Integrity-linked issues have become increasingly serious and attracted considerable attention regarding the sustainability of live streaming; therefore, live streaming anchors and suppliers have become increasingly concerned about their ongoing marketing. Moreover, streaming platforms are also beginning to focus on sustainable development. Determining how live streaming anchor behavior, supplier behavior, and streaming platform strategic decision affect live streaming is essential for achieving the healthy and sustainable development of live streaming ecosystems. However, the game among live streaming anchor behavior, supplier behavior, and streaming platform strategic decisions, which the live streaming ecosystem strongly depends on, has attracted little attention. This study constructed a game model of value co-creation evolution in live streaming ecosystems, with the live streaming anchors, suppliers, and streaming platforms as the main subject. The evolutionary stable strategies of the game were analyzed, and the influence of typical factors on the system was discussed. The results showed that the best evolutionary stability strategy is that live streaming anchors and suppliers choose honesty, and streaming platforms have strict regulation, which is the optimal strategic objective for value co-creation. The appropriate rewards and punishments inherent in a streaming platform can help regulate the behavior of the live streaming anchor and supplier; specifically, great rewards and severe punishments of the streaming platform can be applied to incentivize the honest behavior of the live streaming anchor and supplier. This study provides insights for designing appropriate policies for live streaming platforms to promote sustainable development. The number of live e-commerce users was 469 million, an increase of 5.33 million compared to the previous year .When When x = 1, the strategy is stable, and the live streaming anchor chooses the honesty strategy.When V21 and V22, respectively, and the average expected benefit is We assume that the expected benefits of the supplier’s honesty or dishonesty are The dynamic replication equations for supplier strategy selection and the first derivatives are given below:Then, three situations are discussed based on formula :y = 0, y = 1, and F(y) = 0. According to the stability principle proposed by Friedman (1991), y is the ESS point whenClearly, F(y) = 0 and F′(y) ≤ 0.y ∈ .When y = 0, the strategy is evolutionarily stable, and the supplier chooses the dishonesty strategy.When y = 1, the strategy is stable, and the supplier chooses the honesty strategy.When V31 and V32, respectively, and the average expected benefit is Assuming that the expected benefits of supplier honesty or dishonesty are The dynamic replication equations of live streaming platform strategy selection and first-order derivatives are as follows:Then, three situations are discussed based on formula :z = 0, z = 1, and F(z) = 0. According to the stability principle by Friedman (1991), y is the ESS point when F(z) = 0 and F′(z) ≤ 0.Clearly, z ∈ .When z = 0, the strategy is evolutionarily stable. and the live streaming platform chooses a negative regulatory strategy.When z = 1, the strategy is evolutionarily stable, and the platform chooses a proactive regulatory strategy.When This section constructs and solves the dynamic replication equations of the three-party game among the live streaming anchor, supplier, and live streaming platform to demonstrate the circumstances and methodology of the stable strategy formation of the evolutionary game in the live streaming.F(x) = 0, F(y) = 0, and F(z) = 0. As the stable solutions in multiple-group evolutionary games are strict Nash equilibria, Lyapunov’s first law is used for stability analysis for only eight pure strategic equilibria in the evolutionary system E1 , E2 , E3 , E4 , E5 , E6 , E7 , and E8 .Multiple sets of possible solutions were obtained from According to Friedman’s method, the stability of the equilibrium point of the differential system can be achieved by analyzing the eigenvalues of the Jacobi matrix of the system as follows:Following Lyapunov’s first method , when alCa > 0 and Cs > 0; therefore, only E1 , E2 , E5 , E7 , and E8  present for the five cases of evolutionary stabilization strategies.Combining the settings of the parameter sizes in the model shows that Scenario 1: Expressed by the eigenvalue of stability point E1. The three-party evolutionary stabilization strategy is {dishonesty, dishonesty, negative regulation}. The severity of the punishment is the key to selecting the live streaming platform. If the punishment cannot offset the cost of strict regulation of the live streaming platform, then the live streaming platform will choose the negative regulation strategy. In this Scenario, the live streaming anchor and supplier will not face fines when they adopt collusion strategy. Speculation also contributes toward the live streaming anchor and supplier violating policies, and the system shows value co-destruction.Scenario 2: Expressed by the E2 eigenvalue expression. The limitations affecting the strategy of the live streaming platform are opposed to the constraints in Scenario 1. The live streaming platform tends to strict regulate when the expected gains of strict regulation exceed the costs of strict regulation. In this Scenario, the live streaming anchor and supplier need to measure the loss caused by the live streaming platform penalties when they violate the law. Suppose that the total amount of network externality benefits and incentives that the honesty strategy can obtain is less than the difference between the benefits and fines when the collusion strategy evolves to stabilize the strategy as {dishonesty, dishonesty, strict regulation}. In this case, the violations by the live streaming anchor and supplier, and the mismatch between the strict regulation strategy and the live streaming platform will trigger value co-destruction.Scenario 3: Expressed by the E5 eigenvalue expression. In Scenario 3, the live streaming platform chooses a strict regulation strategy when the anticipated gains of strict regulation are greater than the costs of strict regulation. The variables affecting the supplier’s strategy choice are the same as those in Scenario 2. However, the critical conditions are reversed: the supplier changes its strategy to adopt a positive one. In this Scenario, when the benefit (to the live streaming anchor) of choosing an illegal strategy is greater than the cost of honesty, the live streaming anchor tends to adopt a collusion strategy, and the system evolves into a stable strategy of {dishonesty, honesty, strict regulation}.Scenario 4: Expressed