P < .001) and NLR on the 7th postoperative day  with prognosis of aSAH patients. The results of receiver operating characteristic curve showed that cutoff values of RDW and NLR for predicting the prognosis in aSAH patients were 13.05% and 6.97, respectively. Higher RDW on the 3rd postoperative day and NLR on the 7th postoperative day were possibly associated with poor prognosis of aSAH patients. We should pay attention to the RDW and NLR levels during different hospitalization periods, especially in the short postoperative period. Moreover, the cutoff values for predicting prognosis need to be validated in larger-sample studies.The associations of neutrophil to lymphocyte ratio (NLR) and red cell distribution width (RDW) with the prognosis of aneurysmal subarachnoid hemorrhage (aSAH) patients were confirmed in a few studies. But NLR and RDW levels in most of these studies were on admission. Here we carried 1 retrospective study including 150 patients with aSAH who underwent surgeries in the First Affiliated Hospital of Yangtze University from January 2020 to February 2023 to explore the associations on the preoperative day, the first (1st), third (3rd), and seventh (7th) postoperative days. The level of RDW on the 3rd postoperative day and level of NLR on the 7th postoperative day in patients with poor prognosis were significantly higher than patients with good prognosis. The results of multivariate logistic analysis also confirmed the associations of RDW on the 3rd postoperative day (OR = 16.785, 95% CI: 4.077–69.107,  Previous studies confirmed that NLR was associated with the occurrence, severity, and prognosis of some diseases. NLR was associated with the functional outcomes in patients with cerebral hemorrhage and acute cerebral infarction.,5 On the other hand, thrombosis was also involved in the pathological changes after aSAH. Red cell distribution width (RDW) was a indicator reflecting the heterogeneity of red blood cell size and confirmed to be important and possibly associated with prognosis of patients in many diseases such as ischemic stroke, pulmonary embolism, and tumors.Aneurysmal subarachnoid hemorrhage (aSAH) is a complex and fatal disease. Although breakthroughs have been made in the treatment of aSAH in recent years, the prognosis of some patients is still poor. The important roles of some inflammatory indicators were demonstrated in the progression of cerebrovascular diseases and possibly related to the prognosis in some studies.,11 However, the NLR and RDW levels in most of these studies were on admission. The disease condition was variable during hospitalization. Therefore, we explored the associations on the preoperative day, 1st, 3rd and 7th postoperative days in this study.There were a few studies that explored the associations of NLR and RDW with the outcome of aSAH patients, and suggested that higher NLR and RDW were associated with poor prognosis.We carried one retrospective study including 150 patients with aneurysmal subarachnoid hemorrhage who underwent surgeries in the First Affiliated Hospital of Yangtze University from January 2020 to February 2023. The study was approved by the Ethics Committee of the First Affiliated Hospital of Yangtze University. Patients meeting the following criteria were included: (1) had subarachnoid hemorrhage confirmed by computed tomography (CT); (2) had intracranial aneurysm confirmed by digital subtraction angiography (DSA) and associated with bleeding; (3) received craniotomy clipping or intravascular embolization surgeries after admission. Patients meeting the following criteria were excluded: (1) had subarachnoid hemorrhage caused by arteriovenous malformations and trauma (or did not have aneurysms); (2) suffered from severe systemic diseases, such as severe liver dysfunction, kidney dysfunction, heart dysfunction, hematological diseases, etc.; (3) had Modified Rankin Scale (mRS) score ≥ 3 before admission; (5) were unable to complete follow-up of 3 months. The flow chart was showed in Figure rd, and 7th postoperative days. NLR was the ratio of neutrophil to lymphocyte. The prognosis was determined by the Modified Rankin Scale (mRS) results of 90-d follow-up after morbidity. The patients with scores of mRS 0–2 were defined as having a good prognosis, while mRS 3–6 were defined as having a poor prognosis.Baseline characteristics included gender, age, Hunt-Hess grade, Glasgow Coma Scale (GCS) score, combination of intraventricular hemorrhage, the location of aneurysm, neck length of aneurysm, date of surgery, time length of surgery. The blood cells counts were extracted from the hospital information system including white blood cells, neutrophils, lymphocytes, hemoglobin, and the red blood cell distribution width on the preoperative day, the 1st, 3t test was used for analysis. If the quantitative variables met the normal distribution but variances were not homogeneous, corrected t test was used. If the normal distribution could not be satisfied, Mann–Whitney U test was used for analysis. The qualitative variables were compared by Pearson chi-square test, Continuity correction or Fisher exact test. The multivariate logistic regression model was used for factors associated with prognosis. Covariates that had a P-value >.05 in the univariate analysis were added to the multivariate logistic analysis. ROC (receiver operating characteristic) curve was used for the cutoff values of characteristics for predicting poor prognosis in patients with aSAH. Graphpad prism 6.0  was used for the correlation analysis. All p-values were 2-sided and the statistical significance was set at P < .05.We used SPSS 23.0  for data analysis. If the quantitative variables met the normal distribution and variances were homogeneous, independent-sample P < .05 , P = .001. The preoperative counts of neutrophils in patients with good prognosis and poor prognosis were (9.96 ± 4.00) × 109/L and (12.77 ± 5.16) × 109/L respectively, with significant difference, P = .001. The preoperative counts of lymphocytes had no significant difference, P = .379. The preoperative NLR in patients with good prognosis and poor prognosis were 10.69 ± 9.41 and 11.50 ± 10.25 respectively, with no significant difference (P = .461). The median preoperative RDW in patients with good prognosis was 12.7%, significantly lower than patients with poor prognosis (12.9%), P = .017. There was no significant difference in preoperative hemoglobin between patients with good prognosis and poor prognosis, P = .356  × 109/L, significantly lower than patients with poor prognosis ([12.57 ± 5.70] × 109/L), P = .001. The counts of neutrophils in patients with good prognosis and poor prognosis were (8.72 ± 3.63) × 109/L and (11.14 ± 5.58) × 109/L respectively, also with significant difference, P = .001. The counts of lymphocytes in patients with good prognosis and poor prognosis were (0.95 ± 0.39) × 109/L and (0.83 ± 0.37) × 109/L, with no significant difference, P = .122. The NLR ratio on the 1st postoperative day in patients with good prognosis was 9.86 ± 6.32, significantly lower than patients with poor prognosis (14.37 ± 13.05), P = .001. The median RDW level in patients with good prognosis was 12.90%, significantly lower than that patients with poor prognosis (13.25%), P = .001. The levels of hemoglobin (P = .553) on the 1st postoperative day in patients with good prognosis and poor prognosis had no significant difference  × 109/L, significantly lower than patients with poor prognosis ([11.54 ± 4.40] × 109/L), P < .001. The counts of neutrophils in patients with good prognosis and poor prognosis were (7.47 ± 2.79) × 109/L and (9.78 ± 3.11) × 109/L respectively, with significant difference, P < .001. The counts of lymphocytes on the 3rd postoperative day were (1.05 ± 0.68) × 109/L and (0.79 ± 0.64) × 109/L respectively, with significant difference, P = .013. The median NLR level in patients with good prognosis was 6.46, significantly lower than patients with poor prognosis (10.30), P < .001. The median RDW in patients with good prognosis on the 3rd postoperative day was 12.40%, significantly lower than patients with poor prognosis (13.25%), P < .001. There was no significant difference in hemoglobin level between patients with good prognosis and poor prognosis, P = .063  × 109/L, significantly lower than patients with poor prognosis ([12.54 ± 4.16] × 109/L), P < .001. The counts of neutrophils in patients with good prognosis and poor prognosis were (7.21 ± 2.67) × 109/L and (10.27 ± 3.95) × 109/L respectively, with significant difference, P < .001. The counts of lymphocytes on the 7th postoperative day were (1.26 ± 0.62) × 109/L and (1.09 ± 0.59) × 109/L respectively, with significant difference, P = .033. The median NLR level in patients with good prognosis was 5.63, significantly lower than patients with poor prognosis (9.12), P < .001. The median RDW in patients with good prognosis on the 7th postoperative day was 12.40%, significantly lower than patients with poor prognosis (13.20%), P < .001. There was no significant difference in hemoglobin level between patients with good prognosis and poor prognosis, P = .367  × 10P = .008). On the 1st postoperative day, the proportions of cases with lower quartiles of RDW and NLR were significantly lower than cases with higher quartiles (P = .004). On the 3rd postoperative day, the proportions of cases with lower quartiles of RDW (P = .001) and NLR (P < .001) were significantly lower than cases with higher quartiles. On the 7th postoperative day, the proportions of cases with lower quartiles of RDW (P = .003) and NLR (P < .001) were significantly lower than cases with higher quartiles.As showed in Table P < .001), RDW  on the 3rd postoperative day and NLR  on the 7th postoperative day were associated with the prognosis of aSAH patients , and the Youden index was 0.428. The sensitivity and specificity of RDW ≥ 13.05% on the 3rd postoperative day for predicting poor prognosis were 67.6% and 75.2% respectively , and the Youden index was 0.427. The sensitivity and specificity of NLR ≥ 6.97 on the 7th postoperative day for predicting poor prognosis were 41.2% and 89.9%   cases with hematoma dilation. The higher white blood cells at admission, the lower risk of hematoma dilation. Hematoma dilation was negatively correlated with the count of neutrophils.White blood cells and neutrophils were once considered sensitive biomarkers and proven to be associated with the prognosis of stroke. Tao C et al conducted a retrospective study exploring the associations between counts of blood cells and scores of mRS at 90 days in 336 patients with spontaneous cerebral hemorrhage. The results of multivariate analysis showed that higher white blood cells and neutrophils were associated with poor prognosis. Moreover, linear regression analysis showed that counts of these blood cells were significantly correlated with Glasgow Coma Scale score and cerebral hemorrhage volume at admission. In another study including 192 patients, there were 54 (28.1%) cases had neurological deterioration. NLR was independently correlated with neurological deterioration. NLR was the best variable for predicting poor outcomes. In our study, the associations of white blood cells, neutrophils, lymphocytes and NLR on different days with the 3-month prognosis were explored. The results of univariate analysis showed that counts of white blood cells and neutrophils on the preoperative, the 1st, the 3rd and the 7th postoperative days were all associated with prognosis. The results also showed that counts of lymphocytes on the 3rd and the 7th postoperative days, levels of NLR on the 1st, the 3rd and the 7th postoperative days were associated with prognosis. However, the result of multivariate logistic analysis only confirmed the association between NLR on the 7th postoperative day and the prognosis of aSAH patients.NLR was the ratio of neutrophil to lymphocyte, combining the changes of the 2 blood cells, with more efficacy and accuracy compared to a single indicator for predicting inflammatory response. Sun Y. et al conducted one prospective study in 352 patients with acute cerebral hemorrhage. All patients were divided into different groups based on the NLR levels at admission to evaluate the associations of NLR with hematoma volume, disease severity, and prognosis. After a 3-month follow-up, patients with NLR ≥ 7.85 had higher NIHSS scores and more hematoma volumes, but not poor 3-month prognosis. Lymphocytes and neutrophils were found in the brain in areas of acute ischemic stroke. Post-stroke immunosuppression was also observed after stroke. NLR ≥ 5.9 was associated with pulmonary edema and high fever. Among patients with aSAH, whether a higher NLR level represents a severe or acute stress, or increased neutrophils and decreased lymphocytes originate from immunologic response to acute aSAH remains unknown. Delayed neuroinflammation or systemic inflammatory response syndrome after subarachnoid hemorrhage possibly resulted in a high NLR, which could be the trigger of secondary harmful hematologic or immunologic host responses. Neuroinflammation after acute cerebral hemorrhage could be reflected by acute leukocytosis and