (IV), characterized by higher concentrations of anaerobic bacteria, including Dialister, Prevotella, Atopobium, Gardnerella, Megasphaera, Peptoniphilus, Sneathia, Eggerthella, Aerococcus, Finegoldia, and Mobiluncus.Unlike other anatomical sites, one or more species of Lactobacillus spp. or Lactobacillus crispatus (group I) species are represented, a slightly higher pH, between 4.4 (group III) and 5.0 (group II) is exhibited, suggesting that these communities together may not produce levels of lactic acid equal to those reported for group I or, alternatively, they may have different buffering capabilities.Interestingly, in the communities where the Several studies indicate a correlation between HPV infection and dysbiotic vaginal microbiota. In fact, there is evidence to suggest that persistence of HPV is more probable in those with altered microbiota.A multicentric cross-sectional study, lead by Laniewski et al., on 100 premenopausal women, shows that, similar to larger epidemiological studies, HPV16 is the most prevalent (65% of HPV-positive samples). Other frequent types identified are HPV45, HPV58, and HPV31. Co-infection with multiple HPV genotypes is uncommon, as well as the decreased severity of cervical dysplasia, consistent with previous data from the literature .Lactobacillus decreases, while other bacteria, such as anaerobes Prevotella and Megasphaera, increase significantly. Accordingly, in Laniewski et al., patients with advanced cervical dysplasia and invasive carcinoma display a significantly higher rate of diverse non-Lactobacillus-dominant (NLD) vaginal microbiota (VMB) (p = 0.04). In particular, Lactobacillus iners is reported to increase in HPV-positive women and women with LGD and HGD, being associated with a higher tendency to shift to dysbiotic VMB. In addition, Lactobacillus iners may be connected with a higher risk of cervical dysplasia development [As for the vaginal microbiota, Sikao Wu et al. demonstrate that, with the progression of cervical dysplasia, the composition of the vaginal microbiota microenvironment, has corresponding changes. In particular, in the HSIL and CA groups  elopment .Lactobacillus crispatus-dominated, CST II: Lactobacillus gasseri-dominated, CST III: Lactobacillus iners-dominated, CST IV: Lactobacillus-depleted, and CST V: Lactobacillus jensenii-dominated.Most of the papers analyzed are based on the vaginal microbiome community state types (CSTs) classification  is lower with the increase in the severity of the disease. In the LSIL women, a significant over-expression of Lactobacillus jensenii (p < 0.01) and Lactobacillus coleohominis (p < 0.05) is observed.In Mitra’s study, by examining the microbiota of 169 women, they identify 5 major clusters that show a bacterial community structure superimposable on previous reported vaginal CSTs, finding that the rate of a CST IV vaginal microbiome doubles in patients with low-grade squamous intraepithelial lesions (LSIL), triples in patients with high-grade squamous intraepithelial lesions (HSIL), and quadruples in patients with invasive cancer. Conversely, the frequency of CST I (Lactobacillus spp. depletion (CST IV), are predisposed to have a persistent HPV infection, and a long-term positivity to HPV genotyping.A longitudinal study by Brotman et al. suggests that women with a highly diverse vaginal microbiome, with Lactobacillus, even if it remains the most abundant genre in all groups. This results are in agreement with previous studies [Chen and colleagues also observe that, during HPV infection, there is an increase in vaginal bacterial richness and diversity, despite the status of CINs, causing the shift from vaginal bacterial community structure III to CST IV, and a decrease in the percentage of  studies ,46,47,48p < 0.001), mainly in CST II. When a depletion in Lactobacillus is observed in the HSIL group, enrichment in the bacteria associated with bacterial vaginosis is found, even though no statistical connection is demonstrated between CSTs and grades of squamous intra-epithelial neoplasia (p = 0.49).Conversely, in a study of Mengying Wu et al., cervical intra-epithelial neoplasia (CIN) switches the vaginal bacterial community structure from CST IV to CST II, with the LSIL group dominated by CST I . Microbiota heterogeneity is found to be more marked in CST II and CST IV  to have a connection with HPV infection and/or cervical neoplasia , along with the common bacteria , most of which are usually associated with BV. However, they also discover an association, in HPV-positive women, of some bacterial taxa with the lesser-known aerobic vaginitis microbes (AV) , or other dysbioses .Concerning specific bacterial taxa, Laniewski et al. find novel bacterial genera .Consistent with the study by Laniewski et al., Plisko and colleagues reveal a relation between AV and cervical dysplastic lesions, showing that AV, but not BV, is found to be significantly more often associated with abnormal pap smear cytology. On the other hand, BV is linked with CIN1, but not with CIN2+ with low-grade lesions being promoted by both HR and LR-HPVs. Furthermore, for the first time, it is observed that AV-associated microbiota changes are also linked to histological pre-invasive cervical lesions, and that there is a positive correlation between the severity of AV and that of the cervical HPV-induced lesions: moderate to severe AV is significantly associated with CIN2+, while BV is not , meaning that there is no significant difference in the vaginal microbiota of women with or without intraepithelial lesions or malignancy. For this reason, they speculate that the more severe the lesion, the greater the vaginal diversity, but this effect is not reported to be statistically significant either, which is in agreement with the results reported by Mitra et al. Their findings suggest that vaginal microbial diversity is associated with the advancing severity of CIN, but does not attain significance. The same results are shown by Huang et al. [Despite many pieces of evidence suggesting a role of vaginal microbiota in the pathogenesis of HPV-related lesions, some studies still struggle to show a correlation. For example, in Mengying Wu’s study, CSTs and grades of squamous intraepithelial neoplasia have no statistical connection p = 0., meaningAbove all, we should remember that persistent infection with HR-HPV genotypes represents the most important risk factor for cervical cancer progression and ICC development .Lactobacillus is the most dominant bacterial genus in the vagina, and it maintains the stability of the vaginal microenvironment, preventing the colonization of bacterial vaginosis (BV)-associated bacteria, through maintenance of a low pH and the production of bacteriostatic and bactericidal metabolites, such as lactic acid, H2O2, and biosurfactants and bacteriocins. This is vital for the preservation of the cervical epithelial barrier that inhibits the entry of pathogens, such as HPV, to the basal keratinocytes [p < 0.0001). Other than vaginal microbiota, increased vaginal pH is also correlated with infections, including sexually transmitted ones, and, therefore, could also be associated with sexual activity.While in other parts of the human body having a diversification of microbes is considered a sign of health, in the vagina, a particularly diverse microbiota is often associated with a state of dysbiosis and disease. Under normal circumstances, inocytes . ConsistHowever, there are many other conditions that could modify vaginal pH, such as the use of different vaginal products, hygiene habits , low estrogen levels, recent sexual activity, and the presence of sperm or blood. All of these conditions may be taken into consideration.Recent studies show that CSTs III and IV, characterized by high diversity and low Lactobacilli dominance, are frequently associated with HPV infection and the