by the E7 eigenvalue expression. In Scenario 4, the variables affecting the strategy selection of the live streaming anchor and supplier do not change compared to Scenario 2. However, the critical conditions are reversed when the live streaming anchor tends to perform negatively, the supplier chooses honest behavior, and the live streaming platform takes punitive measures for the live streaming anchor and incentivizes the supplier by sharing network externality benefits with the supplier. The system tends to {honesty, dishonesty, strict regulation}, and resources abuse by the live streaming anchor or the supplier reduces the overall value of the system if the live streaming platform does not meet the expectations of the live streaming anchor and supplier due to docking dysfunction triggered by value co-destruction.Scenario 5: Expressed by the E8 eigenvalue expression. When the cost of strict regulation of the live streaming platform is less than the gain from network externality, and the difference between the cost of the positive strategy and extra gain of the live streaming anchor and supplier is less than the loss of the penalty imposed by the negative strategy, {honesty, honesty, strict regulation} is the system evolutionary stability strategy under the condition that all three parties make reasonable use of resources. All can buttress the other parties’ performance with positive behavior. Value co-destruction transforms into value co-creation. Generally, when the general ecosystem of the industry is more relaxed, the live streaming platform tends not to take a higher penalty for the rapid accumulation of traffic and the fastest speed at reaching the minimum network effect; that is, θ accepts a smaller value. However, as the industry and the scales of its associated live streaming platforms expand, the violations become more frequent. In this Scenario, the live streaming anchors and suppliers receive more penalties for violations, so they are incentivized to be honest. Overall, the live streaming platform that provides appropriate incentives and punishments is the key to achieving ESS. This Scenario is the ideal state and desired ESS point.Ea = 2, Es = 6, Ep = 1, Ca = 5, Cs = 5, Cp = 6, Ia = 5, Is = 4, α = 0.5, β = 0.5, Ra = 3, Rs = 1, λ = 0.3, γ = 0.5, Fa = 6, Fs = 4, θ = 0.5, Da = 2, Ds = 6; for Scenario 2, Ca = 6, Cp = 4, other parameters are the same as in Scenario 1; for Scenario 3, Ca = 5, Cs = 3, Ia = 3, Is = 8, other parameters are the same as in Scenario 2; for in Scenario 4, Ca = 1, Cs = 5, Cp = 2, Ia = 5, Is = 4; other parameters are the same as in Scenario 3; for Scenario 5, Cs = 1, Cp = 1, Fa = 4, Fs = 2, other parameters are the same as in Scenario 4. MATLAB simulated the values of the five Scenarios, and Figs To check the validity of the above model analysis results, the parameters were assigned according to the above settings and constraints in different cases based on . For SceE8  in Scenario 5 is the ideal ESS point. Based on the values assigned to each parameter in Scenario 5 in Section 4.1, it is assumed that the initial values of the strategy selection of the live streaming anchor, supplier, and the live streaming platform are as follows: x = 0.2, y = 0.2, z = 0.2; x = 0.5, y = 0.5, z = 0.5; x = 0.8, y = 0.8, z = 0.8. The experimental results of the numerical simulations for different initial values are shown in Figs When the live streaming anchor and supplier operate in good faith, and the live streaming platform is strictly regulated, then this situation is good for the sustainable development of the live streaming industry; that is, E5 . However, a change in the initial values affects the time the system reaches a steady state.As shown in Figs x = y = z = 0.2), the suppliers may experience instability in their behavioral strategy at the early stage of development because of speculative psychology. This instability is influenced by the strict regulation of the live streaming platform and the honest strategy of the anchor, making the suppliers finally choose an honest strategy. Finally, the system evolves to point E5 . Figs E5  point. We also find that the time for the system to reach stability becomes shorter as the initial probability of the tripartite subject keeps increasing. For example, Using the results in λ be equal to 0.3, 0.6, and 0.9, respectively. The simulation results are shown in Figs Incentive strength refers to the strict supervision of the live streaming platform under the honesty of the live streaming anchor and supplier strategy to apply specific incentives so that the live streaming anchor and supplier can continue this positive strategic tendency. These incentives include traffic recommendations, official training, and live streaming platform subsidies. Fixing the other parameters at constant values, we let the value of λ does not substantially influence the evolutionary trajectories of the live streaming anchor and supplier’s behavioral strategies. Eventually, the live streaming anchor and supplier will choose the behavioral honesty strategy, and compared with the supplier, the live streaming anchor will be the first to make ethical behavioral decisions. When λ is 0.3 or 0.6, the time taken by the live streaming anchor and supplier to converge on the behavioral strategy of honesty is the same, whereas when λ = 0.9, the time to reach stability becomes longer for the live streaming anchor and supplier, which may be influenced by the strategy choice of live streaming platform. λ = 0.3 or λ = 0.6, the live streaming platform will eventually choose strict supervision. The time required for λ = 0.3 to stabilize is shorter than that required for λ = 0.6. When λ = 0.9, the live streaming platform will increase its cost due to excessive reward strength, and its strategy will oscillate. It cannot reach a stable state, which will affect the value co-creation and thus affect the benign and sustainable development of the live streaming industry. Therefore, the critical interval of the reward strength λ lies between . Higher incentives can have a negative impact on live streaming platform revenue. However, live streaming platforms need to be brave enough to shoulder social responsibility and balance their own earnings with social responsibility.As shown in Figs θ on the outcome of the three-party evolution game, it is assumed that the values of penalty intensity θ are 0.1, 0.5, and 0.9 under the condition that other parameters are specified. The results are shown in Figs Penalty strength refers to a certain degree of punitive measures adopted by the live streaming platform for the illegal collusion of the anchor and supplier, such as limiting the flow of suspension, imposing fines, and blocking