decline of lymphocytes. Infiltrated neutrophils could release inflammatory cytokines, and enhanced perifocal edema by promoting capillary permeability, cell swelling, and blood-brain barrier damage. Lymphocytes were the main immune regulatory cells, and their decrease and inactivation were markers of brain injury, which were the results of excessive activity of the sympathetic nervous system. On the other hand, patients with lower lymphocytes and higher neutrophils possibly developed concomitant infections, which also increase the risk of poor prognosis. Unfortunately, we did not pay attention to the characteristics of infected patients.NLR can get affected in systemic inflammatory response syndrome, sepsis, post-surgical state and many other confounders. The patients with abdominal infection had elevated counts of neutrophils before surgery and low lymphocyte counts. Critically ill patients with severe sepsis or septic shock had significantly highest values of neutrophil relative counts and marked lowest values of lymphocyte counts. One systematic review and meta-analysis exploring the association of RDW with prognosis in aneurysmal subarachnoid hemorrhage patients confirmed that higher RDW was associated with a worse functional outcome, a worse discharge and 3-month functional outcome, and a higher in-hospital and 90-day mortality. In another study carried in 8175 community-dwelling adults 45 years or older and exploring the association between RDW and risk of death, the results showed that RDW was a widely available test and a strong predictor of mortality in the general population of adults 45 years or older. In our study, the univariate analysis results showed that the levels of RDW on the preoperative day, the 1st, 3rd and 7th postoperative days were all associated with the prognosis of aSAH patients, further suggesting that RDW might be a highly advantageous prognostic indicator. Moreover, the results of multivariate analysis confirmed the association between level of RDW on the 3rd postoperative day and the prognosis. The results by ROC curve in our study also showed that the cutoff value of RDW for predicting the prognosis in aSAH patients was 13.05%. The sensitivity and specificity of RDW ≥ 13.05% for predicting poor prognosis were 67.6% and 75.2%, both relatively high. Some possible mechanisms by which higher RDW was associated with poor prognosis in aSAH patients were explored. Higher RDW levels were associated with endothelial dysfunction and increased levels of inflammatory cytokines, and reflected the potential pro-inflammatory state.,25 The higher the RDW level, the greater the likelihood of thrombosis formation in patients. The increase of RDW level indicated the increase of immature reticulocytes, which had worse deformability and stronger thrombolytic ability than mature erythrocytes. Deformed erythrocytes could accelerate the process of platelet aggregation, damage blood vessels, and reduce blood flow. Higher RDW levels were also associated with lower antioxidant levels and higher oxidative stress levels, which could weaken the deformability of erythrocytes, increase adhesion between erythrocytes and endothelial cells, and promote thrombosis. In addition, the lifespan of erythrocytes was approximately 120 days. Therefore, RDW possibly reflect the long-term inflammatory state of aSAH patients.There were a few studies on the association of RDW with prognosis of aSAH patients. The results of one study including 274 patients with aSAH admitted to the intensive care unit confirmed a significant association between RDW levels and mortality.The prognosis differences among patients with different quartiles of RDW and NLR in Table Our study have some limitations. First, the dataset had some bias. There were two-thirds or over cases with a good Hunt-Hess grade (I/II) in all patients or in patients with good outcome, one-third or less cases with a poor Hunt-Hess grade (III/IV/V). The good outcome was possibly driven in general by a favorable natural history due to minor SAH in the majority of patients with a good Hunt-Hess grade. Second, the patients with good outcome by far outweighed the patients with poor outcome, so the patients with poor outcome might be underpowered. Third, the number of patients was not large enough, which possibly caused some bias. We will investigate the associations in larger-sample study. Finally, characteriazatioin of patients who developed hospital or out-hospital infections did not get noticed in our manuscript, which was an additional limitation.In summary, higher RDW on the 3rd postoperative day and NLR on the 7th postoperative day were possibly associated with poor prognosis of aSAH patients. We should pay attention to the RDW and NLR levels during different hospitalization periods, especially in the short postoperative period. Moreover, the cutoff values for predicting prognosis need to be validated in larger-sample studies.We are thankful to all the medical staff in the Neurointensive care unit and the statistician, in The First Affiliated Hospital of Yangtze University.Conceptualization: Jie Min, Yongfeng Zhao, Xian Wang.Data curation: Xian Wang.Formal analysis: Yongfeng Zhao.Methodology: Jie Min, Jian Zhao.Project administration: Xian Wang, Jian Zhao.Writing – original draft: Jie Min, Yongfeng Zhao.Writing – review & editing: Jie Min, Xian Wang.


Human papillomavirus (HPV) is associated with 5% of all cancers globally at a range of body sites, including cervix, anus, penis, vagina, vulva, and oropharynx. These cancers claim > 400,000 lives annually. The persistent infection of HPV and the function of viral oncogenes are the primary causes of HPV-related cancers. However, only some HPV-infected persons or infected lesions will progress to cancer, and the burden of HPV-associated cancer varies widely according to gender and the part of the body infected. The dissimilarity in infection rates at different sites can explain only a small part of the differences observed. Much responsibility likely sits with contributions of specific epithelial cells and the cellular microenvironment at infected sites to the process of malignant transformation, both of which affect the regulation of viral gene expression and the viral life cycle. By understanding the biology of these epithelial sites, better diagnosis/treatment/management of HPV-associated cancer and/or pre-cancer lesions will be provided. Forty yOne of the most distinctive characteristics of HPV is the genotype-specific preference for distinct anatomical sites (tropism) of particular HPV types, where they cause lesions with distinctive clinical pathologies . TheGiven the impact of high-risk HPV as a cancer causative agent, it is reasonable that the study of HPV has been driven by the association of the high-risk Alphapapillomavirus types to anogenital and oropharyngeal cancers . By compSome of the notable functions of the high-risk E6 protein are its interaction with and degradation of TP53 and BAK, which result in the inhibition of growth arrest and apoptosis after DNA damage, as well as an accumulation of mutations , 15. E6 Given the malignant transformation of the infected lesion, which produces no virus progeny, the development of cancer from lesions does not contribute to the virus fitness and is considered to be a collateral consequence of the infection . FurtherAlthough high-risk HPV infections are common, with the majority of people being infected at least once in their lifetime, most will develop only subclinical or transient lesions that spontaneously regress, and invasive cervical cancer will only develop in some persistent infections/lesions. Also, it is now being accepted as the natural history of HPV infection that HPV infection is not always cleared completely but becomes a controlled infection below the limits of detection (latent infection) , 35, whiClinically, HPV infection in the cervix is classified depending on its risk of developing precancer and cancer. For example, a subclinical infection can be HPV-positive, but no cervical abnormalities are detected. Though the detection of high-risk HPV is considered as the risk of developing precancer and cancer, the lack of detectable cervical lesions is considered as there being no immediate risk of cancer. Such subclinical infections have become more common since the HPV test was introduced as part of primary cervical cancer screening. CIN1, which is referred to as the LSIL  designation used in the Bethesda system, also does not show a significantly higher risk of precancer lesions . As therHistopathologically, the classification of CIN1-3 reflects the extent to which basal-like cells (undifferentiated cells with a high nuclear/cytoplasmic ratio and nuclear atypia) have replaced the cervical epithelium. Since the discovery that cervical dysplasia is an HPV-infected lesion, our understanding of cervical dysplasia has become much more advanced. Low-grade lesions (CIN1) typically represent a "productive infection", where the level and pattern of viral gene expression are properly controlled to complete the stages of its viral life cycle and to produce virus progeny. In contrast, high-grade lesions represent "non-productive" or "abortive infection", where the expression of viral genes is dysregulated  and no infectious virus is produced. Virologically, the extent to which basal-like cell replacement has occurred reflects the extent of viral oncogene expression.The deregulated expression of high-risk E6 and E7 proteins is the primary risk of the development of cancer, and it explains the difference in the risk of developing cancer among cervical dysplasia. Deregulation of viral gene expression may be caused by viral genome integration, which is often observed in high-grade premalignant lesions and cancer , 43. Suc(i) Tobacco smoking has shown a significant increase in the development of cervical cancer and its precursor lesions . The mol(ii) The long-term use of oral contraceptives has been hypothesized to be associated with development of precancer and invasive cervical cancer. The regulatory region of HPV genome contains hormone response elements that can be stimulated by estrogen and affect the proliferative capacity of the infected cells. There is also some evidence of cooperation between estrogen and HPV in the development of cervical cancer in humans and in model systems. However, the interpretation of studies of oral contraceptives for risk of cervical cancer has been impeded by limitations in design  confounded by Pap smear history , and the formulation and dose of hormonal contraceptives. In summary, the long-term (5 yeas o more) use of oral contraceptives can increase the risk up to twice as much , with li(iii) The microbial microenvironment in cervical tissue is considered to determine the integrity of homeostasis and protective mucous layer of cervical tissue by maintaining healthy immunity and metabolic signalling. This can affect HPV infection or risk of persistent/dysregulated infection by changing viral gene expression. The role of chronic inflammation is less certain, with very limited data about Chlamydia trachomatis infection . The inf(iv) There have been numerous studies which examined the association between risk for cervical cancer and dietary intake or nutrient concentrations, including vitamin C, folate, β-Carotene, retinoic acid. Most of the studies were small and high rates of spontaneous regression of lesions hampers sufficient power to test the efficacy of the selected agents. In addition, most of these studies have methodological limitations in analysis, lack of adequate consideration of HPV infection, and confounding factors . Due to these limitations in observational studies and supplementation trials, there has been no strong evidence of a role for any one micronutrient in the development of cervical cancer. Limited data, though, suggests that higher folate is possibly protective against the risk of malignant transformation.(v) Familial clustering of cervical cancer has been explored as a potential risk marker of inherited genetic susceptibility for cervical cancer. In general, these studies failed to differentiate sufficiently the effects of genetic susceptibility from those of environmental and behavioural factors, with indications of a familial risk, and requires further investigation. As the control of HPV infection (spontaneous regression of LSILs and even HSILs) is mediated by cellular immune responses against virus-specific antigens, it is reasonable that the polymorphisms of HLA Class I and II genes lead to variations in the antigen recognition site that may confer susceptibility or resistance to HPV infection and malignant transformation. HLA recognition is considered to be HPV type-specific, and HLA polymorphisms can also explain that HPV type-specific prevalence varies according to geographic area and ethnicity. Indeed, epidemiological studies in different populations have found various relationships between risk for malignant transformation and HLA polymorphisms. However, it has not been utilised for diagnosis/treatment/management of cervical pre-cancer lesions.Compared to the infection of carcinogenic high-risk HPV types, non-infectious co-factors play a minor part in cancer development. Many potential co-factors, which may affect the risk of infection or risk of persistent/dysregulated infection, have not been evaluated rigorously enough in epidemiological studies (see ).