development of cervical disease . In thes2O2, lactic acid, and other products is reduced because of fewer producers, and the barrier of cervical and vaginal epithelial mucosa becomes more fragile, the microflora stimulates the production of pro-inflammatory cytokines, leading to the disruption of the epithelial intimal barrier [While the level of H barrier .Lactobacillus-dominant VMB leading to chronic inflammation and HPV persistence and a progression in infection, with the possible development of cervical cancer [HPV infection itself is thought to maintain the altered acidic environment of the vagina, and it might lead to changes in the lower genital tract microbiota by inducing host mucosal immune response and genital inflammation. As a result, the vaginal environment is subverted, promoting the abnormal adhesion of HPV in the vagina, causing a local microecological imbalance and destroying the local immune function of the cervix, while simultaneously increasing the adhesion, invasion, and colonization of an abnormal microenvironment. This forms a vicious cycle in lower genital tract tissues, with elevated vaginal pH and non-l cancer ,54.As for pathogenic mechanisms, Plisko et al. also hypothesize that the inflammatory characteristics of AV and HPV-induced cervical dysplastic lesions are crucial for the progression of cervical lesions in invasive cancer. Indeed, one of the critical mechanisms of HPV carcinogenesis is inflammation, shown by increased inflammatory interleukins (IL) in subjects with progressive CIN . Also, iLactobacillus competes with pathogens, and inhibits their growth by secreting lactic acid, bacteriocins, and H2O2 and, furthermore, by activating the complement system, triggering a local immune response. As mentioned earlier, when pathogens colonize the vaginal epithelium, this leads to a waterfall effect with an elevated pH and the adhesion of pathogens, such as HPV, to the epithelium, causing disruption and the activation of a host immune response with chronic genital inflammation.In VMB there is a complex balance between microorganisms. Many studies investigate the role of the inflammatory response in patients with cervical intraepithelial neoplasia or cervical cancer. In most of them, a chronic inflammatory response with the deregulation of pathways and the overexpression of cytokines, is found only in CIN2-3 or ICC. In accordance with this, the HR-HPVs, which are proven to be the main motive of cervical cancer, down-regulate cellular tumor suppressors (pRB and p53) and epidermal growth factor receptors (EGFR), through HPV oncoproteins  integrating into the cellular DNA and stimulating some inflammation pathways. E6 and E7 are also demonstrated to have an association with elevated levels of NF-κB [p = 0.02), HPV+ subjects , and women with high-grade cervical dysplasia . ICC, but not low-grade or high-grade cervical intraepithelial neoplasia, shows a significant increase in the inflammatory score system contrary to HPV− subjects (p < 0.05) [Laniewski et al. evaluate the expression of specific mediators of the immune system in cervico-vaginal washings; the authors report that pro-inflammatory and chemotactic cytokines such as IL-36γ, TNFα, RANTES, MIP-1α, MIP-1β, RANTES, and IP-10; hematopoietic cytokines, as well as Flt-3L and GM-CSF; cytokines of adaptive immunity such as IL-2, IL-4, and sCD40L; and an anti-inflammatory cytokine IL-10, are significantly expressed in ICC. The authors then underline that the genital inflammatory pathway is significantly activated in ICC patients (60%) compared to HPV− patients  . As for Sneathia and Fusobacterium spp. either contribute to shift Th1 immunity to Th2, increasing the levels of IL-4 and TGF-β1, or directly intervene in the E-cadherin/β-catenin signaling pathway in cervical HPV-infected cells, leading to the activation of the transcription factor NF-kB in the nuclei [Likewise, Audirac et al. report that some agents of the cervical microbiota communities may cause a modification of cytokine expression in the cervical microenvironment, especially during the progression of CIN into ICC. e nuclei .Consistent with previous studies, there is evidence that HPV-associated cervical lesions  show a cIn order to perform a literature review, we followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines . The medTwo authors (M.F.P. and M.L.V.) independently researched reference lists of recognized manuscripts, aiming to perform an integration of the literature. Interventional, observational, prospective, and retrospective studies were considered. Exclusion criteria were the following: single case reports, book chapters, and conference abstracts. In addition, publications written in a language other than English, and in vitro or in animals experimental trials were excluded. The bibliographic research provided a preliminary group of papers, which were carefully analyzed. Two investigators (M.D. and B.G.) independently evaluated the risk of bias to confirm the validity of the selected papers and overcome possible selection, performance, detection, attrition, and reporting bias, according to the Cochrane Handbook for Systematic Reviews of Interventions ,61,62. FRecent studies in the literature suggests that vaginal microbiota may play a possible role in the onset and progression of cervical intraepithelial neoplasia, in particular high-grade CIN, even if, for some authors, this correlation does not attain significance. Therefore, what is yet to be examined is the interaction between all of the factors that are involved in this relationship, in order examine if it is significantly relevant or not.First of all, the insurgence of CIN, and its progression in high-grade lesions, inevitably requires the presence of previous HPV integration in the cervical tissue, and the activation of oncogenic protein expression. In addition, data in the literature underline that HR-HPVs are the main etiological factors involved in the development and progression of CIN and cervical cancer. Instead, the motivation of why only in some cases HPV infection is cancerous, whereas in other women it is suppressed, may be explained by immunological response system diversity. After infection, in fact, there are many factors involved in the acquisition and persistence of HPV leading to the development of cervical lesions, and immunological response may probably be more significantly responsible in the clearance of HPV infection.Among extrinsic factors to consider are hormonal contraceptives, recent intercourse, hygiene practices, and smoking, while, among intrinsic factors, the most important is ethnicity, which may also mirror in different genetic settings and in the composition of vaginal microbiota biofilms, resulting in the insurgence of vaginal dysbiosis, as BV and the lesser-known AV, which seem correlated with HPV infection and persistence. In consideration of this, additional biological analysis is required to confirm the role of the vaginal microbiota in the pathogenesis of CIN and cervical cancer. In addition, it is important to investigate the cause–effect relationship between them, in order to understand if the changes in the microbiota microenvironment are the cause, or the consequence, of HPV infection.Delftia, that may be used as predictors of the evolution of HPV lesions, and to investigate the pathogenesis of chronic inflammation that leads to cervical cancer. It is already known, in fact, that the inflammatory response is associated with CIN2+ lesions. Finding predisposing factors in HPV+/CIN1 lesions may be of use in preventing the progression of the disease.Furthermore, it could be crucial to find potential biomarkers, such as Sneathia and The information about vaginal microbial composition, biomarkers, and chronic inflammation may grant the opportunity for the development of new therapeutic strategies for CIN.