the number. If the punishment is not sufficiently strong, the violation cost of the live streaming anchor and the supplier will be significantly reduced, which may increase the probability of the live streaming anchor and supplier participating in collusion. To study the influence of the penalty intensity θ in the punishment of speculative behavior by the live streaming platform, the evolution rate of all three subjects  accelerates and the time to reach stability becomes shorter. From θ = 0.1, the live streaming platform is less punitive for violations, suppliers will initially have a fluke mentality, and their preliminary choices will tend to be dishonest strategies. If the live streaming platform does not take timely guidance measures at this time, then suppliers’ violations will have a negative demonstration effect. If the live streaming platform and live streaming anchor can guide the supplier to establish the correct values at this time, then the supplier will eventually choose the honesty behavior strategy after evolution. However, it will take longer to reach stability, and there may be a choice of violation strategy in the middle. Figs Figs β accordingly. Keeping the other parameters constant and changing the values of α and β so that the values of α are 0.1, 0.5, 0.9, and β are 0.1, 0.5, and 0.9, the results are shown in Figs Network externality benefit refers to the benefit spillover effect shared between the live streaming platform and the live streaming anchor, the live streaming platform and supplier when the live streaming platform has strict regulations, and the live streaming anchor performs their responsibility, or the supplier actively participates in governance. Each subject could change the size of the benefit distribution coefficient through active strategies. For example, when the live streaming platform has less manual intervention and traffic regulation, fans have higher loyalty to the live streaming anchors. This strong social engagement from private domain traffic gives live streaming anchors more space to play. The high and low involvement of the live streaming platform in the traffic distribution mechanism makes α larger and smaller, respectively. Similarly, the level of involvement of the live streaming platform in the evaluation mechanism of suppliers changes the size of β. When β = 0.1, the external revenue allocation weight of the supplier is significant and the supplier with the high revenue gained will quickly choose the honesty behavior strategy. As β increases, the external revenue allocation weight of the supplier gradually becomes smaller, the gains obtained keep decreasing, and the time required for suppliers to make an honesty behavior strategy becomes longer, such as in β = 0.1, the network externality gain allocation factor of the live streaming platform is small, the network externality gain of the live streaming platform is also small, and the live streaming platform may have a speculative mentality in the behavior strategy selection. In addition, the evolution time required for the live streaming platform to tend to stabilize the strategy at this time is the longest, and when β = 0.9, the network externality gain of the live streaming platform is the largest. Additionally, the shortest time required for the live streaming platform to converge to a strict regulatory stabilization strategy.By constructing a tripartite evolutionary game model with the anchor, the supplier, and the live streaming platform, this study initially analyzed the current solutions for value co-creation in the live streaming ecosystem. In addition, the influences of three typical parameters on the live streaming ecosystem, including incentive strength, punishment intensity, and network externality benefit distribution, were investigated in this study. According to the results and analyses presented in Section 4.3, the three parameters influenced on the three participants under different strategy choices. In terms of the intensity of rewards and penalties, the probability of live streaming anchors and suppliers choosing a breach of trust or rent-seeking behavior strategy decreases as the intensity of rewards and penalties increases, which is accordance with previous findings , 38. MosThis study considers value co-creation in the live streaming platform ecosystem based on perspectives in previous research. Although value co-creation theory has been used in a variety of areas, such as healthcare, social media, travel, public services and retail –37, reseThis study considered people’s limited capacity to be rational and built a tripartite evolutionary game model with the live streaming anchor, the supplier, and the live streaming platform. In addition, this paper analyzed the evolutionary stabilization strategies and investigated how the live streaming platform can control the violations existing in live streaming through a reasonable policy strategy to reduce the problems of false promotion and false traffic, purify the environment of live streaming, maintain the possibility of purifying the live webcast environment, maintain social credit, and protect the legal rights of citizens. The authenticity and validity of the research content were verified using numerical simulation analysis. The following results were obtained:The difference in the initial probability had no effect on the stabilization strategy of the system but rather only affected the time at which the system became stable. When live streaming platforms are strictly regulated, live streaming anchors and suppliers will be attracted to honesty driven by prospective profits, resulting in honesty behaviors being stable in the market. ESS is only influenced by the values of parameters that satisfy the stability conditions. The larger the initial value is, the faster the evolutionary trajectory reaches the ESS .The strength of the reward and punishment can affect the size of the co-creation value. Live streaming platform behavior will appear shocking when the strength of the reward is too large, thus hindering the importance of the co-creation effect of the ecological system of the live streaming platform. As the incentive strength rises from 0.3 to 0.6, the time required for live streaming anchors and suppliers to reach a stable strategy is shortened and the rate increases; however, the strategy of the live streaming platforms adjusts more slowly and takes longer to stabilize. This may be related to the negative impact of incentive strength on live streaming platform revenue. However, the live streaming platform side needs to be brave enough to shoulder the social responsibility in its revenue and cultivate a good live streaming ecology. When the incentive