(i) TobThe primary risk of HPV-associated cancer development is a function of HPV infection. High-risk human papillomaviruses infect a wide range of epithelial sites, however, the frequency of HPV-associated cancer among the sites of bodies shows huge diversity Fig.  due to tThe infection of high-risk HPV types is associated with virtually all (> 99%) cervical cancers. In some developed countries, the number of cases other HPV-associated cancers is increasing. The incidence of HPV-associated head and neck cancer is also increasing and is greater than the incidence of cervical cancer. However, cervical cancer is the most prevalent (> 80%) cancer among all HPV-associated cancers worldwide. While cervical cancer reaches over 600,000 cases per ear worldwide, HPV-associated cancer of the vulva, vagina, and penile cases reaches around 50,000 per year . The cerHead and neck squamous cell carcinoma (HNSCC) develops at different anatomic sites of the upper aerodigestive tract including the nasopharynx, paranasal sinuses, oral cavity, oropharynx, hypopharynx, and larynx. Since HPV was discovered in cervical cancer, HPV has also been recognised as a causative agent for HNSCC, especially for oropharyngeal squamous cell carcinoma (OPSCC). The main difference when compared with cervical cancer is that OPSCC can be observed as two (HPV-negative and HPV-positive) diseases. The socioeconomic status of patients, molecular profiles, clinical characteristics, and especially the prognosis of the tumours, tend to differ between HPV-positive and HPV-negative OPSCC. High-risk HPV-associated OPSCC makes up a substantial proportion of all OPSCC cases and this number is rising, mainly in the Western world. This is most likely due to changes in sexual behaviour , 63. TheHPV infection is also associated with a small number of other HNSCCs, however, the contribution of each site varies hugely  accounts for only 4% of cases, while in contrast, 47% of cases of OPSCC is HPV-associated). HPV-positive OPSCC often arises from the tonsillar crypt epithelium. Interestingly, reticular tonsillar crypt epithelium would provide easier access to HPV than other stratified squamous epithelium, and it suggests that its malignant transformation reflects the different biology of infected tissue at each anatomical site. The tonsillar reticular crypt epithelium might not be permissive for productive HPV infections that support the viral life cycle to generate viral progeny, but may be permissive for abortive/non-productive infection, where viral gene expression is dysregulated and likely to progress to malignant transformation . This is consistent with the observations that large studies on non-malignant tonsil samples have detected no or low HPV prevalence, and LSIL-type productive lesions, which are the most common infected lesion at cervix, have not been identified as a cancer precursor at the tonsillar crypt.High-risk HPV has been detected in 90% of anal cancers with HPV16 detected in 80% of those cases , 65. TheTwo distinct pathways to invasive squamous cell carcinoma (SCC) exist in the vulva with HPV being detected in one-third of vulvar SCC . This diHigh-risk human papillomavirus infection causes HPV-associated cancer, with a great diversity of its incidence among the sites of the body infected. It appears that differences in infection rates at different sites are only a small part of the reason for this. Specific epithelial stem cells and the cellular microenvironment at these sites must be influencing the control of viral gene expression and contributing differently to the process of malignant transformation. In contrast to our understanding of high-risk HPV protein functions, our knowledge of the biology/molecular mechanisms of the different epithelial sites is very limited, and further studies are required to provide better diagnosis/treatment/management of HPV-associated cancer and/or pre-cancer lesions.


Cervical cancer is a major public health challenge, accounting for substantial morbidity and mortality. Human Papilloma Virus (HPV) vaccination is the recommended primary public health intervention for HPV infection prevention. However, there’s limited evidence on the level of knowledge, attitude, and practices of adolescent girls regarding HPV vaccination in Kampala city, Uganda. This study assessed the knowledge, perceptions, and practices of adolescent girls aged 10-14 years towards HPV vaccination program in Kampala, Uganda to generate evidence to guide programs targeted at improving uptake of the vaccine.A convergent parallel mixed methods study was conducted in Kampala, Uganda. A structured questionnaire was used to elicit data from 524 adolescent girls. In addition, 6 Focus group discussions, and 24 key informant interviews (teacher and parents) were conducted. Multistage and purposive sampling techniques were used to select quantitative and qualitative participants respectively. Quantitative data were entered using epidata, cleaned and analyzed using Stata v14 while qualitative data were analyzed using thematic content analysis in atlas ti version 8.Overall, only 8.6% (45/524) of the girls had completed the HPV vaccine schedule of two dozes, 49.2% (258/524) of the girls had low knowledge about the HPV vaccine and teachers and parents affirmed this lack of knowledge among adolescent girls especially concerning the target age group, dosage, and vaccine interval. About 51.9% (272/524) of girls had negative perceptions towards HPV vaccination. Parents expressed negative perceptions, beliefs, superstitions, and safety concerns of the vaccine.Girls residing in rural areas  had lower knowledge levels compared to those in urban areas. Girls whose mothers were healthcare providers , girls with high knowledge levels  and positive perceptions  had a higher prevalence of being fully vaccinated.Girls generally had low levels of knowledge, negative perceptions, and poor uptake of HPV vaccination. We recommend sensitization campaigns in schools and communities to improve awareness, perceptions, and practices of stakeholders towards HPV vaccination. Cervical cancer is the fourth most prevalent cancer in women globally, with an estimated 570,000 new cases and 311,000 deaths in 2018 . FurtherHuman papillomavirus (HPV), a group of extremely common viruses is responsible for causing cervical cancer. HPV is mainly transmitted through sexual contact and the majority of cases are infected shortly after onset of sexual activity . DespiteStudies elsewhere have shown that knowledge and perceptions can predict uptake of health services including HPV vaccination, –10. In aOur study utilized a convergent parallel mixed-methods design for data collection. This design involved the collection and analysis of both quantitative and qualitative data, and thereafter the two were compared and integrated. This allowed us to gather a more comprehensive understanding of the factors influencing knowledge and perceptions of HPV vaccination among girls in our study population. Data collection was conducted in Kampala between February and April 2021. Kampala is the capital and largest city in Uganda with an estimated population of 1,507,114, of which 724,326 are males  and the The Kish Leslie (1965) formula was used to determine sample size . A prevaAll the five divisions in Kampala were considered. In each division we sampled 105 adolescent girls to come up with the sample size required. A list of primary schools in each division was obtained from Kampala Capital City Authority (KCCA), the urban authority body of the city. We then used random sampling to select four schools from each division’s list using the Ms Excel randomizer. This gave a total of 20 schools. The sample size of 521 was divided among the 20 schools and each was to give 26 pupils to meet the sample size as indicated in Fig. Qualitative respondents were purposefully selected from each division and included the following: adolescent girls aged 10–14 years, their teachers, and parents. The teachers and parents were selected based on their knowledge and experience, level of influence and involvement in the health and well-being of the adolescent girls, and willingness to share information. Furthermore, we selected teachers based on their legal registration status at the respective schools and key roles in the girls’ lives as health advisors in the school administrative structure . Adolescent girls for the FGDs were selected based on their experiences receiving or not receiving HPV vaccination and their willingness to share information. For representativeness of views and gender inclusivity, among the key informants, the study purposively engaged equal numbers of males and female teachers and parents.A semi-structured questionnaire was used to obtain data from adolescent girls aged 10–14 years. The tool contained questions on the knowledge about the vaccination program, perceptions towards the HPV vaccine and vaccination and practices towards HPV vaccination, as adopted from the African Women Awareness of cancer (AWACAN) tool for breast and cervical cancer ; and modFGD guides and KII guides were used to explore the knowledge, perceptions, and practices of the pipuils and key informants towards the HPV vaccination program. The FGD and KII guides were developed through a process of review of existing literature on HPV vaccination and in consultation with experts in the field of reproductive health in the Ministry of Health and qualitative research methods at the Makerere University School of Public Health (MakSPH). The guides were designed to explore the key themes and topics related to knowledge, perceptions, and uptake of HPV vaccine among adolescent girls and their parents/guardians and teachers. The guides were pretested with a small group of participants to assess their clarity, relevance, and comprehensiveness before the actual data collection. The FGD and KII guides were also modified iteratively during the data collection process based on the emerging themes and issues from the previous interviews/discussions.The research team engaged parents in the study during morning or evening hours as they dropped off and picked up pupils from school while, teachers and adolescent girls were engaged during appropriate breaks such at break tea or lunch time. After meeting the identified stakeholders, the study team did self-introductions, explained the purpose of the study, and then sought informed consent before engaging interviews. We conducted FGDs with the adolescent girls because we wanted to gather their perspectives on knowledge, perceptions, and uptake of the HPV vaccine in a group setting, where they could interact and share their experiences with one another. Each FGD consisted of 7–12 adolescent girls. This allowed for a more comprehensive understanding of their attitudes and beliefs towards the vaccine. On the other hand, we conducted key informant interviews (KIIs) with the adults because they were seen as more knowledgeable and experienced about the vaccination process and the factors that influence uptake. Additionally, it was easier to schedule interviews with the adults than to arrange for them to attend group discussions, especially considering their busy schedules. All qualitative interviews were moderated by a female well-trained research assistant with vast experience in qualitative research and work with young adolescent girls. The moderator was assisted by a note taker to ensure no information was missed out. The surveys lasted approximately 30 min, the KIIs 40 min while the FGDs took about an hour.The main outcome variables included; the level of knowledge, perceptions and uptake of HPV vaccination among adolescent girls. Knowledge was measured using a set of 8 questions. For each question, a correct response was awarded a score of 1 and any wrong response was awarded 0. This made the total score 8 in case a respondent passed all the question and 0 if they failed all. The knowledge score ranged between 0 (minimum mark observed) to 8 (maximum mark observed). A respondent’s score therefore depended on how many questions they passed. The mean score was 4.05(SD: 2.41), the median score was 5 and range (8 − 0) was 8. The median score (5) was taken as a cut-off point. Those who obtained a score ≥ 5 were considered as knowledgeable and those who scored below the median score were considered as having a low level of knowledge. The same approach had been applied in other studies to assess the level of knowledge on HPV vaccination , 19.