Squamous cell carcinoma of the external auditory canal (EACSCC) is an uncommon tumor and responsible for no more than 0.2% of all the head and neck malignancies. Although there is remarkable research evidence exhibiting that high-risk human papillomavirus (HPV) accounts for considerable head and neck malignancies, its role in the pathogenesis of EACSCC is yet to be determined.INK4a and Ki‑67.We evaluated 16 patients with EACSCC treated at our department. We employed PCR to assay for high-risk subtypes of HPV. Two pathologists reviewed the histopathological staining via hematoxylin and eosin along with immunohistochemical staining of p16INK4a immunostaining. Besides, 3 (37.5%) individuals were HPV positive as per p16INK4a PCR results. In addition, all of the p16INK4a-positive specimens were diagnosed as moderately differentiated carcinomas.Detection of HPV DNA was done via PCR in 3 (18.75%) patients, and 8 (50%) positive (+) cases were determined via p16INK4a immunostaining is a suspectable approach for diagnosing HPV for EACSCC. In addition, HPV might enhance an elevated proliferation rate in EACSCC, illustrated via expression of Ki-67.Expression of Ki-67 was related to HPV status. This is the first report implicating high-risk HPV in squamous cell carcinoma of the external auditory canal. However, p16 External auditory canal squamous cell carcinoma (EACSCC) is rare, occurring in one to six cases in about 1,000,000 persons per year . The gloEACSCC, manifesting histologically as a progressive squamous dysplasia, emanates from aggregated genetic and epigenetic alterations triggered by the exposure to solar ultraviolet light and inflammatory stimuli of long-standing chronic suppurative otitis media . In addiINK4a IHC (immunohistochemistry). As a cyclin-dependent kinase repressor, the expression of p16INK4a is influenced by HPV-associated oncoproteins as E7 [INK4a in normal cells, gets disrupted via a decrease in the activity of pRb in growing squamous epithelial cells that are expressing HR-HPV E7 [INK4a is utilized as an HPV-linked surrogate biologic signature in numerous study methods since the procedure is well-developed and saves time [INK4a staining for detecting infection with HPV usually does not provide accurate results [INK4a over-expression might also be seen in tubal metaplasia along with atrophic cells and in normal columnar cells from the cervix, resulting in poor specificity [INK4a over-expression does not take place exclusively in HPV-triggered but additionally in an estimated 5–10% of HPV-negative HNSCC [INK4 expression result in diverse interpretations of "p16INK4a-positivity" [INK4a for diagnosing HPV infection in HNSCC.Infection with HPV is frequently detected via p16ns as E7 , 10. TheR-HPV E7 . Hence, ves time , 13. How results , 15. Firve HNSCC , 18. Moritivity" –21. ThusINK4a and Ki-67 in a cell implies epithelial cell transformation that might progress to cancer [INK4a has two diametrically opposed biological roles in cell cycle regulation. Conversely, p16INK4a expression could serve as a pivotal modulator of cellular senescence [INK4a expression can be triggered upon signaling of HPV E7 oncogene [INK4a-positive cells.Ki-67 constitutes a nuclear antigen as well as a biological signature of cellular proliferation, which is expressed in all stages of the cell cycle except G0 . p16 ando cancer . p16INK4nescence . On the oncogene . Thus, pINK4a /Ki-67 as a marker for detecting HPV-induced transformed cells in EACSCC.The objectives of this research work were twofold: first, to investigate the incidence of HPV in EACSCC; second, to explore the clinical performance of p16Sixteen individuals with EACSCC were enrolled in this research work, with a mean age of 57 years, ranging from 1 to 76 years, with a male/female ratio of 10:6. The histopathologic assessment of all samples was confirmed and verified by two board-certified pathologists. The research work was granted approval by the Institutional Ethics and Review Committee of the Faculty of Clinical Medicine, Eye and ENT Hospital, Fudan University, Shanghai, China.INK4a anti-Ki67, was introduced overnight. The slides were inoculated for 10 min with secondary antibody . The immunoperoxidase labeling was done; diaminobenzidine  was utilized as a chromogen for visualizing docking of the antibody. Finally, we stained the sections with Harris's haematoxylin, followed by clearing and mounting.Immunohistochemical analysis was done on five μm-thick paraffin sections of EACSCC and adjacent normal skin tissue. The following deparaffinization with xylene, and subsequent rehydration via 96% ethanol, blocking of the activity of endogenous peroxides was done with 3% hydrogen peroxide for 10 min and rinsed in PBS. Exposing of the antigens was done by boiling the sections in citrate buffer (pH 6.0) for 15 min. After that, the primary antibody, anti-p16INK4a, the presence of nuclear staining with or without cytoplasmic staining was regarded as a positive result. The tumoral p16INK4a expression trends were documented and categorized as per following criteria: (1) diffuse ; (2) focal (numerous p16INK4a-positive cells not harboring criteria for diffuse p16INK4a expression).For p16Ki‑67 expression was stratified into four groups as per the distribution along with the fractions of cells with positive nuclear staining as shown: Score 0, < 10% of the cells; score 1, 10‑29% of the cells; score 2, 30‑69% of the cells; score 3, ≥ 70% epithelial cells .Total genomic DNA was extracted from fresh frozen tissue of all samples using the TIANamp Genomic DNA Kit . HPV detection and genotyping were done using the Human Papillomavirus (HPV) Genotyping Real-Time PCR Kit . The Luminex bead-based genotyping approach allows for the diagnosis of 15 high-risk forms  and six low-risk types , according to the manufacturer’s instructions. The HPV genotypes were categorized as HR‑HPV or LR‑HPV based on the scheme opined by Dunne et al.SPSS software  was used for statistical analysis. Fischer's tests and non-parametric Mann–Whitney's test were used for univariate analysis. P < 0.05 was considered to indicate a statistically significant difference Table .INK4a staining  patients were HPV + based on PCR results Table . Only inng Table . In 5 , followed by HPV-18 (33.3%). Low-risk type HPV DNA (HPV-6/11) was not found in all specimens.INK4a expression absence was seen in 8 of 16 (50%) EACSCC  EACSCC Table . The difINK4a protein). Furthermore, 3 of 16 patients were found to have detectable HPV 16 and HPV 18 DNA. Our data align with the other research conducted by Masterson et al. [To investigate the presence of HPV DNA, we studied 16 cases of surgically removed primary SCC in the external auditory canal. We carried out a dual investigation: a molecular biology study (using PCR to diagnose the presence of HPV DNA) and immunohistochemical profiling (to investigate the expression of p16INK4a was an insensitive signature of HPV condition with a low estimation significance for HPV infection [INK4a staining might be an inadequate HPV detection approach because we reported a high rate of failure in cases that were positive for p16INK4a but were actually HPV−. Through this method, the number of false-positive HPV+ cases would be 50 percent higher. Presently, ultraviolet along with ionizing radiation constitutes the only evidence-centered supported risk factors for TB SCC [INK4a protein in human keratinocytes [INK4a protein was exceeded in one-half of the EACSCCs. Notably, a strong relationship between p16INK4a over-expression and the degree of tumor differentiation is seen. These facts suggest that p16INK4a should be necessary to induce tumor progression and de-differentiation.It is reported that p16nfection . This sur TB SCC . In addiinocytes –32. On tinocytes . In our Ki‑67 constitutes a nuclear protein that is linked to RNA transcription along with the progress of cell cycle . The preINK4a immunostaining seems to be insufficient for HPV detection in EACSCC. However, a p16-dependent cascade might be implicated in tumor de-differentiation of EACSCC. Furthermore, we found an impressive link of the presence of HPV to Ki-67 expression. Because the number of patients in our study was limited, a larger study is necessary to address this issue.In summary, our data show that high-risk HPV 16 and HPV 18 are related to the SCC of EAC. p16