reaches 0.8, the live streaming anchor still chooses the integrity strategy, but the strategy choices of the supplier and the live streaming platform adopt an oscillation phenomenon. The system cannot reach a stable state, and the value probability of co-destruction increases. Moreover, as the penalties increase, live streaming anchors and suppliers will choose integrity strategies more quickly, and the supplier is relatively sensitive to the punishment intensity.An increase in the revenue distribution coefficient has a positive effect on the value co-creation of the ecological system of the live streaming platform. The live streaming anchor is relatively the least sensitive to changes in distribution coefficients.The study of evolutionary game in terms of value co-creation in the ecosystem of live streaming platforms provides the following suggestions.Establish a scientific reward and punishment mechanism, reasonably allocate the network externality gain coefficient, and promote value co-creation. Although an increase in reward and punishment strengths can improve the evolutionary efficiency of the live streaming anchor and the supplier tending to an honesty strategy and reduce the probability of collusion events, when the reward strength is too high, the live streaming platform cost burden is too heavy. Such conditions prevent the live streaming platform from operating sustainably and benignly, leading to unstable strategic choices for each subject. At the same time, excessively high penalties will require the live streaming platform to pay more human and financial resources, which will also limit the governance efficiency of the live streaming platform. Moreover, increasing the revenue allocation coefficient of the network externality will also promote the stability of the positive strategies of each subject. When the revenue allocation coefficient is too small, the live streaming platform will consider a negative regulatory strategy because of the pursuit of an increase in user scale and traffic. Therefore, the live streaming platform needs to grasp the rewards and punishments further, build a scientific and reasonable reward and punishment system, improve the revenue distribution system based on traffic regulation and evaluation mechanisms, motivate the live streaming anchor to perform an excellent job by actively performing their duties to provide better live streaming content and product services, thus obtaining traffic support and avoiding unfair competition and monopoly phenomena using punishment to maintain the sustainable of the live streaming ecosystem.Building a value co-creation governance system that regulates the behavior of the live streaming anchor. The live streaming anchor is a crucial subject in the ecological system of the live streaming platform. The live streaming platform should appropriately strengthen punishments, optimize punishment measures, and establish a credit scoring system for the live streaming anchor. In the long run, for healthy and sustainable development of the live streaming ecosystem, the live streaming platform should improve the entry threshold for the live streaming anchor and strengthen the pre-broadcast audit mechanism to enhance implementing incentives.Establish a supplier blocklist system and withdrawal mechanism. To establish a supplier qualification verification mechanism, name registration and qualification audits of suppliers must be accurate, industry associations must be relevant, and administrative departments must share or exchange information related to the administrative penalties of suppliers. The supplier information changes should be updated promptly and a blocklist system and withdrawal mechanism of the supplier must be established. In the event of violations of laws and regulations or live streaming platform rules and other circumstances, warning reminders are required for the deadline for rectification and other measures. The accounts of suppliers who commit severe violations of the law or have destructive social impacts should be placed on the blocklist and canceled if necessary.Strengthen the social responsibility of the live streaming platform, clarify the leadership responsibility of ecosystem value co-creation governance, and reduce the risk of value co-destruction. The strategy choice of the live streaming platform side has a crucial influence on the behavior of the anchor and the supplier. Although when the live streaming platform chooses to supervise strictly to a certain extent, which will increase the supervision cost and the risk of decreasing user scale and user traffic, its strategy choice of strict supervision can guide live streaming anchors toward adopting ethical behaviors and give up behaviors that will hinder value co-creation, such as false promotion. This strategy can guide and encourage suppliers to give up substandard and dishonest behaviors. Therefore, the live streaming platform itself should take the initiative to assume social responsibility, play the leading role, strengthen public opinion against false promotion, enhance user education, guide the live streaming anchor and the supplier to use resources rationally and interact efficiently, catalyze the aggregation of all subjects in the live streaming platform to face the problems together, and stimulate bilateral users in the live streaming platform to fulfill their responsibilities. There are three key parameters in this problem, including the strength of the reward and punishment and the network externality benefit distribution. The live streaming anchors, suppliers, and live streaming platforms will make different strategic choices based on parameter changes. Thus, by adjusting these parameters, value co-creation can be achieved in the live streaming ecosystem. These results can also inform government agencies or live streaming platforms to consider these three key parameters when designing policies/business models.This study has certain limitations: (1) The subjects involved were only the tripartite groups of the live streaming anchors, suppliers, and live streaming platform; however, consumers also influence the live streaming ecosystem during the live streaming process. (2) Although the relevant influencing factors were simulated and analyzed in this study, more factors should be studied, such as the embeddedness of the live streaming anchor and the supplier. Therefore, future studies can solve these problems by constructing a four parties game model including the consumers and analyzing the embeddedness of the live streaming anchor to make the results more consistent with reality and to enhance their objectivity and usability.S1 File(DOCX)Click here for additional data file.