≥ 8 were considered as having positive perceptions and those who scored below 8 were considered as having a negative perception.A 3-point Likert scale  was used to measure perceptions of girls on HPV vaccination using a set of 13 statements. For each question, a desired perception was awarded a score of 1 and the undesired perception and neutral was awarded a zero. The minimum score observed was 0 and the maximum score was 13. The perceptions score therefore ranged from 0 to 13 depending on how the respondent agreed or disagreed to the statements. The mean score was 7.83(SD: 3.01), the median score was 8 and the range (13 − 0) was 13. The median score (8) was then taken as a cut-off point and those who obtained a score Conversely, uptake of the HPV vaccine was measured by asking the girls if they have ever received the HPV vaccine. Girls responded Yes or No depending on whether they have ever been vaccinated against HPV or not and a follow up question on number of dozes received was asked.The independent variables included socio-demographic factors such as; age, religion, education level, parents’ occupation, and sex debut. Age was collected as a continuous variable and later categorized into (10–12 and 13–14) years. Education level was categorized into primary five, six, and seven. The education levels in Uganda include pre-primary, primary, secondary and post-secondary. Primary level has seven classes, (primary one to seven), each class running for a period of one year. The official school going age bracket for pre-primary level is 3–5 years; 6–12 years for primary level, 13–18 years for secondary level and 19–24 years for post-secondary school level. However, due to the impact of the COVID-19 pandemic to the education system, we were able to recruit girls aged 13–14 at primary level . ReligioBoth qualitative and quantitative tools were translated to Luganda and the researchers ensured to only recruit research assistants who were conversant with Luganda since it was the commonest language spoken by the prospective respondents. Research assistants with a good command of English were recruited to conduct interviews, however, the interviews were conducted in a language most comfortable to the respondent. Research assistants were trained on the research protocol and ethical issues surrounding the study. To ensure the appropriateness of the questions, reliable and accurate information, pre-visits to the study setting and pretesting of the study tools were done. The tool was pre-tested from schools in Wakiso district, an equally urban area with similar characteristics as those in the sampling frame in Kampala. Furthermore, the research assistants were supervised by a team led by the Principal Investigator during the actual data collection exercise. The supervisors ensured that the tool is checked and field edited, if necessary, to ensure completeness of data before data entry.Quantitative data was field edited for consistency and omissions. Data materials were secured under lock and key and were only accessed by the study team. The data was entered using Epidata, edited for consistency, and then exported to STATA 14 for statistical analysis. Descriptive analysis was done to generate the mean and the standard deviation for continuous variables and proportions for categorical variables. Frequency tables were used to present these results. The modified Poisson regression model was used for further analysis to establish the associations between the independent and outcome variables at both bivariate and multivariable analysis. Modified Poisson regression has been shown to provide more accurate estimates of the strength of the association in situations where the outcome is common (greater than 10%) –24. At tAll qualitative interviews were digitally recorded with permission from respondents and transcribed verbatim. The transcripts were proofread before importing them into a qualitative data management software- atlas.ti6. Data coding and analysis were conducted subsequently. An initial codebook using a sample of transcripts was developed. The developed codebook was then applied to the entire atlas project with any emerging codes being added in the process. Thematic analysis was used and results were presented using themes with typical quotations from different qualitative interviews to summarize knowledge and perceptions. Content analysis was used for practices and these were summarized using a matrix.A total of 524 girls were interviewed. The mean age of the respondents was 12.9 years (SD ± 0.8). Close to two-thirds, 63.9% (335/524) of the respondents were aged 13–14 years, 97.1% (509/524) were in primary seven. and 95.8% (502/524) were residing in an urban area. Close to a third, 31.7% (166/524) of the respondents were catholic, and less than a tenth, 7.1% (37/524) had ever had sexual intercourse  of the respondents had low levels of knowledge on HPV vaccination, 70.6% (370/524) were aware of the HPV vaccine, 66.6% (349/524) believed that the HPV vaccine reduced the risk of cervical cancer, and 68.7% knew the target age group for the HPV vaccine. More than half, 54.8% (287/524) knew the recommended number of HPV vaccine doses and 52.9% (277/524) did not know the correct interval between the HPV vaccines doses. About 17.0% (89/524) of the respondents wrongfully mentioned that the vaccine is only for sexually active people  came here last time, they took girls for the first dose then they came for the second dose. I don’t know if that was all. I am not sure whether 3 or 4, they did not tell us or the girls. So I don’t think the girls know” “Well, those nurses who came said they wanted children of 14 years and in some cases, they said they wanted people who were still producing. So, I don’t know who is recommended but I think sexually active women are recommended.” “Some key stakeholders also expressed during key informant interviews that they were not sure of some of the information related to the vaccine, including the target age group and recommended number of doses and also believed the girls may not know.You can receive it (HPV vaccine) when you are about 9 to 12 years. It is preferred that you receive the vaccine when you are a virgin. It is given before sexual contact. The vaccine is given to females and males, and they are given two doses. The second interval is after six months after the first interval.” “However, in the FGDs with adolescent girls, some respondents were knowledgeable about the vaccine, target population for the vaccine, when it’s preferable to receive the vaccine, number of doses and interval between the doses. A girl expressed that;At a multivariable analysis, area of residence and religion were significantly associated with level of knowledge of girls on HPV vaccination. The prevalence of a high knowledge level was 0.38 times higher among respondents who were residing in a peri-urban area  compared to those living urban areas. Furthermore, the prevalence of a high knowledge level was 38% higher among respondents from the Pentecostal faith  compared to those from the catholic faith  had negative perceptions towards HPV vaccination.Only 49.1% (257/524) agreed that the HPV vaccine would be beneficial to a teenage girl or boy’s future health, and 36.3% (190/524) disagreed that HPV vaccination is not necessary because a Pap test can be done to rule out cervical cancer. About 27.9% (146/524) of the girls agreed that if other people knew they received the HPV vaccine, they would be embarrassed and 17.4% (91/524) believed that it would be difficult and embarrassing for them to ask for the HPV vaccine because it is associated with a sexually transmitted infection. Close to three quarters, 72.7% (381/524) agreed that they would be willing to recommend the HPV vaccine to others and 80.5% (422/524) agreed that they would be willing to receive the HPV vaccine if offered for free (Table They (Parents) have got all sorts of beliefs originating from people out there. People tell parents that, “you people, your children are going to be barren because of the vaccine.” So, somebody thinks about that and has to pick between cancer and infertility for their children. When girls are drawn into all this, it affects their perceptions too” “Yeah, usually there are superstitions within the community and among girls that the government does not want the girls to produce many children” “Some parents are against the vaccine because of their political views. Some are anti-government and think that whatever program the government brings could be detrimental to their children’s health.” “For me I wanted but when I brought the letter home, (consent form) and gave it to my parents, my mother told me that I should not go to school that day; that she talked with the teacher on my behalf.” “Negative perceptions toward HPV vaccination were also recorded especially among parents and these are passed down to the girls. A respondent was quoted expressing that some parents are against vaccinating their children due to the beliefs that the vaccine will make the children barren/infertile. Furthermore, some respondents believed that the government was aiming at wiping their children out through HPV vaccination.P -value = 0.000) higher among girls with a high knowledge level compared to those with a low level of knowledge  had not received a single shot of HPV vaccine, 27.5% (144/524) had ever received the HPV vaccine and only 8.6% (45/524) had completed the vaccination schedule of two dozes. Majority of the vaccinated girls 84.7% (122/144) received their first shot while in the age range of 10 to 12 years. The most mentioned reasons for failure to receive the vaccine was not being aware of the HPV vaccination, 48.2% (183/524) and where to access the vaccine, 28.4% (108/524) “I haven’t taken her for HPV vaccination. We’ve all been reluctant to do so because we have concerns about the side effects” “Parents also expressed that they had not supported their daughters to go for HPV vaccination mainly because of fear of side effects. In contrast, teachers mentioned that in support of HPV vaccination program, they provide information on HPV, encourage children to get immunized, and support healthcare workers in whichever way they could.At multivariable level, girls’ knowledge level, perceptions regarding HPV, and their mothers’ occupation were significantly associated with receiving the HPV vaccine. The prevalence of HPV vaccination was 94% higher among girls whose mothers were healthcare providers by profession  compared to those whose mothers were civil servants. Girls with high level of knowledge on HPV vaccination  reported a higher prevalence of HPV vaccination compared to those with a low knowledge level. Girls with positive perceptions toward HPV vaccination  had a higher prevalence of HPV vaccination compared to those with negative perceptions ” “Sensitization is lacking. People are not aware that this vaccine is available and the healthcare facilities that offer this vaccine are not very common. You find the health centers or medical facilities that have those vaccines are fewer, which might affect uptake. People think that they are supposed to pay some money to access the vaccine and they don’t know that it’s free of charge. It’s now up to the government to do some bit of sensitization. You find that most parents are illiterate. The illiterates may not understand the benefits, so we need more sensitization about it.” “Respondents further affirmed that knowledge regarding HPV vaccination among the different key stakeholders, including parents and teachers is really low due to a lack of sensitization on the matter and it could be affecting uptake of the vaccines. In this regard, key informants were quoted saying;This study aimed to assess stakeholders’ knowledge, perceptions, and practices towards the HPV vaccination program in Kampala. The findings revealed that 49.2% of the girls had low knowledge of HPV vaccination. Girls residing in peri-urban areas had lower knowledge levels compared to those in urban areas. About 52% of girls had negative perceptions towards HPV vaccination and only 8.6% (45/524) of the adolescent girls had received both doses of the vaccine. Girls whose mothers were healthcare providers, girls with high knowledge levels and those with positive perceptions had a higher prevalence of those vaccinated with at least one doze. Qualitative findings supported the quantitative analyses.Almost half (49.2%) of the girls had low levels of knowledge regarding HPV vaccination. Similarly, a knowledge gap was revealed among other key stakeholders  on the causes and risk factors for HPV and HPV vaccine. A low knowledge level among parents and teachers is likely to impact the girls’ knowledge, attitudes, and practices concerning HPV vaccination. These findings concur with those in a study conducted in Lira district which showed that adolescent girls had poor knowledge levels (56.1%) . Other sAfter adjusting for confounders, girls residing in peri-urban areas were 0.4 times less likely to have a high level of knowledge compared to those in urban areas. This could be because girls in urban areas are exposed to various sources of information which could be lacking or limited in rural areas. A review of the literature has supported these findings. For instance, a study conducted by Chen, Orom  revealedRegarding perceptions toward HPV vaccination, this study found that 51.9% of the girls had negative perceptions towards HPV vaccination. The negative perceptions could be explained by the lack of knowledge and limited sensitization