IntroductionHuman papillomaviruses (HPV) are known to be the main culprit of cervical cancer. It is the fourth most common cancer among women worldwide. In recent years, it has begun to spread more widely in Saudi Arabia. Saudi Arabia's Ministry of Health recently added HPV vaccination for women to the list of recommended vaccinations.AimThis study aimed to assess Saudi females' knowledge regarding HPV infection, screening, and the available tools for prevention in Qassim, Saudi Arabia.Subject and methodsThis is a descriptive cross-sectional study conducted among women living in the Qassim region of Saudi Arabia. A self-administered pre-structured questionnaire was distributed among the targeted women using an online platform. The questionnaire includes socio-demographic characteristics  knowledge questionnaire and a questionnaire about the attitude toward HPV protection. The minimum required sample size was 385. This means 385 or more measurements/surveys are needed to have a confidence level of 95% that the real value is within ±5% of the measured/surveyed value. All females aged 18 years and above who are living in the Qassim region of Saudi Arabia were included in our study. Males are excluded from this study. All data analyses were performed using Statistical Product and Service Solutions (SPSS) .ResultsOf the 387 women involved, 52.2% were aged between 18 and 25 years old. The prevalence of women who have heard of HPV was 49.1%. Fifty-four percent were willing to accept HPV vaccination if offered. The overall mean knowledge score was 3.56 (SD 2.51) out of 11 points. Poor knowledge levels constituted most of the women (71.1%), 24.5% had moderate knowledge and only 4.4% were considered good. Increased awareness was more prevalent in younger women, more educated, those who underwent Pap smear, and those who were willing to accept HPV vaccination.ConclusionThe awareness of women toward HPV infection was deficient. Younger women who had a better education and who are willing to receive HPV vaccination were more likely to demonstrate better awareness levels toward HPV infection as compared to the rest of the women. More research is needed to establish the level of awareness among women in our region. Human papillomaviruses (HPV) are known to be the main culprit of cervical cancer, along with other diseases. They are mainly transmitted through sexual activity. HPVs are categorized into high-risk types and low-risk types. Low-risk genotypes (HPV 6 and 11) result in genital warts, a common benign condition of the external genitalia that leads to high morbidity. High-risk types 16 and 18 are responsible for 70% of cervical cancer and precancerous lesions.Cervical cancer is ranked as the fourth most common cancer among women around the globe, with an estimated 604,000 new cases and 342,000 deaths in 2020 . In SaudHPV vaccination was originally proposed for usage in the United States of America (USA) for female teenagers and youthful adults in 2006 and male teenagers and youthful adults in 2009 . The preThe HPV vaccination has just been added to the official immunization schedule for women in the Kingdom of Saudi Arabia. However, the absence of national cervical cancer screening programs poses a significant health concern in the country, as early diagnosis of precancerous lesions by Pap screening is a major component in preventing cervical cancer . TherebyThis was a descriptive cross-sectional study conducted among women living in the Qassim region of Saudi Arabia. A self-administered pre-structured questionnaire was distributed among the targeted women using an online platform. The questionnaire includes socio-demographic characteristics  knowledge questionnaire and a questionnaire about the attitude toward HPV protection. The minimum required sample size was 385. This means 385 or more measurements/surveys are needed to have a confidence level of 95% that the real value is within ±5% of the measured/surveyed value. All females aged 18 years and above who are living in the Qassim region of Saudi Arabia were included in our study, males are excluded from this study.The knowledge of women regarding HPV has been assessed using an 11-item questionnaire with "yes" coded with 1 and “no/I don’t know” coded with 0 as the answer options. The total knowledge score has been calculated by adding all 11 items. A possible score range from 0 to 11 points had been generated. A higher score indicates higher knowledge levels of HPV. By using 50% and 75% cut-off for scores to determine the level of knowledge, participants were considered as having poor knowledge if the score was less than 50%, 50% to 75% were considered as moderate, and above 75% was considered a good knowledge level.Descriptive statistics were used to describe the overall group of respondents including numbers and percentages , and mean and standard deviation (continuous variables). The differences in the scores of knowledge according to the socio-demographic and the attitude toward HPV protection had been conducted by using the Mann-Whitney Z-test. Statistical collinearity was measured using the Shapiro-Wilk test as well as the Kolmogorov-Smirnov test. The awareness score follows the non-normal distribution. Therefore, the non-parametric test was applied. Two-tailed analysis with p<0.05 was used as the cutoff for statistical significance. All data analyses were performed using Statistical Product and Service Solutions (SPSS) .This study involved 387 women. Table Table When measuring the differences in the score of knowledge according to the socio-demographic characteristics and the attitude of women toward HPV protection Table , it was Our study evaluates the knowledge of women regarding HPV infection, screening, and prevention. The findings of this study showed that the overall knowledge of women about HPV infection and prevention was lacking. Approximately 71.1% were categorized as poor levels. A total of 24.5% were moderate and only 4.4% were categorized as having a good level of knowledge . These findings are consistent with the literature wherein most women demonstrated a lack of understanding regarding the basic facts of HPV, infection, screening, and prevention . In AseeThe results of our study indicate that younger women (≤25 years) showed better knowledge levels than older ones (>25 years). This mirrored the study that was published in 2021, which reported that respondents who were younger (18 to 25 years) exhibited better knowledge of HPV . ContradIncreased awareness was more prevalent among educated women. This is consistent with the other two studies, both studies found education levels as one of the key factors for HPV awareness ,16. FurtThe lack of awareness of our respondents stemmed from the specific questions related to the basic facts about HPV. For example, those who have heard of HPV were just almost half of our samples (49.1%), women who were aware that HPV can be detected through a Pap smear were also below half of our subjects (45.2%) and they had a poor understanding that HPV can lead to cervical cancer (34.6%) or can cause genital warts (21.4%) and relatively few women believed that HPV infection does not detect in blood samples 10.3%). In addition, only 16% were aware of the condition that HPV screening should be done regularly every three years. In a study done in Riyadh, Saudi Arabia, women showed a better awareness level that HPV was transmitted sexually (78.9%), or that it caused genital warts (81.7%) and cervical cancer (81.9%) [.3%. In aMoreover, we discovered that while more than half of our subjects (51.4%) disagreed that HPV vaccination is intended for those who had the disease, however, only 17.3% were against the opinion that the vaccination is for females only. Likewise, 42.9% viewed periodic examinations may have been better than HPV vaccination. Only one-third of the respondents believed the availability of HPV vaccines and the proportion of women who indicated its safety was suboptimal (33.9%).Only 18.6% of our subjects had undergone Pap smear tests. We further observed that only 2.1% had ever gotten an HPV vaccination, although 54% showed a willingness to accept the HPV vaccine if offered. In Aseer Region, Saudi Arabia, a survey involving 1,116 showed a similar proportion of women had Pap smear test screening (27%) while in Makkah, Saudi Arabia a negative attitude was documented among married women, wherein the majority did not perform the Pap smear test regularly and most of them never had a PAP smear done adding that the lack of knowledge was the main reason for not having it ,17.The study's limitation was that it only obtained its information from the Qassim region. This gives us a more concentrated result as opposed to one that is more generalized. We recommend that in future research, data be collected from primary health centers, schools, and universities in a broader range of Saudi Arabia regions in order to ensure a more generalized result.The awareness of women toward HPV infection was deficient. Younger women who had a better education and who are willing to receive HPV vaccination were more likely to demonstrate better knowledge levels toward HPV infection as compared to the rest of the women. There is a need to improve awareness regarding HPV among women in our region. A more constructive program designed to bridge the gaps in knowledge is vital to achieving better understanding among the public. Awareness campaigns through social media, health educational campaigns, posters, pamphlets, and TV ads could provide a big boost to promote HPV infection, HPV vaccinations, and HPV screening programs. More research is needed probably at a national level with a bigger sample size to establish the level of awareness of women regarding HPV infection, screening, and prevention.