Cost savings from the use of generic drugs versus brand-name drugs are well known. Both private and public prescription drug plans encourage the use of generic drugs through a variety of mechanisms. The magnitude of cost savings for a given generic drug is dependent on the degree to which the generic market is competitive. Should the competitive structure become compromised, higher prices and reduced cost savings may result. An alleged conspiracy between Mylan Laboratories and its active-ingredient suppliers in 1997 was associated with an increase in seller concentration in the generic lorazepam market. The Federal Trade Commission (FTC) alleged that Mylan raised costs to consumers by $120 million because of price increases for generic lorazepam from March through December 1998 and for generic clorazepate from January through December 1998. In November 2002, a settlement with Mylan was approved by the FTC, and a federal district court required Mylan to pay $147 million, including $28.2 million to state agencies including Medicaid.To (a) describe the seller concentration in the national Medicaid generic lorazepam market over a 19-year period from January 1991 through December 2009, (b) estimate the excess payments for generic lorazepam by Medicaid between 1998 and 2009, and (c) investigate potentially increased utilization and prices of 2 substitute pharmaceuticals: branded lorazepam (Ativan) and generic alprazolam (another widely used intermediate-acting benzodiazepine).Using Medicaid State Drug Utilization Data from the Centers for Medicare Medicaid Services, we calculated the 4-firm concentration ratio (CR4) and the Herfindahl-Hirschman Index (HHI) for the Medicaid generic lorazepam market, along with pre-rebate reimbursement for pharmacy claims, number of claims (utilization), and average pre-rebate reimbursement per claim (average “price”) for generic lorazepam, from 1991 through 2009. Medicaid’s excess payments were estimated under 2 different assumptions regarding what the average generic lorazepam price would have been in the absence of the alleged conspiracy. To find counterfactual prices, the average per-claim reimbursement for lorazepam for the 4 quarters prior to the alleged conspiracy, $6.80, was inflated using (a) the quarterly change in the average per-claim reimbursement for generic alprazolam and (b) the Consumer Price Index (CPI) for all urban consumers, all goods. Potential impact of the alleged conspiracy on the branded lorazepam and generic alprazolam markets was investigated.The average pre-rebate reimbursements per claim for generic lorazepam were $10.25, $23.12, and $8.48 in 1991, 1998, and 2009, respectively. For the same 3 years, CR4 = 52.80, 76.02, and 86.74, while HHI = 905.71, 2,166.25, and 2,233.36. Medicaid’s excess payments from 1998-2009 were estimated at approximately $625-$657 million. The data also suggest the possibility of small impacts on the utilization of branded lorazepam and the price of generic alprazolam.Prior to the alleged conspiracy in 1997, average pre-rebate reimbursement per claim for generic lorazepam was declining, while seller concentration was rising. After a jump in average payment per claim in the years immediately following the alleged conspiracy, prices have gradually returned to their pre-1998 levels. However, the generic lorazepam market was more concentrated in 2009 than prior to the alleged conspiracy.