among the girls as highlighted by the key informants in this study, hence contributing to the negative perceptions. These findings corroborate those in another cross-sectional study which revealed uncertainties about the need for the vaccine among the girls in terms of perceived risk, and safety concerns of the vaccine . This, tSimilarly, other stakeholders portrayed negative perceptions towards HPV vaccination. The study found negative beliefs, superstitions, and safety concerns among parents, which are likely to influence their willingness to get their daughters vaccinated. As indicated in the literature, knowledge level can greatly impact individuals’ perceptions. Therefore, the negative beliefs portrayed in this study could be due to a lack of awareness among these stakeholders as remarked by some of the key informants. The study findings concur with those in other cross-sectional studies which also found that parents and teachers had negative attitudes toward HPV and the vaccine , 30.Girls with a high level of knowledge regarding HPV were more likely to have positive perceptions compared to those with a low level of knowledge. Knowledge has been shown to predict individuals’ attitudes toward a behavior. This could be because an increase in the level of knowledge leads to a better understanding of the inherent risks related to HPV and cervical cancer, which consequently translates into positive attitudes towards the uptake of the vaccine. Therefore, change of perceptions towards the uptake of the HPV vaccine may require continuous sensitizations.The study estimated the level of uptake of HPV vaccination (receiving two doses of the vaccine) at 8.6%, which is significantly way below the targeted coverage of 80% . This loFrom the qualitative findings, teachers exhibited good practices toward HPV vaccination. They expressed supporting healthcare workers in HPV vaccination through availing information to the girls and parents to enhance their willingness to take up the vaccine. In contrast, the study revealed poor practices among parents concerning vaccination. The difference in practices among teachers and parents could be because teachers are exposed to health-related information while at school which could lead to a better understanding of the need for vaccination and consequently influence their practices .The study also indicated that the prevalence of HPV vaccination were higher among girls with positive perception towards the HPV vaccine, and girls whose mothers were healthcare providers by profession. The positive perceptions including, perceived susceptibility to HPV, the severity of cervical cancer, benefits, and efficacy of the HPV vaccine could motivate the girls to take up the vaccine. Such girls would therefore have better practices concerning HPV vaccination. However, these findings do not corroborate those in a study conducted in England which found no significant correlation between attitude and HPV vaccination uptake .This study reached a large number (524 adolescent girls aged 10–14 years) which makes the findings more reliable and generalizable. In addition, the study included a qualitative component which further triangulated the quantitative findings. The practices were however assessed based on self-report and self-reported responses are prone to recall bias and social desirability bias. In our case, we did not ask to look at the vaccination cards of the adolescents and therefore could not ascertain whether the girls were actually vaccinated or not. We however minimized the bias in responding by emphasizing the importance of the study and the need for them to answer questions correctly. Our sampling strategy involved selection of all five divisions in Kampala and each division producing 104 students to come up with the sample size required. This may have affected representativeness of HPV vaccine uptake. However, given that all the five divisions have government hospitals where HPV services including vaccination are given for free, we believe that this sampling strategy is representative of Kampala City. Additionally, this was a cross-sectional study design and is limited in its ability to establish temporality between the exposure and outcome variables. There’s a need for more rigorous studies with longitudinal designs to better understand the relationships between HPV knowledge, attitudes, and vaccine uptake.Adolescent girls in this study had relatively low levels of knowledge about the HPV vaccine, more than half had negative perceptions towards the vaccine, and less than a tenth had completed the vaccination schedule. The low uptake was attributed to lack of awareness on HPV vaccination and where to access the vaccines. This indicates a critical need for awareness raising about HPV vaccines among all stakeholders. Girls residing in urban areas had better knowledge levels compared to those in peri-urban areas. Girls with high knowledge levels had positive perceptions towards vaccination and positive perceptions were associated with uptake of at least one doze of the HPV vaccine. Qualitative findings further affirmed low knowledge levels, negative perceptions, and beliefs about the vaccine among adolescents and parents.


Classical oropharyngeal squamous cell carcinoma (OPSCC) caused by alcohol consumption and smoking and HPV-associated OPSCC caused by human papillomavirus (HPV) infection have different etiologies, incidences, and prognoses. Therefore, the 8th American Joint committee on Cancer (AJCC) and Union for International Cancer Control (UICC) TNM classifications propose distinguishing HPV-associated OPSCC from classical OPSCC and classifying it as an independent disease. Therefore, this review provides an overview of HPV-associated OPSCC from the perspectives of epidemiology, carcinogenesis, development, diagnosis, treatment, and prevention. The incidence of HPV-associated OPSCC is increasing. Although HPV vaccination has been shown to be effective at reducing the incidence of cervical cancer, it is still unclear how it affects the incidence of HPV-associated OPSCC. Additionally, the prognosis of patients with HPV-associated OPSCC is extremely favorable compared to that of patients with classical OPSCC. Therefore, patients with HPV-associated OPSCC may undergo reduced-dose therapy, although attempts to reduce treatment intensity should be carefully planned to ensure they do not compromise oncological outcomes, and large-scale trials aimed at reducing treatment intensity are ongoing. Human papillomavirus (HPV) contributes to the carcinogenesis of various cancers, including cervical, oropharyngeal, and anal cancers. Cervical cancer has long been known to be an HPV-associated cancer. Therefore, HPV vaccination has been introduced to prevent cervical cancer in developed countries. However, the association of HPV with oropharyngeal cancer was recognized only recently, and the full picture of HPV-associated oropharyngeal cancer has been gradually unraveled. Classically, oropharyngeal squamous cell carcinoma (OPSCC) was believed to be caused by alcohol or tobacco use. However, Mork et al.  reportedOver the past two decades, smoking and alcohol consumption rates have continuously decreased in developed countries. Consequently, the incidence of classical OPSCC related to alcohol consumption and smoking has also begun to decrease. In contrast, the incidence of HPV-associated OPSCC has increased with an increase in HPV infection rates. HPV-associated OPSCC now accounts for approximately 70% of all OPSCC cases in the United States , 3. SimiSince HPV-associated OPSCC shows markedly favorable oncological behavior compared to classical OPSCC , the latIn this review, we provide an overview of HPV-associated OPSCC from the perspectives of epidemiology, carcinogenesis, development, diagnosis, treatment, and prevention.In Europe and the United States, HPV vaccination has resulted in a decrease in cervical cancer, which is the most common HPV-associated cancer. The incidence of cervical cancer decreased by 1.6% annually between 1999 and 2015. Conversely, the incidence of OPSCC increased by 2.7% annually in males and 0.8% annually in females during this time period , 11. On Sexual behavior is a well-recognized risk factor for HPV-associated OPSCC, and a strong association between the number of lifetime oral sex partners and the incidence of this disease has been reported , 12. ThiMost HPV-associated OPSCCs occur in the palatine and lingual tonsils. This suggests that the specialized histological structure of tonsil tissue is associated with HPV infection. Tonsil tissue consists of a reticulated epithelium lining the crypts. In the reticulated epithelium of the tonsil, epithelial disruption leaves the basal cell layer and basement membrane exposed to viral deposition without mucosal trauma . By contIntegration of HPV DNA into the human genome is also an important step in the carcinogenesis of OPSCC . The oncNeck masses and sore throats are the most common initial symptoms of OPSCC. Patients with HPV-associated OPSCC are more likely to notice a neck mass as the first symptom, whereas patients with classical OPSCC usually complain of sore throat, dysphagia, or odynophagia as the first symptoms .HPV-assoHPV-associated OPSCC is more sensitive to chemotherapy and radiotherapy than classical OPSCC, and many studies have reported that HPV-associated OPSCC has an extremely favorable prognosis compared to classical OPSCC . In the Given these findings, recent TNM classifications have proposed distinguishing HPV-associated OPSCC from classical OPSCC and classifying HPV-associated OPSCC as an independent disease. Since p16 immunostaining has high sensitivity and specificity for high-risk HPV, such as HPV-16 and HPV-18, and a significant correlation with HPV in situ hybridization , p16 immIn clinical practice, early OPSCC is treated either by transoral surgery or radiotherapy, and advanced OPSCC is treated by open surgery with reconstruction with or without postoperative CRT or definitive chemoradiotherapy (66–70 Gy radiotherapy with concurrent platinum-based chemotherapy). Transoral robotic surgery (TORS) is a minimally invasive surgical technique for early-stage OPSCC developed by Weinstein et al. in 2007 . Since tAs patients with HPV-associated OPSCC are more likely to live longer than patients with classic OPSCC and therefore experience late adverse events of definitive chemoradiotherapy , there iRadiation therapy with cetuximab, dose reduction of postoperative radiotherapy, dose reduction of radiotherapy in CRT, and a combination of radiotherapy with dose reduction and immune checkpoint inhibitors have been assessed to reduce treatment intensity in patients with HPV-associated OPSCC. The RTOG1016 trial was designed to test the non-inferiority of bioradiotherapy (BRT) using cetuximab to CDDP-based CRT , but BRTTreatment de-escalation for patients at a high risk of recurrence should be avoided, even those with HPV-associated OPSCC. In the NRG Oncology HN002 study , 60 Gy oHPV vaccination has largely been promoted to prevent cervical cancer. Accordingly, in Western countries with high rates of vaccination, the incidence of cervical cancer has decreased –45. By cSince the average age at which patients are affected by OPSCC is significantly higher than that of patients with cervical cancer, it would take several decades to verify the efficacy of HPV vaccination in preventing HPV-associated OPSCC. Therefore, there is currently no clear evidence that HPV vaccination prevents HPV-associated OPSCC. However, oral HPV infection might be used as a surrogate. Studies on oral HPV infection rates among Japanese healthcare workers have shown that HPV infection rates are higher among men in their 30s and older . In addiSeveral countries have implemented national HPV vaccination programs for girls and boys. Australia was one of the first countries to implement a program for boys and girls and demonstrated high levels of vaccine administration beginning in 2013, with 75.9% and 80.2% vaccination rates for boys and girls, respectively, in 2017 . TherefoSeveral studies have estimated the future incidence of HPV-associated OPSCC based on vaccine coverage. Damgacioglu et al.  estimateHPV-associated oropharyngeal carcinoma is one of the few carcinomas increasing in incidence. In Japan, the incidence of HPV-associated OPSCC is expected to continue to increase over the long term, partly because of the prolonged suspension of the HPV vaccination program. It is important to prevent HPV-associated OPSCC by promptly establishing a public vaccination program including both boys and girls and by educating the public about HPV infection and HPV-associated cancers. Because the prognosis of patients with HPV-associated OPSCC is favorable, large-scale phase III clinical trials are needed to develop treatments aimed at reducing treatment intensity. Promising clinical trial results have been reported on reducing radiation doses in low-risk patients and reducing the intensity of postoperative radiotherapy after transoral resection. Careful decisions must be made to ensure that a reduction in treatment does not decrease oncological outcomes.