The detection rate of vaginal BV in the experimental group was 10.36% (29/280) compared with the detection rate of 5.0% (7/140) in the control group, and the difference was statistically significant . This indicates that women with high-risk HPV infections for 6 months or longer have a higher incidence of vaginal microecological imbalances than healthy individuals and aggressive vaginal microecological screening. It is necessary to carry out the program. Detect and treat possible abnormal conditions in time to prevent the further onset of the disease.Many microorganisms live in the vagina of healthy women. They interact with and compete with the microenvironment in the female vagina to form a dynamic balance of the microenvironment in the female vagina. However, imbalanced vaginal microecology can lead to vaginal resistance to pathogenic microorganisms. Poor capacity can cause women to develop infections of the reproductive tract. This article analyzes the vaginal microecological status of women with high-risk HPV infection for more than 6 months and healthy women and explores the risk factors that cause long-term high-risk HPV infection for timely detection and regulation of possible vaginal microecological imbalance in women with high-risk HPV infection for more than 6 months to prevent further development of cervical lesions in such patients. This article covers women with a sexual life history who attended the gynecology department of a hospital from January 2020 to September 2021. There were 280 patients in the experimental group: positive high-risk HPV; and there were 140 patients in the control group: negative high-risk HPV test. The correlation between vaginal microecology of CIN patients and patient prognosis according to the subject's vaginal microecology test results and prognosis of various levels of cervical lesions was analyzed. The experiment proved that the detection rate of normal vaginal microecology in the experimental group was 12.14% (34/280) compared with the detection rate of 29.29% (41/140) in the control group, and there was a trend of decrease, and the difference was statistically significant ( HPV is the main pathogenic factor of cervical cancer. Studying its carcinogenic mechanism and factors affecting the occurrence and development of cervical cancer plays an important role in the prevention and treatment of cervical cancer. Multiple HPV infections can increase the risk of cervical lesions in patients, accelerate the progression of cervical lesions, and affect the sensitivity of cervical cancer patients to treatment and affect the prognosis of patients. This effect is associated with accelerated HPV recombination during multiple HPV infections, the formation of new types of HPV with greater pathogenicity and invasiveness, faster disease progression, and increased disease malignancy. Therefore, it is of particular importance to study the correlation between the various clinical features of cervical lesions caused by multiple HPV infections and whether multiple HPV infections promote HPV recombination and its mechanism.High-risk HPV infections are mainly transmitted through sex. Cervicitis and CIN caused by infection are the most common cervical problems in clinical practice. If they are not treated in time, they may progress to cervical malignant tumors. Although the health departments around the world attach great importance to high-risk HPV infection and invest a lot of resources in the prevention, screening, and vaccine development of high-risk HPV, the detection rate and mortality of cervical cancer are still on the rise. The virus in most infected people does not last for more than 6 months. Most women can quickly get rid of high-risk HPV by relying on the self-cleansing capacity of the vaginal microecosystem and the self-repair of cervical epithelial cells. However, some women are still unable to get rid of high-risk HPV infections in their bodies. The continued action of high-risk HPV ultimately leads to the development of CIN and cancer.P < 0.05), but the testing equipment was immature at the time of the study, resulting in insufficient accuracy [The cervix is always surrounded by the vaginal microecological environment. The cervix and vagina infected with high-risk HPV are inextricably linked to the vaginal microecological environment. Whether there is a clear difference between the vaginal microecological status of people with high-risk HPV long-term infection and healthy people, domestic and foreign scholars have not yet reached a consensus. In order to assess the vaginal microecological status of high-risk HPV infection cervical intraepithelial neoplasia, 241 high-risk HPV-infected CIN patients diagnosed and treated by the Cervical Disease Center were selected as the experimental group, and 168 women who underwent physical examination in the outpatient clinic were studied. The HPV-negative and normal cytological examination results were used as a control group to compare the differences in the vaginal microecological status between the two groups. However, the lack of experimental data in his study led to small differences in the sample set, resulting in inaccurate results [accuracy .In the past, clinical studies on patients with high-risk HPV infections mostly focused on the relationship between HPV infection and simple vaginal inflammation. Few studies were conducted on the overall status of vaginal microecology in women with high-risk HPV infections over 6 months. This article focuses on the relationship between HPV multiple infections in cervical cancer patients and the clinical manifestations of cervical cancer, revealing the effects of multiple HPV infections on the clinical features of cervical cancer. By investigating the mechanism of this effect and discovering and controlling potential imbalances in the vaginal microeconomics in time, further development of cervical lesions and multiple HPVs in the future can be prevented. It may provide a more theoretical basis for the prevention, evaluation, and follow-up of patients with infectious diseases.β-glucuronidase, which reflects the overgrowth of aerobic bacteria in the vagina and indicates possible VVC infection; or TV acetylglucosaminidase, etc., which are all negative. A healthy vaginal microecological environment has a protective function for the human body [It is generally believed that a healthy vaginal microecological status is the following: the density of the colonizing bacteria in the vagina is between level II and level III, the diversity of the flora is between level II and level III, and lactobacilli occupy an absolutely dominant position in terms of quantity. No infection of pathogenic microorganisms such as BV, candidal vaginitis (VVC), and trichomonal vaginitis (TV) was found in the vagina, the number of white blood cells is less than 10/high-powered field of view, the pH value in the vagina is ≤4.5, which reflects that lactic acid Bacillus functions are positive for hydrogen peroxide; sialidase, which reflects BV infection; leukocyte esterase, which reflects the massive destruction of vaginal white blood cells due to inflammation; Lactobacillus is the dominant bacteria that colonize the vagina of healthy women, and it is also the main force to maintain the vaginal microecological balance. It accounts for more than 95% of the total vaginal microbes, and 80 to 90% of women can isolate the bacteria in the vagina. The decrease in the number of lactobacilli reduces the resistance of the vagina to pathogenic microorganisms, resulting in the persistence of high-risk HPV infections. The specific mechanism of action still needs more in-depth research , 7. It iThe concept of vaginal microecology was first proposed by the Obstetrics and Gynecology Infectious Diseases Cooperation Group of the Chinese Medical Association of our country and has been popularized and used for more than 10 years. Traditional vaginal secretion testing methods can only diagnose vaginitis with a clear pathogenic infection and are limited by the time of specimen collection, the quality of specimens, and the inspector's testing experience. The accuracy of the test results is not high, and the efficiency is low. In recent years, the female vaginal microecological detection system has the advantages of fast, simple, multiple results, clear morphological staining, comprehensive functional detection, etc., and the detection rate of pathogenic microorganisms is higher than that of traditional microscope detection methods, and it can be combined with the morphological and functional characteristics of the test samples to obtain more accurate and comprehensive inspection results , 9. The x1, x2, x3, the intensity of the neuron's action can be expressed as w1, w2, w3 by weight, plus a bias term b, which is sent into the cell for processing together [In order to imitate the working mechanism of biological nerve cells, scientists have designed artificial neurons. Assuming that a neuron has three input data together , 13. ThuThe output value can be expressed asf represents the neuron function. According to the difference in solving the problem, the activation function is usually different; otherwise, it will affect the convergence of the algorithm. The commonly used activation functions are as follows, one is the Sigmoid activation function, the other is the Tanh activation function, the third is the ReLU activation function, and the fourth is the Maxout activation function. The mathematical formulas of Sigmoid function and Tanh function are shown inThe symbol There are many similarities between the Sigmoid curve and the Tanh curve: Regardless of whether the input value is large or small, the Sigmoid function can control the value between 0 and 1; for extremely large negative numbers, the output value is 0, and for extremely large positive numbers, the output value is 1. Tanh is derived from Sigmoid, and its output value is between -1 and 1, with an average value of 0 , 15. Thek is the number of feature maps output by Maxout:The advantage of this activation function is that it can process data quickly and only needs to set a threshold. However, the ReLU function also has a relatively large drawback—the ReLU neuron processes large