In Italy, cervical cancer represents the fifth most prevalent cancer in women under 50 years of age and is one of the most commonly detected lesions globally. Given the developing burden of the disease and the availability of both primary and secondary prevention measures, their accurate surveillance is of paramount importance. The aim of this study was to evaluate the trends in cervical cancer screening adherence in the period between 2020 and 2022, as well as to evaluate positive tests, identifying the most frequently associated genotypes and the vaccination coverage. The study sample was made up of 6880 women from the health district of Messina. We highlighted that there was a high proportion of positive results in the investigated period, with a high prevalence of HSIL. Moreover, HPV vaccination coverage was clearly inadequate, as was adherence to screening, both far away from WHO goals. This finding is probably linked to inadequate communication and awareness of the issue in the population and to the lack of data relating to tests carried out privately. In accordance with existing data in the literature, the introduction of the HPV-DNA test in Sicily made it possible to identify women positive for the genotypes most frequently involved in the etiopathogenesis of neoplastic lesions (genotypes 16 and 18), as well as for those in the “others” category, which should be investigated because some of them could have an impact on carcinogenicity and, for this reason, a future vaccine including them could represent a new prevention weapon. Cervical cancer is the second cancer most prevalent in the world, after breast one, with 500,000 new cases and 250,000 deaths every year . In ItalIn Sicily, the regional database of cancer registries observed, for the period of incidence considered 2007–2011), an average annual rate of 6.1 × 100,000, with about 160 new cases on average per year, while mortality data from the Register of Names of Regional Causes of Death (ReNCaM) record an average of about 30 deaths a year. The median 5-year survival is just under 70% [–2011, anIn particular, in the Sicilian Local Health Unit (LHU), the last regional report on the percentage of women who reported having carried out a preventive PAP test in the last 3 years found it was around 69%  . In 2015By applying the correct protocol, the overall costs of screening based on the HPV-DNA test are estimated to be lower than those of conventional cytological screening with a 3-year interval, even if the cost per single round of screening is higher. For these reasons, the Italian 2014–2018 National Prevention Plan provided that by 2018, all regions would switch to the HPV-DNA test as a primary screening test . The HPVMoreover, a descriptive study of hospital discharge records (HDRs) was conducted in Italy between 2008 and 2018. Overall, 670,367 hospitalizations due to HPV-related diseases occurred among Italian subjects. In addition, a significant decrease in hospitalization rates for several types of cancer such as cervical, vulval, vaginal and oropharyngeal cancer, as well as genital warts, was observed. This indicated the positive impact of HPV vaccination coverage and cervical cancer screening on hospitalizations due to cervical cancer ,14. The aim of this study was the evaluation of women’s adherence to screening during and after the pandemic period, testing the local rate of positive tests, identifying the most frequently associated genotypes and, at the same time, the evaluation of vaccination coverage, with particular attention to the onset of L-SIL or H-SIL after the immunization program in the cohort of patients born after 1996 (year of introduction of the HPV vaccine in the Sicilian region).This retrospective study was conducted thanks to cooperation between “Universitary hospital G. Martino”  in Messina and the Screening Management Center of the LHU in Messina, in the period from January to July 2023. The sample consisted of 6880 women aged between 25 and 64 years who had been contacted for cervical cancer screening according to the guidelines of the Sicilian region . In detaNowadays, 2 types of HPV-DNA tests are available: low- and high-risk (HR) HPV-DNA test. The former is a test that allows you to identify each high-risk  and low-risk  HPV virotype. It is recommended in (a) women aged 30–35 every 3–5 years; (b) after a positive HR HPV-DNA test; (c) in the presence of cytologic abnormalities associated with low-risk virotypes. The latter is used for the exact identification of high-risk genotypes  and for generic screening for twelve high-risk virotypes . The type used in the Sicilian region is the latter, and so the results of our investigation could be positive/negative for genotypes 16 and 18 or positive/negative for high-risk types.Continuous variables were expressed as mean ± standard deviation; categorical variables were expressed as absolute and percentage frequencies. Statistical analysis was performed with R software.From the data analysis, it appears that the increase in the effectiveness of cervical screening in the province of Messina (percentage of women aged 25–64 from the target population who received a letter of invitation from the year 2020 to 2022) was equal to 28% in the investigated period .In the sample (n = 6880 women), the rate of adherence to screening in the province of Messina was evaluated in the three-year period under examination. In 2020, the compliance rate was 12.32%, with a growing trend in subsequent years, rising to a 16.85% adherence rate in 2022. In the sample investigated, 23% of screening test results were positive  with different distributions in the various age groups and, in particular, with a higher prevalence in the 25–33 age range  .The most prevalent lesions in our sample were HSIL , and so we recorded an increasing trend compared to previous years.In addition, the rate of positive HPV-DNA test results by type of lesion was analyzed see . From the total number of women screened with both PAP tests and HPV-DNA, we then extrapolated and further analyzed the sample of women who continued the diagnostic process with a second-level investigation; specifically, we analyzed a sample of 110 women screened in 2021 (average age 34.4 ± 9.6 SD) and 174 in 2022 (mean age 39.3 ± 9.2 SD).In this cohort, we evaluated the frequency of the various lesion types and their possible correlation with high-risk genotypes by year. The results are shown in Among the genotypes identified, the most prevalent one was 16 (36% in 2021 and 30.45% in 2022), followed by HPV 18 (7.5%). In the remaining part of cases, other high-risk strains were found in 36.2% of cases, and other low-risk genotypes in 12.6% of cases .Colposcopy with subsequent biopsy and histological examination, carried out as a confirmatory investigation, was negative for the presence of lesions in 19% and 20% of cases in 2021 and 2022, respectively, while in the remaining cases, a high-grade lesion (CIN3) was found in 12% of the 2021 cohort and in 14% of that of 2022; an intermediate-grade lesion (CIN2) in 14% (2021) and 22% (2022); and, finally, a low-grade lesion in 45% (in 2021) and 39% (2022) cases (CIN1). Only in 1% of the cases did we find cervical cancer.The HPV 16 genotype was not only one of the most frequently isolated, but also the one associated with the most severe histological lesions.Moreover, a high number of lesions (CIN1-3) associated with the “Others” category of genotype was found both in 2021 and 2022, with percentages ranging from 1% to 8% in 2021 and from 2% to 18% in 2022. We then analyzed data on vaccination coverage in the two cohorts examined by evaluating the percentage of subjects vaccinated at the time of diagnosis. We recorded that the rate of vaccinated people was 9% in 2021  and 27% in 2022 .Regarding the number of doses received at the time of diagnosis, it should be emphasized that the majority of subjects examined had received three doses , while age at vaccination was less than 35 years in 80% of subjects examined in 2021 and in 55% of subjects in 2022, respectively (>35 years—20% in 2021 and 45% in 2022).In our study, data analysis showed that the adherence of cervical screening in the province of Messina was equal to 28% in the investigated period; this figure is markedly lower than that for the rest of the South (68.8%) and, unfortunately, still far from that for regions in Northern Italy ,15,16. Furthermore, the adherence data collected highlighted a poor adherence to HPV screenings, probably linked to multiple factors, one of which, at least initially, was the COVID-19 pandemic. In fact, as can be seen from the results presented, there was an upward trend in 2022 compared to the previous two years.The low adherence rate that we found (12–16.85%) differs from the WHO recommendations, which require that 70% of women worldwide be routinely screened for cervical disease with a high-performance test and that 90% of those who need it receive adequate care. Indeed, together with vaccinating girls against human papillomavirus (HPV), the implementation of this comprehensive strategy could prevent more than 62 million deaths from cervical cancer in the next 100 years . Integration of vaccination and screening programs for the prevention of cervical cancer: interventions to redefine and implement new screening protocols for women vaccinated before the screening start age” funded by the National Center for the Control of Infectious Disease (CCM) 2016 program, the initial screening age will be raised to 30 years with the introduction of HPV tests. In most regions, there were cohorts of women vaccinated at 12 who reached the age of 25 already in 2021. In other regions, initial screenings of cohorts vaccinated at 12 will take place in 2022. Based on the 2020–2025 National Prevention Plan, regions have the mandate to prepare specific “Free Programs” as regards cancer screening, one of which in particular relates precisely to the screening of vaccinated cohorts. Vaccination status is defined by matching the names of women invited for screenings with regional or corporate archives of vaccinated women. As demonstrated by the 2016 CCM study, this may be incomplete: for example, girls who resided in other regions or provinces are not vaccinated. According to the WHO , moreoveIn our study, the positivity rate for both types of test was 23% and, in particular, 13% were positive for the HPV test; this is a worrying value in light of the fact that HPV prevalence in the general population in Italy has a lower rate (8%) . In factIn our sample, the prevalent lesion was HSIL; an increase in HPV prevalence was found both in the total sample and in women with abnormal cytology. In particular, in the examined three-year period, we recorded a prevalence of 15% in the case of ASCUS , 80% in the case of LSIL , 86% in the case of ASC-H, 92% in the case of HSIL  and 70% in the case of adenocarcinoma. Biopsy analysis allowed our study to evaluate the genotypes most implicated in the various types of lesions; first, according to the data collected at a national and global level, the genotype most frequently associated with the lesions was HPV 16; subsequently, in contrast to nationally conducted studies, in second place we found the “Others” category and not HPV 18. In fact, type-specific prevalence studies in Italy pointed out that the most frequent type of virus is HPV 16. The prevalence of HPV 16 in the healthy population is approximately 5% (2–10%), while the prevalence of HPV 18 is lower, with an average value of just over 1% (0–6%).In our study, we also evaluated the prevalence of the various types of HPV in cytological lesions in the two-year period 2021–2022. As can be seen in It is important to underline that, in the period under study, there was a fair number of lesions, even of an advanced degree, associated with the “others” genotype category, supporting the implication of these genotypes and hence the need to test more and to create a provincial dataset to evaluate their prevalence in our city. Finally, we wanted to evaluate the percentage of vaccinations in screened women in the subsample: the percentage of vaccinated women in 2022 was 27%; in 80% of cases, the age at vaccination was less than 35 years, with a cycle of at least two doses only found in 20% of the sample .This figure is also important in light of recent scientific evidence which demonstrated that HPV vaccination was associated with complete viral