Anellovirus (AV) and seven complete HPV genomes were assembled. Bacteriome results unveiled that vaginal community state types (CST) distribution was independent of HPV or AV status, although bacterial phyla distribution differed between groups. Furthermore, TLR3 and IFNαR2 levels were higher in the Lactobacillus no iners-dominated mucosa and we detected correlations among RIG-like receptors (RLR) associated genes and abundance of specific anaerobic bacteria. Collectively, our data show an intriguing connection between HPV and AV infections that could foster cervical cancer development. Besides that, TLR3 and IFNαR2 seem to create a protective milieu in healthy cervical mucosa (L. no iners-dominated), and RLRs, known to recognize viral RNA, were correlated to anaerobic bacteria suggesting that they might be related to dysbiosis.The relationship among microbiome, immunity and cervical cancer has been targeted by several studies, yet many questions remain unanswered. We characterized herein the virome and bacteriome from cervical samples and correlated these findings with innate immunity gene expression in a Brazilian convenience sample of HPV-infected (HPV+) and uninfected (HPV-) women. For this purpose, innate immune gene expression data were correlated to metagenomic information. Correlation analysis showed that interferon (IFN) is able to differentially modulate pattern recognition receptors (PRRs) expression based on HPV status. Virome analysis indicated that HPV infection correlates to the presence of  Humavia different adaptor proteins, lead to engagement of transcription factors such as NFκB, IRF3, IRF7 that regulate cytokine and IFN production , impairing cytokines and IFN-I production  has several protective components, including antimicrobial peptides , and immune and epithelial cells . Besidesoduction , 10. Patoduction . These goduction –15. Furtoduction –18, but Lactobacillus crispatus, L. gasseri, L. iners, anaerobic bacteria  and L. jensenii, respectively , while a decreased concentration of Lactobacillus other than L. iners, increased diversity and anaerobic dominance typify a transitional mucosa (CST III-dominated) or dysbiosis (CST IV-dominated), the latter also known as bacterial vaginosis (BV) state : CST I, II, III, IV and V, dominated by ectively , 19. A hV) state , 15, 20.V) state –24. SeveV) state , 25, 26 V) state .Anellovirus. Also, bacteriome results suggested that phyla distribution is different depending on HPV and Anellovirus status. When gene expression was assessed, we observed that type I IFN expression is correlated to TLR9 expression in HPV+ women, while it is correlated to TLR4 expression in HPV- samples. Moreover, when bacteriome data were assessed in conjunction with gene expression data, TLR3 and IFNαR2 levels were higher in the Lactobacillus no iners-dominated mucosa and we also detected correlations among RIG-I, MDA5, TRIM25, RNF125 and abundance of specific anaerobic bacteria.An initial characterization of the vaginal bacteriome of Brazilian women indicates that women under reproductive age analyzed in the study grouped into the five CSTs. Nevertheless, a predominance of CST III (36.5%), CST I (30.5%) and CST IV (27.4%) was found. In this population, factors like smoking, number of partners, demographic region of origin and vaginal douching were risk factors for dysbiosis, while ethnicity, educational level and condom use had no impact on CST dominance . Besides22.1Study participants were enrolled at Instituto de Ginecologia (IG), Universidade Federal do Rio de Janeiro (UFRJ), Brazil. This was a convenience sampling from studies of characterization of sexually transmitted infections  and cerv2.2Total genomic DNA (gDNA) was extracted from cervical epithelial cells with the HiYield™ Genomic DNA Mini Kit (Blood/Bacteria/Cultured Cells) . Circular DNA was enriched by rolling circle amplification (RCA) with the Illustra TempliPhi Amplification kit . We prepared sequencing libraries using two nanograms of purified RCA product and the Nextera XT DNA Sample Preparation kit . One HPV- patient was excluded from downstream analyses due to lack of sufficient gDNA. Samples were sequenced in an Illumina MiSeq platform (2 x 100 nt reads).Retroviridae virus family was excluded to avoid biased analysis since most reads from this family were removed during the disposal of human reads. Virus relative abundance was calculated by dividing the number of reads assigned to each virus family per the total of trimmed reads submitted to BLASTX. Reads were also assembled to HPV reference genomes obtained from PAVE database (http://pave.niaid.nih.gov) using Geneious R11  and the consensus sequences representing the HPV complete genomes were extracted.Reads with Phred quality score below 28 were trimmed with Sickle  and the 2.32, 0.2 mM dNTP mix, 0.2 µM each primer, 1.5 U Taq Platinum polymerase , ~12.5ng of template and final volume was adjusted with RNase/DNase free water . The cycling conditions carried out were recommended by the manufacturer of the 16S library. PCR products were visualized by electrophoresis in 1.5% agarose gels and bands of the expected size were purified with the Illustra GFX PCR DNA and Gel Band Purification Kit . Five microliters of purified 16S rRNA PCR products were then used along with Nextera XT DNA Sample Preparation kit  to prepare sequencing libraries with distinct barcodes each one. Libraries were diluted to a final concentration of 10 pM and samples were sequenced in an Illumina MiSeq platform (2 x 100 nt reads).Total gDNA from cervical cells of 21 patients (10 HPV- and 11 HPV+) were used to PCR-amplify the 16S rRNA gene variable V3-V4 regions, generating an amplicon of ~460 bp, with the 16S Metagenomic Sequencing Library Preparation kit . PCR reactions were carried out using 1X Taq PCR buffer (minus Mg), 1.5 mM MgClThe BCL2FastQ2 Conversion Software  was used to demultiplex data and convert BCL files to FASTQ file formats. Reads were subjected to FastQC  for quality analysis, were denoised, filtered and joined using the Divisive Amplicon Denoising Algorithm DADA2  using QIUnsupervised hierarchical clustering based on Bray-Curtis dissimilarity and average linkage was applied to define clusters according to abundance and taxa diversity of each sample. The clusters found were associated with vaginal microbiome CSTs classified in previous studies . Cluster2.4-ΔCt formula.Gene expression analyses were carried out as previously described . Briefly2.5post-hoc test to compare three groups. Odds ratio (OR) was calculated using the free online statistical software MedCalc [https://www.medcalc.org/calc/odds_ratio.php]. Correlation analyses were carried out by Spearman’s correlation in SPSS (IBM Corporation), where r corresponds to correlation coefficient. Degrees of correlation were adapted from Schober and colleagues  or median and interquartile range for numerical variables. Differences between groups were calculated in IBM SPSS Statistics for Windows, version 25.0 . For categorical variables we used chi-square or Fisher exact test; for numerical variables we used Mann-Whitney U test to compare two groups and Kruskal-Wallis test, with Dunn’s multiple comparisons as a lleagues  as folloTo compare gene expression levels according to anellovirus status we used Mann-Whitney U test in SPSS (IBM Corporation), and according to CSTs we used Kruskall-Wallis test in GraphPad Prim 9 .33.1Patients were grouped based on their HPV status into HPV- and HPV+ women. HPV+ women were older, but in both groups the number of married, high school educated and smokers or past smokers were similar Table 1.3.2We evaluated the association between expression of studied genes and HPV status, classifying expression levels in high or low based on the median expression of each gene . We observed associations between HPV+ status with IFNα2, TLR7, TLR3 and MDA5 expression using Fisher Exact Test and decided to perform OR analyses. These assessments indicate that HPV infection is linked to high IFN-I expression  and to low levels of PRRs that recognize RNA   and Anelloviridae  . Overall, we detected two viral families, l reads) Table 3.Anellovirus infection . In addition, AV+ women had more partners in life (AV- (median/IQR) = 2.9/2; AV+ = 10/5; p = 0.028) and presented more HPV reads in NGS analysis (AV- (median/IQR) = 12/0; AV+ = 2638/152; p = 0.002). Nevertheless, no differences in gene expression were detected between AV- and AV+ women .Anellovirus (AV) reads were detected in seven women (six out of seven are HPV+) and we decided to group study participants by status of this viral family  and performed OR analyses. First, we observed that being infected by HPV favors 3.4Firmicutes (74.7%), followed by Actinobacteria (12.7%), Bacterioidetes (5.1%) and Fusobacteria (5.1%). On the other hand, in HPV+ samples the same phyla were present, but at different percentages: Firmicutes (68.3%), Actinobacteria (27.3%), Fusobacteria (2.5%) and Bacterioidetes (0.7%) ((p< 0.0001) when comparing AV- (Firmicutes (65.8%), Actinobateria (27.2%) and Fusobacterium (3%)) and AV+ samples (Firmicutes (85%), Fusobacterium (6%) and Bacterioidetes (5.7%)) . In HPV- cervices, the predominant phyla were L. crispatus dominant, n = 3; 14.2%), CST III , CST IV  and CST V  (L. no-iners (grouping CST I and V), L. iners (CST III) and anaerobic (CST IV). Women within the L. iners group reported the use of condoms more frequently and had their first sexual intercourse earlier than women in the anaerobic group (p = 0.049). On the other hand, women within the anaerobic group smoke/smoked more than L. iners group (p = 0.026). In this sense, smoking increased the chance of anaerobic cervical dominance compared with the L. iners group    Figure 2Sphingomonas genus  as well as between AV abundance and Fusobacterium genus , while AV abundance showed a strong positive correlation with the presence of Shuttleworthia genus . We also performed correlations between the 61 bacterial genera found in the samples and the expression of assessed genes. We found 37 moderate correlations involving 22 genera and 9 innate immunity genes . Some samples were also infected by multiple HPV types. Bacteriome analysis identified 12 phyla that were differently distributed depending on HPV or AV presence. Overall, behavioral factors and innate immune gene expression were associated to L. iners and anaerobic dominated mucosa. Expression of RLRs and their regulators correlated to several genera abundance which in turn, correlated to each other, suggesting that this receptor family triggers innate immune signaling that may impact vaginosis.Cervical cancer development is associated with HPV infection, which progression depends on the cervical milieu. Herein, we tried to elucidate part of the interplay between host innate immunity and the microbiome (virome and bacteriome) using a Brazilian convenience sample. First, we assessed whether social and immune factors could impact HPV infection and found that IFN-I expression correlated to that of different innate immunity genes depending on HPV status. Virome data from cervical samples revealed two viral families: via UCHL1 induction.Known risk factors for cervical cancer development , 42 wereIn this study, the number of sexual partners and number of HPV reads were correlated to AV reads, reinforcing idea that these viruses are sexually transmitted –54. TherL. no-iners ; L. iners  and anaerobic (dysbiosis). In our samples, smoking was the only behavioral factor linked to CST IV (anaerobic), but a larger sample size is needed to perform a more robust analysis. The association between cervical microbial diversity (CST III and IV) and HPV infection/cervical dysplasia has been described   . Interesoly(I:C) , 63. Lite.g., short and long dsRNA, 5′-triphosphorylated RNA, ssRNA and poly(I:C)) and their role in DNA and bacterial recognition is emerging  can be found below: PRJNA933917 (SRA).The studies involving human participants were reviewed and approved by Ethical Committee of Instituto de Puericultura e Pediatria Martagão Gesteira, UFRJ . The patients/participants provided their written informed consent to participate in this study.AB, AG, EM and MS: conceptualization and writing original draft. AB, CP, AM, YF, and GA: sample collection and processing. AB, CP, AG, and EM: gene expression experiment and analysis. AB, LG, JS, GC: virome and bacteriome experiment and analysis. MS, AG, and EM: funding acquisition. AB, AG, EM, LG, JS, GC and MS: writing, reviewing, and editing. All authors contributed to the article and approved the submitted version.