gradients and updates the parameters, which may easily cause the neuron to not activate the data . The corMaxout does not have a fixed curve and can fit any convex function, so the fitting ability is relatively good, and it contains the advantages of the ReLU function while avoiding its defects. Other activation functions include threshold functions and linear functions. Different functions have different characteristics, which constitute a neural network with different functions nowadays , 18.Similar to brain tissue being composed of countless nerve cells, ANN is also composed of many artificial neurons. x1, x2, x3}, the weights corresponding to the hidden layer are {w11(1), w12(1), w13(1)}, {w21(1), w22(1), w23(1)}, {w31(1), w32(1), w33(1)}, and the bias value is {b1(1), b2(1), b3(1)}. The hidden layer has 3 output values {c1(2), c2(2), c3(2)}, the weight corresponding to the output layer is {w11(2), w12(2), w13(2)}, and the bias value is b1(2). The output result is represented by Z, f(h) represents the activation function, and the mathematical formula to express this neural network isThe input layer of a feedforward neural network structure contains multiple input neural nodes that represent the input values, and the input layer transfers data to the hidden layer for further processing. The processed values of the hidden layer cannot be directly observed during the operation of the neural network. The neuron has multiple inputs, but there is only one output value that can be sent to multiple neurons as input , 22. Thel layers are roughly similar. Taking cil to represent the output value of the i neuron of the l layer and wijl − 1)( and bil − 1). Suppose the total error is E, the error of zi is ei, and the expected output is ti. Then the total error can be obtained byThe reverse conduction algorithm can continuously optimize the connection weights and biases between each artificial neuron according to the expected output information; that is, the algorithm can propagate the difference between the expected value and the network output to the input layer layer-by-layer, to modify the weight of the connection. When the error signal is transmitted back to a certain layer, the signal will be allocated to the processing unit of this layer as the basis for modifying the weight of the unit. Backpropagation will continue until the number of iterations or learning times set by the network, or the output error is reduced to the range that the network designer can accept –26. The w5 weight in the network can be used for illustration. The calculation formula is shown in Equation 11), the w2, w3, w4 is similar. Since then, according to the chain calculation rule, the partial derivatives of weights w1 and b1 are calculated as shown inBy analogy, the update method of other weights w, b) is the weight parameter of the neural network,  represents the data set {(x(1), y(2)) ⋯ (xm)} to be trained by the network and contains m training samples, J is the corresponding loss function of a single training sample , and δil + 1).It can be seen from The 280 cases of the experimental group and 140 cases of the control group were tested for the diversity of vaginal flora. The results are shown in χ2 = 1.176, P > 0.05).It can be seen from 280 cases of the experimental group and 140 cases of the control group were tested for vaginal dominant bacteria; the results are shown in χ2 = 0.62, P > 0.05).It can be seen from 280 cases of the experimental group and 140 cases of the control group were tested for vaginal secretion white blood cell count. The results are shown in χ2 = 0.004, P > 0.05).It can be seen from The 280 cases of the experimental group and 140 cases of the control group were tested for vaginal pH. The results are shown in χ2 = 5.372, P < 0.05).It can be seen from 280 cases of the experimental group and 140 cases of the control group were tested for vaginal microecological enzymes. The results are shown in χ2 = 5.182, P < 0.05). The detection rate of sialidase positive in the vaginal discharge of the experimental group was 29.29% (82/280), while the detection rate of the control group was 14.29% (20/140), and the difference was statistically significant. It was χ2 = 8.233, P < 0.05. The detection rate of positive leukocyte esterase in vaginal discharge in the experimental group was 71.79% (201/280) compared to the detection rate of 55.0% (77/140) in the control group, and the difference was statistically significant . Compared with the control group, the vaginal discharge in the experimental group had a higher detection rate of β-glucuronidase and acetylglucosaminidase, but the difference was not statistically significant.The 280 cases of the experimental group and 140 cases of the control group were comprehensively evaluated for the vaginal microecological status, and the results are shown in χ2 = 17.23, P < 0.05)). The detection rate of vaginal BV in the experimental group was 10.36% (29/280) compared with the detection rate of 5.0% (7/140) in the control group, and the difference was statistically significant . The detection rate of VVC, TV, vaginal flora abnormality, and dysfunction in the experimental group was higher than that of the corresponding control group, but the difference was not statistically significant.It can be seen from The age composition of cervical lesions of each grade is different. Therefore, by comparing the relationship between high-risk HPV and cervical lesions of each grade, the age factor should be controlled. The 280 high-risk HPV-positive patients were divided into three groups: ≤35 years old, 36 years old~, and >50 years old. The results are shown in P < 0.05). It shows that there are significant differences in the high-risk HPV load of different grades of cervical lesions. And as the level of cervical lesions increases, the load of high-risk HPV also increases significantly. In addition, in patients with chronic cervicitis, CINII~III, and cervical cancer, there was no significant difference in the high-risk HPV load of each age group (P > 0.05), indicating that there is no correlation between age and high-risk HPV load. Only in CINI patients, there was a statistically significant difference in the load of high-risk HPV in each age group (P < 0.05). With the increase of age, the load of high-risk HPV decreased.It can be seen from χ2 tests, and the difference was statistically significant . This indicates that the high-risk HPV infection rates for different grades of cervical lesions differ significantly and that the high-risk HPV infection rates increase significantly with the level of cervical lesions.In the chronic cervicitis group, 45.4% of high-risk HPV cases were negative, and the remaining 54.5% of high-risk HPV-positive cases were 34.5%, 10.7%, and 9.4%, respectively. In the CINI group, 28.0% of cases were high-risk HPV-negative, and 72.0% of high-risk HPV-positive cases were 28.4%, 24.3%, and 19.3%, respectively. 7.7% of CINII-III cases were high-risk HPV-negative, and 92.3% of high-risk HPV-positive cases were 30.0%, 40.5%, and 21.8 with low-, moderate-, and high-level loads, respectively. In the cervical cancer group, 4.1% of cases were high-risk HPV-negative, and 95.9% of high-risk HPV-positive cases were 27.5%, 41.9%, and 26.5%, respectively. The load distribution of high-risk HPV in each grade of cervical lesions was compared by multiple sample rate β-glucuronidase-positive and acetylglucosaminidase-positive detection rates are all higher than those of the control group. There is no statistical significance (P > 0.05). The shortcomings of this article are the following: Because vaginal microecological testing is a new type of testing method, most medical institutions have not introduced the testing equipment, and the patients' acceptance of this testing method is not high, resulting in the failure of this study to be carried out on a large scale and many times. Center clinical trials can lead to inaccurate statistical results. Although there is a certain relationship between changes in female vaginal microecological status and long-term high-risk HPV infection, the specific mechanism remains unclear, and further studies are needed. Functional vaginal microecosystems play an important role in combating high-risk HPV infections. In the future, it will be used to increase the quantity and quality of lactic acid bacteria in the vagina, restore normal levels of hydrogen peroxide, and regulate vaginal pH when diagnosing and treating high-risk HPV infections. I can do it. And methods of killing pathogenic microorganisms improve the cure rate of high-risk HPV infections in many ways.Multiple HPV infections can increase the risk of cervical lesions. On the one hand, it may be related to the interaction between HPV subtypes. On the other hand, more and more reports about HPV recombination suggest that multiple HPV infections may increase the risk of HPV recombination, producing HPV that is carcinogenic. This article analyzes the correlation between the vaginal microecological status of high-risk HPV-infected CIN patients and the patients' prognosis. Compared with healthy women, women with high-risk HPV infection for more than 6 months have the vaginal flora density of women with high-risk HPV infection for more than 6 months. The detection rates of abnormalities, abnormal flora diversity, dominant bacteria, vaginal secretion white blood cell count > 10/high-powered field of view, and