Regression analysis revealed a significantly higher proportion of SCT in pregnant women with G6PDd than those without G6PDd . The interaction between malaria and SCT was observed to be associated with anemia outcomes among the G6PDd women . Our findings show that anemia is a common condition among G6PDd women attending ANCs in northern Ghana, and its outcome is impacted by malaria and SCT. This warrants further studies to understand the impact of antimalarial treatment and the blood transfusion outcomes in G6PDd/SCT pregnant women.During pregnancy, women have an increased relative risk of exposure to infectious diseases. This study was designed to assess the prevalence of the co-occurrence of glucose-6-phosphate dehydrogenase deficiency (G6PDd) and sickle cell trait (SCT) and the impact on anemia outcomes among pregnant women exposed to frequent infectious diseases. Over a six-year period (March 2013 to October 2019), 8473 pregnant women attending antenatal clinics (ANCs) at major referral hospitals in Northern Ghana were recruited and diagnosed for common infectious diseases , G6PDd, and SCT. The prevalence of all the infections and anemia did not differ between women with and without G6PDd (χ Human glucose-6-phosphate dehydrogenase (G6PD) is an X-linked housekeeping enzyme that catalyzes the first limiting step in the pentose phosphate pathway to generate reducing power in the form of NADPH, which is particularly necessary within the RBC cytoplasm to control its intrinsic oxidative stress ,2. The eG6PDd distribution overlaps in the regions where malaria is predominant . Thus, tUnlike G6PDd, sickle cell disease (SCD) is an autosomal recessive disorder, in which individuals inherit both abnormal beta-globin (HBB) alleles located on chromosome 11 11p15.5). While about four million people are known to have SCD, forty-three million have sickle cell trait (SCT) .5. While and otheIn summary, G6PDd and SCD/SCT are the most prevalent genetic polymorphisms in malaria-endemic areas and are characterized by high red blood cell turnover ,21,22,23Study Area: The study was conducted in the ANC unit of the Bolgatanga Regional Hospital (Upper East Region), the Tamale Teaching Hospital (Northern Region), and the Wa Regional Hospital (Upper West), all in Northern Ghana . These tStudy Design: The study design employed was a hospital-based cross-sectional type. Information on the gravidity, parity, age, and trimester of the visit of participants was obtained by means of a guided questionnaire via an interview. At the ANC, the pregnant women were bled twice; capillary samples were first obtained and used for rapid diagnostic testing to assess the seroprevalence of malaria, HBV, syphilis, and HIV; venous blood samples were subsequently collected at the laboratory and used for the quantification of hematological parameters . The study subjects consisted of the first ANC attendees (pregnant women who gave their consent) in any trimester of pregnancy. The pregnant women were recruited as and when they appeared at the ANC, and the numbers obtained within the sampling period was used for the study. Women with documented chronic alcoholism and those with known chronic degenerative diseases with excluded from the study. The pregnant women diagnosed with the infections G6PDd, SCT, and/or anemia did not show any disease symptoms during the study period; despite that, they were referred by the midwife/nurse for appropriate treatment at the health facility.Estimation of hemoglobin (Hb): The Hb levels among the study subjects were estimated via full blood count (FBC). Five (5) mL of venous blood was collected into an EDTA tube. Using a sample roller, the venous blood was mixed with the anticoagulant  for a minute. The Sysmex XS-500i  analyzed the blood sample while the Hb values were obtained from the FBC data. The anemia degree defined throughout this study follows the WHO criterion of Hb < 11 g/dL for pregnant women .Sickling Test: The sickle cell slide test was used to determine the sickle cell status of the participants . This teGlucose-6-phosphate Dehydrogenase (G6PD) Test: The methemoglobin reduction test  was usedPlasmodium falciparum histidine-rich protein 2 (PfHRP-2) and Plasmodium spp. lactate dehydrogenase (pLDH), guaranteeing detection under HRP-2 deletion. As P. falciparum is the main Plasmodium species in Ghana, the RDT kit chosen was specific for P. falciparum. P. malariae and P. ovale only account for 3.6 and 1.0%, respectively, of the diagnosed malaria in Ghana [Malaria Testing: A rapid diagnostic test (RDT)  was used to diagnose malaria disease in the blood samples collected from the recruited study subjects (pregnant women). The RDT system detects in Ghana . The reain Ghana ,28.Hepatitis B Testing: The HBV surface antigen RDT kit  was used for testing hepatitis in whole blood. This kit is specific for and sensitive to the identification of the hepatitis surface antigen, as specified by the manufacturers.HIV Testing: A pretest counselling session was held for the pregnant woman before the HIV test was conducted. The HIV first response RDT kit  was used for initial testing on whole blood, while the OraQuick test kit  was used for confirmatory testing on oral fluid.Treponema pallidum) kit (INNOVITA (Tangshan) Biological Technology Co., Ltd., Tangshan, China), following the manufacturer’s instructions. This kit is highly sensitive and specific in identifying Treponema pallidum species, as specified by the manufacturers.Syphilis Testing: This was performed on whole blood using the TP  and test (T) lines in the result window. A test was considered negative when only one color band was observed at the control (C) line. An invalid test showed no color band at either the “C” or the “T” line, or it showed a color band at the “T” line. Invalid tests were repeated with new test kits.p = 0.158. For all the tests of association, p < 0.05 was considered statistically significant.The data were entered into a Microsoft Excel spreadsheet, which was imported unto SPSS (version 28) for analyses. Summary output tables of the percentage distribution were produced for the categorical variables. The association of G6PD status with background characteristics, infections, anemia, and sickling status was evaluated using the Pearson chi-square test and Fisher’s exact test. Multinomial logistic regression analysis was used to predict the associations of G6PDd with the infections  and the hematological indicators (SCT and anemia). Univariate analysis of variance was applied to evaluate the effect of interaction between infections and SCT status on the outcome of anemia among G6PDd women. Prior to the univariate analysis, Levene’s test was used to test for homogeneity of variance between the interacting factors, with The background characteristics of the pregnant women are summarized in The prevalence of G6PDd, sickling positivity, and anemia was 8.6%, 7.2%, and 44.8%, respectively, for all the participants screened . The pre2 < 3.0; p > 0.05 for all comparisons, 2 < 3.6; p > 0.05 for all comparisons, 2 = 15.7; p < 0.001, p < 0.011, Age, gravidity, and parity had no significant association with G6PDd among the pregnant women , which trigger hemolysis in G6PDd individuals, is well documented . Though G6PDd and SCD are two common genetic conditions in malaria-endemic regions and are associated with protection against malaria . Thus, tMisdiagnosis is another potential challenge; there is a possibility of misdiagnosing G6PDd among sickling positive or SCD individuals. G6PD enzyme activity is elevated in reticulocytes, while it decreases as the RBCs age ,6. ConsiLaboratory findings in patients with G6PDd and SCD show that reduced hemoglobin and hematocrit, a high level of reticulocytes, elevated lactate dehydrogenase (LDH), and increased unconjugated bilirubin are associated with increased hemolysis . MonitorThe strength of the present study relies on the large dataset used, from which a convincing and sound conclusion was derived. However, some limitations are acknowledged, such as the lack of molecular genetic studies to identify the alleles associated with G6PDd/SCT co-occurrence that could have been misclassified in some cases of G6PDd, but with very a minor impact according to the large number of samples used in the study and given that it would only occur in cases concurrent with submicroscopic malaria that were undetectable by RDT. Thus, according to recent data , cases o


Fusobacterium spp., Porphyromonas, Prevotella, and Campylobacter, are currently being considered as potential microbiomarkers for HPV-positive CC. However, the composition of the CVM in CC is inconsistent; thus, further studies are needed. This review comprehensively discusses the complex interplay between HPV and the CVM in cervical carcinogenesis. It is postulated that the dynamic interaction between HPV and the CVM creates an imbalanced cervicovaginal microenvironment that triggers dysbiosis, enhances HPV persistence, and promotes cervical carcinogenesis. Moreover, this review aims to provide updated evidence on the potential role of bacteriotherapy, particularly probiotics, in the treatment of CC.Uterine cervical cancer (CC) is a complex, multistep disease primarily linked to persistent infection with high-risk human papillomavirus (HR-HPV). However, it is widely acknowledged that HR-HPV infection alone cannot account for the formation and progression of CC. Emerging evidence suggests that the cervicovaginal microbiome (CVM) also plays a significant role in HPV-related CC. Certain bacteria, such as  Uterine cervical cancer (CC) remains a major public health problem, with a significant number of new cases and deaths annually . PersistLactobacillus spp., which benefits the host through symbiotic relationships . ,70. 