False negative (FN) results in cervical cancer (CC) screening pose serious risks to women. We present a comprehensive literature review on the risks and reasons of obtaining the FN results of primary CC screening tests and triage methods and discuss their clinical and public health impact and implications. Misinterpretation or true lack of abnormalities on a slide are the reasons of FN results in cytology and p16/Ki-67 dual-staining. For high-risk human papillomavirus (HPV) molecular tests, those include: truly non-HPV-associated tumors, lesions driven by low-risk HPV types, and clearance of HPV genetic material before sampling. Imprecise disease threshold definition lead to FN results in visual inspection with acetic acid. Lesions with a discrete colposcopic appearance are a source of FN in colposcopic procedures. For FAM19A4 and hsa-miR124-2 genes methylation, those may originate from borderline methylation levels. Histological misinterpretation, sampling, and laboratory errors also play a role in all types of CC screening, as well as reproducibility issue, especially in methods based on human-eye evaluation. Primary HPV-based screening combined with high quality-assured immunocytochemical and molecular triage methods seem to be an optimal approach. Colposcopy with histological evaluation remains the gold standard for diagnosis but requires quality protocols and assurance measures. The fundamental objective of screening is to distinguish healthy persons from those in an early, asymptomatic phase of certain diseases by the use of available, validated, inexpensive, non-invasive, and widely accepted screening tests . As no iThe aim of this work is to discuss the risks and reasons of FN results of cervical cancer (CC) screening tests and triage methods, as well as their implications on screening process.A great majority of cervical neoplasia is driven by infections with oncogenic types of human papillomavirus (HPV) , and theDue to a long period of precancerous lesions development and the possibility of their accurate identification and effective treatment, CC screening has been introduced in many countries ,18. ScreExfoliative cervical cytology remains the basic test for CC screening in developed countries. However, as the discovery of the role HPV in the aetiology of CC emerged , the posFor decades, the Pap smear remained a basic screening test for the secondary prevention of CC in the more affluent countries and has been proven to be very effective in the reduction of CC incidence  and mortPublished studies reported specificity of cytology at 86–100% . HoweverAmong the reasons for FN cytological results are: (1) misclassification of an abnormal slide as normal, which results in no referral for further diagnostic examinations; (2) sampling errors; (3) true lack of abnormal cells on a slide due to lesion location—submucosal or deep in the endocervical canal; and (4) rater-dependence, with low reproducibility.Interpretation of a slide as normal, despite the presence of abnormal cells, was pointed out as a reason for FN diagnoses in over 50% of slides in many studies. In a pooled analysis by DeMay, 655 no intraepithelial lesions or malignancy (NILM) slides were evaluated, and 340 of them were reclassified as abnormal (51.9%) , which wSmall number of abnormal cells on the slide was shown to impede proper evaluation ,50. In aSampling issues are a common reason for FN cytology results. In studies on the reevaluation of NILM slides preceding cancer diagnosis, a fraction of slides were subsequently rated as unsatisfactory for evaluation, according to the Bethesda system ,53, withSome FN slides truly do not contain abnormal cells. This may be related to the absence of CC precursor lesion on the cervix at the time of sampling and extremely short time of cancer development and invasion , as wellAdenocarcinoma (ADC), a histological subtype of CC, was identified significantly more frequently than squamous cell carcinoma (SCC) among women with FN cytological results, compared to those with screen-detected cancer, due to their common location in the endocervical canal and more difficult sampling ,59,60,61Overall, the lower sensitivity of cytological screening for the detection of ADC than SCC is a complex issue and may result from a true lack of abnormal glandular cells on the slide due to: (1) the location of the lesions and less efficient sampling , (2) difCytology is an examination with limited reproducibility. As shown by Stoler et al. in the ALTS study (ASC-US/LSIL Triage Study) on almost five thousand cytological slides, the kappa coefficient was only moderate  for an atypical squamous cell of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) cytologies . Sørbye Depending on the oncogenic potential of specific HPV genotypes, the International Agency for Cancer Research (IARC) classified 12 HPV types , as associated with a high risk (HR) of CC development. Eight types  are considered probably carcinogenic due to the more common occurrence in CC cases than in cohorts with normal cytology .As a primary prevention against HPV infection, prophylactic HPV vaccines are currently available, and many countries worldwide have decided to implement HPV vaccination into national immunization programs . The bivOnly two likely carcinogenic genotypes (66 and 68), according to IARC classification, are included in the standard HPV tests used in the primary screening. Due to the rare occurrence of remaining genotypes among CC cases , a possible slight increase in sensitivity at the expense of a high decrease in specificity, extending the spectrum of HPV types included in validated screening tests, is not recommended ,82. According to the meta-analysis by Koliopoulos et al., the sensitivity of HR HPV tests for the detection of CIN2+ lesions in group of women aged 30 or more was 93.9%, compared to 72.2% for cervical cytology (both LBC and Pap). HPV tests were shown to be statistically significantly more sensitive for CIN2+ detection than both conventional cytology and LBC, with relative sensitivities of 1.52 and 1.18, respectively. Specificity was significantly lower for HPV testing (with relative specificities of 0.94 for Pap test and 0.96 for LBC). Similar results were obtained for CIN3+ detection . On account of the increasing number of HPV tests on the market without proper validation, some criteria ensuring safety and high-quality performance were urgently needed . In 2009The use of HPV tests for detecting HR HPV types in screening significantly reduced the problem of FN results, compared to cytological-based screening, due to the significant increase in sensitivity . DespiteTrue lack of HPV DNA in the sampleThe homogenous group of SCC, which accounts for about 75–85% of all CC cases worldwide ,87, is hAccording to the International Endocervical Adenocarcinoma Criteria and Classification, two groups of ADCs were extracted, depending on the HPV infection status: (1) non-HPV-associated  and (2) HPV-associated , mucinous intestinal, mucinous signet ring, and invasive stratified mucin-producing carcinoma) . DetaileIn general, HPV(−) CC cases were shown to be associated with higher age, worse prognosis, diagnosis of ADC, and higher risk of relapse and distant metastases ,97,98,99Histological MisclassificationsHistological misclassifications seem to explain a part of the HPV(−) CCs phenomenon. Endometrial cancer, which is not, or barely, related to HPV infection , may be In the study by the Cancer Genome Atlas Research Network, 8 out of 178 primary CC cases were reclassified as endometrial-like (5%); a total of 7 of them were HPV(−) . In a stCervical Cancer Developed from Infection with Low-Risk Oncogenic TypesPublished studies confirmed the relationship between the HPV types classified by WHO as probably, or possibly, carcinogenic (which are not being detected by primary HPV screening tests) and the development of a fraction of CC cases. Those cases are usually single-infected, which makes them impossible to diagnose with contemporary HPV screening tests. In the ATHENA trial, with over 46 thousands women involved, 55 HPV(−) CIN2+ cases were found . After rHit and Run TheoryA hit and run theory may bring another explanation for HPV(−) CCs phenomenon. The hypothesis is broad and related not only to CC. It states that some types of cancer need the viral infection to initiate the oncogenesis; however, as the mutations are accumulated, the need for oncogenic factor presence no longer exists, and the initial infection clears . SubsequAccording to the work by Tjalma et al. on over 6000 CIN2+ women, comparing HPV prevalence in high-grade CIN and invasive CC, the rate of HPV(−) high-grade CIN cases was lower than the rate of HPV(−) invasive CCs  . DiffereSampling Errors, Laboratory ErrorsAside from the test-specific factors, other common mistakes may be responsible for FN results. Sampling errors may result in a lack of the necessary amount of HPV DNA material; a similar problem is vital in cytological screening . As selfAnother classical reason for obtaining false-negative results is laboratory error, following inappropriate laboratory procedures, poorly trained staff, no validation or quality assurance of procedures, and the use of screening tests with no analytical and clinical validation ,115.In settings where financial resources and the number of well-trained health workers are limited, the WHO recommends using VIA with the immediate treatment of suspected treatable lesions as a screening method. VIA involves the application of a 3% to 5% solution of acetic acid on the cervix and observation of the acetowhite lesions using the naked eye after at least 1 min since application. If necessary and appropriate, treatment with cryotherapy or loop electrosurgical excision procedure (LEEP) should be provided. This approach is aimed at ensuring a high negative predictive value, reducing the loss to follow-up, and treating all women in need using scant finances .The sensitivity of VIA screening for CIN2+ detection ranged widely between 42% and 92% in 26 studies, where all subjects underwent confirmatory testing, with a pooled sensitivity of 80% . SimilarThe phenomenon of FN VIA results is not widely discussed in the literature, due to the lack of long-term follow-up of women in developing countries. Besides, the gold standard of colposcopy with biopsy is not always performed in all screened women; in many studies, only VIA(+) participants underwent confirmatory testing . The potVIA, as an unquantifiable method based on the human eye evaluation only, is strongly observer-dependent and has low reproducibility. The results of the study in rural Nigeria showed that, depending on the provider, 0–21% of women were suspected to have cancer, and 0–25% of women were assessed as VIA(+), despite the homogenous background population . In the Additionally, the definition of the disease threshold may impact the accuracy of VIA, as it is not strict. As stated in guidelines by JHPIEGO, abnormalities looking similar to “raised and thickened white plaques or