9,70ormation ,81. The ormation . In lineormation  identifiLactobacillus spp. and an increase in CVM diversity in HR-HPV-positive women than in HR-HPV-negative women. For instance, Brotman et al. [Lactobacillus spp., including L. crispatus, L. jensenii, and L. gasseri, compared to HPV-negative groups. In particular, Lee et al. [Lactobacillus than their uninfected twins. Furthermore, they suggested that Sneathnia spp. could be identified as a microbiological marker of an HR-HPV infection. Also, Lebeau et al. [Lactobacillus survival, the Lactobacillus concentration was significantly decreased. In other words, the escape of HPV from the immune response results in an imbalance of microbiota in the female vaginal flora.In addition, many studies have reported the association between the CVM and HR-HPV infection ,84,85,86n et al.  identifie et al.  comparedu et al.  followedLactobacillus CVM was predominant in SIL women compared to HPV-negative and HPV-positive non-SILs. S. oralis and unclassified OTU265 differed between HPV-positive and HPV-positive LSILs. Also, this study revealed several unclassified OTUs, such as OTU880, OTU893, and OTU883, predominant in the HPV-positive HSIL group. In a longitudinal study, Usyk et al. [L. iners and L. crispatus, one cluster containing high levels of G. vaginalis, and other CSTs without a significant group. L. iners were associated with HPV clearance, whereas G. vaginalis correlated with CIN2 progression after the first visit. Prevotella amnii and Anaerococcus prevotii, two common causative agents of BV, were linked to disease progression on the second visit. L. iners may have two opposite functions in the cervicovaginal microenvironment: one can promote health, and the other is associated with dysbiosis and CIN susceptibility [L. crispatus in low-risk CIN, whereas L. iners was dominant in medium-risk CIN.HPV-persistent infection can interfere with the cervical microenvironment, cell proliferation, angiogenesis, and tissue differentiation. Any changes in regulatory factors in these processes may cause cervical neoplasia. However, HPV infection alone does not explain the development of CC. Indeed, some aspects, such as the milieu of mucosal secretion, epithelial surface integrity, immune regulation, and local microbiota, may influence HPV carcinogenesis . Notablyk et al.  evaluatetibility  identifiL. crispatus changed into L. iners after HPV infection in the normal cervical microenvironment. When epithelial lesions became SILs, Sneathia and other Fusobacterium spp. were the most abundant. Fusobacterium necrophorum was highly represented in HPV-positive ICC cases and associated with increasing anti-inflammatory cytokines, such as IL-4 and TGF-β1. The authors concluded that Fusobacterium spp. might play a role in shifting Th1 immunity to Th2 or by having a direct effect on the E-cadherin/β-catenin signaling pathway in cervical HPV-transformed cells. However, this finding differed from Wu et al. [Fusobacterium was not an excellent prognostic marker for CC, although the abundance of this bacteria was the highest among the groups. The potential marker genera for CC in this study were Porphyromonas, Prevotella, and Campylobacter. Together, the CVM composition in CC differed from case-to-case; therefore, a large and well-designed study on the microbiome in CC with different HPV statuses is necessary for clinical applications.Concerning ICC, evidence about the relationship between the microbial community and ICC was also observed in several studies . Audiracu et al. , who fouLactobacillus-dominant communities and in high-grade dysplasia. 3-hydroxybutyrate, eicosenoate, and oleate/vaccinate are the metabolic features of CC [Streptococcus, Prevotella, Megasphaera, Atopobium, and Sneathia [Recently, multiomics has become an increasingly important area in cancer research. Several multiomics studies were carried out and contributed various insights into the underlying mechanism of the complex interaction between CC and its microorganisms. IIhan et al.  evaluatees of CC . AnotherSneathia .In the early stages of CC, there are multiple choices for treatment, such as surgery , radiation, and neoadjuvant chemotherapy. In the locally advanced stage, chemoradiation is the best approach. However, 30–40% of the patients do not respond entirely, and some patients face side effects from this therapy . TherefoLactobacillus, Bifidobacterium, Lactococcus, Streptococcus, and Enterococcus. Also, some strains belonging to Bacillus and Saccharomyces are utilized nowadays [Recently, bacteriotherapy has emerged as a promising platform for cancer treatment. One of the most common representatives of bacteriotherapy is probiotics. Probiotics contain a number of live microorganisms that provide several benefits to their host. There are many sources of probiotics in the human diet, such as fermented milk products  and mainly vegetables (cabbage and cucumber) ,101. Thenowadays .The past decade has seen the rapid development of probiotics for CC ,14,103. L. crispatus, L. jensenii, and L. gasseri on CaSki cells, Wang et al. [Lactobacillus could inhibit CC cell proliferation through the regulation of HPV oncogenes and cell cycle-related genes. Another study showed that L. gasseri could promote apoptosis in HeLa cells through its exopolysaccharides. Also, L. gasseri could affect the anti-inflammation of HeLa cells by reducing TNF-α and increasing the cytokine IL-10 [Lactobacillus spp. from different microenvironments resembled inhibitors of HPV 16 and 18 by restoring E-cadherin and suppressing MMP9. Another strain, Bifidobacterium adolescentis SPM1005-A, has potential antiactivity by inhibiting E6 and E7 oncogene expression in SiHa cells [Various studies have assessed the efficacy of probiotics on CC cell lines. For instance, on the effect of g et al.  reportedne IL-10 . Moreovene IL-10  found thHa cells .Lactobacillus casei) for six months had higher HPV clearance than the control group; however, the difference was not significant [Lactobacillus rhamnosus and Lactobacillus reuteri daily. Although the trial effects did not influence HPV clearance, they significantly decreased the rates of mildly abnormal and unsatisfactory cervical smears. Likewise, Dellino et al. [L. crispatus M247 had reduced HPV-related cytological anomalies compared to the control group. However, HPV clearance was not significantly different between the probiotic group and the group without probiotics. In contrast, Pierro et al. [L. crispatus M247; furthermore, in some cases, the CVM shifted the CST status to CST I.There are also reports on the special role of probiotics in HPV infection and CC in human trials . In 2013nificant . In a wenificant , 121 HPVo et al.  found tho et al.  reportedLactobacillus have been studied and developed to prevent vaginal dysbiosis, such as BV or vulvovaginal candidiasis  is safe and can promote an increased abundance of Lactobacillus and decreased pH and Nugent score in women with symptomatic BV after four visits. Until now, there have been limited studies using probiotic vaginal administration in CC treatment, but some vaginal suppositories have reported high efficacy in HPV-positive women. A vaginal L. rhamnosus BMX therapy was implemented in 117 women with HPV and BV or vaginitis concomitantly after using an antibiotic (metronidazole) or antifungal (fluconazole) long-term. The study revealed that vaginal L. rhamnosus BMX could change abnormal cervical lesions and increase HPV clearance after long-term therapy [In addition to oral probiotics, vaginal suppositories containing didiasis  [110,111 therapy .Also, lacidophilin, a bacteriocin produced by lactic acid bacteria, has been considered a promising therapy. Lacidophilin can be an antibacterial factor by generating lactic acid and destroying the bacterial membrane . The comThe combination of probiotics and chemotherapy is currently being reported as a new approach to cervical cancer (CC) treatment. Previous studies have shown that using probiotic supplements can reduce the side effects or toxicity of chemotherapy . For insL. casei and α-GalCer (an anticancer and NK T-cell stimulator) subcutaneous injections in a mouse model of CC. They found that splenocyte proliferation, lactate dehydrogenase, nitric oxide, and IFN-γ levels increased more in the combination therapy group than in the control group. Compared to Gardasil injection in the mouse model, the heat-killed preparation of L. casei and α-GalCer had a similar effect. These results could promise a new therapy for CC management [A recent study reported the combination of a heat-killed preparation of nagement .Lactobacillus acidophilus LA-5 and Bifidobacterium animalis subsp. lactis BB-12 in 57 CC patients with diarrhea after radiotherapy. The study found that diarrhea symptoms in the mild-to-moderate group and the severe group were significantly reduced after three weeks of using probiotics.Diarrhea is the most common side effect of radiotherapy in treating cervical cancer (CC) patients . PreviouLactobacillus abundance [VMT may improve CVM imbalance when transplanting vaginal fluid from a healthy person with high bundance . Lev-Sagbundance  reportedOverall, these findings suggest a significant role for probiotics in fighting CC. Nevertheless, further studies are required to understand the function of bacteriotherapy and its underlying mechanisms, particularly during CC treatment.Lactobacillus and changes in the immune system and metabolism, which induce dysbiosis, enhance HPV persistence, and promote cervical carcinogenesis. However, this research is only beginning to understand the role of microorganisms in carcinogenesis. Further studies combining new molecular tools, or multiomics, should be considered to identify underlying mechanisms and provide insights into the interaction of microorganisms with immune and metabolic responses. Notwithstanding the relatively limited evidence, probiotics offer new opportunities for future therapy that could change the response to cancer treatment. Hence, continuing well-designed studies about the antitumor function of probiotics in managing CC patients is necessary.This review suggests a strong association between HPV infection and CVM status in cervical diseases. Accordingly, the dynamic interaction can cause an imbalanced cervicovaginal microenvironment, such as depletion of