acetowhite epithelium, usually near the SCJ (squamocolumnar junction)” are of clinical significance when using VIA for screening, as well as “cauliflower-like growth or ulcer; fungating mass”, which should be considered a cancer suspicion . CryotheColposcopy is one of the most commonly used triage methods to screen positive women in cytology and HR HPV-based screening . ColposcDespite the numerous attempts to standardize the procedure for the proper training and certification of colposcopists, the accuracy of the procedure is far from perfect, and FN results are common. According to the meta-analysis by Mitchell et al., the pooled rate of positive cone biopsies, widely accepted as a gold standard, among colposcopically-directed negative biopsies in women with cytological abnormalities reached approximately 4% . In a prThe rate of CIN2/CIN3 detected by colposcopy with targeted biopsy reached 56% in 2112 women with cytological abnormalities . On the Likewise in cytology, colposcopic examination, as a method based on the evaluation by human eye, is subjective and observer-dependent. Poor agreement between colposcopists was reported in the literature ,135 withSensitivity of colposcopy is believed to depend on colposcopists’ experience; however, many studies contradict this statement ,132,141.The issue of colposcopically-targeted and random biopsies taken during colposcopy was widely discussed in the literature. Since the severity of lesion did not always correspond with the most visually advanced area of the colposcopic appearance, random biopsies were suggested, in order to elevate colposcopy sensitivity ,148. TheA part of the FN colposcopies phenomenon may also occur due to histological misclassifications. According to a study by Ismail et al. based on 100 colposcopic biopsies, as evaluated by 8 experienced colposcopists, the overall agreement was moderate but highly dependent on lesion severity, ranging from a kappa of 0.175 for CIN1 and CIN2 to 0.832 for invasive cancer . In a siDS for the simultaneous detection of p16 and Ki-67 proteins within the same cell became a widely investigated triage procedure, due to its high sensitivity, relatively high specificity, and possibility to perform on the residual sample after the HPV test and LBC . In the DS was shown to have a higher sensitivity in detecting precancer lesions and provide longer negative reassurance in HPV(+) women than negative cytology, with 5 years relative risk of CIN2+ detection of 0.7 ,157. In Published evidence pointed out difficulties in slide interpretation as a reason of improper DS result. As the threshold for positivity is usually set at one dual-stained cell on the slide, the correct exclusion of the positivity of each visible cell is crucial. Cells presented in groups on the slide, or with a scant cytoplasm and weak p16 staining, seemed to be challenging for readers ,162, as The issue of factors influencing overall positivity of a DS slide is by far better investigated in the literature than the matter of strict FN DS reports. It was shown by Benevolo et al. that the longer time between sampling and immunostaining or between immunostaining and evaluation of the slide, the higher probability of its positive interpretation. Other factors like referral cytology result, presence of CIN2+ or HPV mRNA detection were also naturally shown as associated with slide positivity . To our ®  and QIAsure Methylation test kit (FAM19A4 and miR124-2 genes) and two on cervical scrapes: PAX1 DNA detection kit (PAX1 gene) and ZNF582 DNA detection kit (ZNF582 gene) [The test measuring methylation levels of specific genes involved in the process of carcinogenesis was shown as a promising method of precancerous lesions detection in HPV(+) women. Many potential biomarkers have been verified to find appropriate ones ,169,170.82 gene) . The per82 gene) ,173 and 82 gene) ,175 and In published studies virtually all CC cases were properly identified by methylation tests based on these genes. In a cross-sectional study of 519 CC cases from 25 countries in 5 continents, 510 (98.3%) had positive methylation test result and—worth noting—the set of samples consisted of both cervical scrapes and cervical tissues and covered SCCs and ADCs as well as adenosquamous cell carcinomas and other rare CC histotypes . AdditioThe negative result of methylation test in women with CIN2 or CIN3 may indicate low risk of progression to cancer in these persons. De Strooper et al. used the length of preceding HPV infection in women with precancerous lesions as a proxy for advancement of the lesion and risk of progression to cancer. In his study all CIN2+ lesions preceded by HPV infection lasting >5 years were detected by the methylation test (51/51) and only 8/19 (42%) of early CIN2/CIN3 lesions (with <5 years since prior HPV infection) were detected . SimilarThis explanation of lower performance of methylation test in less advanced lesion is in line with observation that CIN2/CIN3 lesions were more commonly methylation negative in women below 30 years old ,183. IndSamples with methylation level near the cutoff value seem to be less reproducible. Bonde et al. observed that 80 samples among 983 selected for both intra- and inter-laboratory agreement analysis had discordant results (8.1%), with 77 of them being close to the methylation positivity threshold . Additionally, the only case of CC undetected by methylation test in this study had borderline methylation level . This phIn order to address still unmet needs in CC screening some novel methods have been proposed. HPV testing on first-void urine samples seems to be an acceptable method for reaching underscreened women, with sensitivity and specificity comparable with respective indicators of HPV test performed on clinician-taken samples ,186. HR To balance sensitivity and specificity of screening process both indicators of the test as well as interval between screens should be taken into consideration. More frequent testing increases sensitivity and reduces FN rate at the expense of decreasing specificity and rising number of FPs. Risks of both CC and precancer occurrence after a negative screening test result were investigated in studies with large sample size and long-term follow-up to assess negative predictive value of the test and establish acceptable intervals . NeverthNo screening or triage test ideally distinguishing healthy and diseased people exists, neither for CC screening nor for any other type of screening. Sensitivity of 100% is beyond the reach of current possibilities and both policymakers and screening organizers should be aware of it. Women must be informed that participation in screening significantly reduces the risk of both incidence and mortality of CC but it does not eliminate it completely. Emphasis should be placed on the use of certified and validated screening tests, only to ensure highest possible quality. Additionally, quality assurance procedures and quality control should be in place.It is impossible to avoid FN results in cervical cancer screening but due to their serious consequence efforts should be undertaken to limit their burden. The whole set of reasons of FN results may be divided into broadly understood errors and test deficiencies. To minimize human factor extensive and regular training should be introduced, however, it does not always guarantee satisfactory effects and may be difficult to run in some settings. According to the “WHO guidelines for screening and treatment of cervical pre-cancer lesions for cervical cancer prevention”, HPV testing is considered superior to both cytology-based and VIA screen-and-treat approach by means of overall effectiveness of screening program . HPV-basAlthough HPV-based screening is very sensitive, false-negative results may occasionally occur . As suppA relatively low specificity of HPV-based screening, however, leads to an excessive number of referrals for colposcopy, colposcopies performed, and treatment procedures executed. Accurate triage methods are, therefore, crucial for avoiding both overdiagnosis and overtreatment. Some actions were taken to support proper decision when using subjective screening and triage tests; this may both limit the number of FN and FP results. Colposcopy still serves as a gold standard in many countries with screening programs implemented worldwide. A few decades ago, cervicography, based on taking pictures of the cervix, which were subsequently evaluated by trained colposcopists, was proposed as a useful tool . More reDS obtained approval by the U.S. Food and Drug Administration with the indication for HPV(+) women with HPV genotypes other than 16 and 18, in order to determine the need for colposcopy referral, as well as for HPV(+) women with HPV 16 or 18 detected, to guide their management . There hAmong the investigated triage procedures, the methylation of FAM19A4 and miR124-2 genes provided promising results, in terms of high sensitivity for cancer detection, and allowed for the use of self-sampled vaginocervical material. However, the rate of FN methylation results is higher for less severe CIN2 and CIN3, which are the main targets of CC screening. According to the aforementioned studies, the methylation test correctly identifies cases with long-lasting HPV infection; however, further studies are required, in order to assess the potential for the progression, regression, and persistence of HPV(+) methylation-negative HSIL cases. To conclude, FN results are unavoidable in screening, and this phenomenon can only be minimized by careful quality assurance. Human error can be reduced, but not excluded, by both training and use of AI methods. In light of the published studies and despite some inevitable adverse effects, HPV-based screening seem to be the most efficient, in regard to FN rates. The triage methods of HPV(+) women are still being investigated. Both DS and methylation of FAM19A4 and miR124-2 genes seem promising for the future; however, colposcopy with appropriate protocols and quality measures incorporated will probably still serve as a reliable triage examination, until further studies will be performed to elaborate on the most optimal protocol for proper risk stratification and management of HPV(+) results.