elimination in 86% of cases. Analyzing the differences between healed and unhealed, it was highlighted that the vaccinated, while producing anti-HPV antibodies, had a significantly lower antibody rate, about seven times, compared to the healed. The study states that the time to virus elimination for people who already have the infection is, on average, about two years, in the meantime exposing these subjects to the risk of infertility, multiple miscarriages, condylotomies and HPV-related tumors. In the subjects investigated, vaccination proved to be able to induce the elimination of the virus in 86% of cases, through an increase in the rate of circulating anti-HPV antibodies. They also highlighted how a cut-off value equal to or greater than the threshold value of 1:125, obtained from the analysis of the ROC curve, can determine a greater clearance rate, thus confirming the therapeutic efficacy of the vaccine. Therefore, in cases of low antibody production, it is possible to hypothesize the use of a second vaccination cycle or an additional dose to obtain a level of antibodies capable of guaranteeing the curative effect.In conclusion, we can state that the analysis of the results brought out an inadequate adherence rate, certainly far from the values present in other situations in Northern and Central Italy, confirming the data present in the literature, and far too low to achieve the elimination of cervical cancer by 2030.This finding is probably linked to inadequate communication and awareness of the issue of screening in the population. Another determining factor could be represented by under-reporting relating to the number of screening tests carried out privately.From this perspective, possible strategies to combat this issue could be to create a synergy between the local authorities and gynecologists, to recover data relating to the remaining portion of the population that presumably performs the tests privately, to involve GPs  as a tool for action in light of the relationship of trust established with patients and finally to strengthen educational and communication campaigns. Finally, it is clear that the COVID-19 pandemic certainly impacted adherence to screenings, due to the fear of contagion that the global population experienced in the three-year period under study, the loss of efficiency in routine activities due to dealing with the emergency as well as due to the initial restrictive measures.Self-sampling could, in such cases, represent an essential tool in overcoming possible difficulties in accessing the screening sampling clinics . Self-collection has already been proposed in various European countries  and in dA strength of our study is certainly its retrospective nature, which allowed us to record real and unreported data, unlike other types of studies present in the literature; however, the limitation linked to the lack of data from privately performed screenings and then the impossibility of further analyzing all HPV genotypes present in the population under examination should be noted.Finally, we can state that many interventions could be useful for increasing not only screening uptake but also vaccine coverage, especially for at-risk categories such as pregnant women, frail patients and healthcare workers. This last category has a fundamental role in fighting vaccine hesitancy and more interventions must be implemented to increase the knowledge and expertise of many categories of HCWs in terms of vaccine use in general population. Public health specialists are the main figures driving and serving as an example to increase prevention strategies ,27,28,29


Its genotypes are classified into two main classes, high-risk genotypes and low-risk genotypes, according to their capacity to induce cancers. The low-risk class (types 6 and 11) is associated with anogenital and genital lesions. The high-risk class is responsible for up to 4.5% of all new cancer cases yearly. The aim of this study was to evaluate the incidence of HPV-related hospitalizations and its trend in a southern Italian region for the years 2015–2021. (2) Methods: This was a retrospective study performed in the Abruzzo region, Italy. All admissions for the period 2015–2021 were extracted from the hospital discharge record (HDR). (3) Results: During the study period (2015–2021), a total of 5492 hospitalizations attributable to HPV infection occurred in the Abruzzo region, Italy. A significant number of admissions were related to cervical cancer (3386 cases) and genital warts (638 cases). The trend declined for all diagnoses except for penile cancer admissions. Considering the first year of the pandemic (year 2020), a decrease in the standardized incidence of the majority of the diseases considered was reported, particularly among cases of cervical cancer. (4) Conclusions: HPV-related hospitalizations decreased in Abruzzo during study period. These results could be useful to LHAs and policy-makers in improving vaccination coverage and screening adherence. The human papillomavirus (HPV) is the most common agent related to sexually transmitted infections in the general population ,2.The number of HPV genotypes that have been identified is almost 200 ,4, and tHPV genotypes are classified into two main classes, high-risk genotypes and low-risk genotypes, according to their capacity to induce cancers. The low-risk class, mainly represented by types 6 and 11, is associated with anogenital warts and low-grade genital lesions . On the Although Pap tests and HPV-DNA screening tests are able to early detect cervical cancers, there is a lack of screening tools to prevent other types of HPV-related cancers. Therefore, the most important strategy for facing off against HPV-related diseases is vaccination . A safe The World Health Organization aims to eradicate cervical cancer by 2030 if nearly 90% of female adolescents are vaccinated against HPV . To meetThis was a retrospective study performed in the Abruzzo region, Italy. Abruzzo is a southern Italian region that counts over 1.3 million inhabitants and whose healthcare services are organized into four local health authorities (LHAs) . Data reCondyloma acuminatum (078.11);Head and neck cancers ;Anal cancers ;Cervical cancers ;Cancers of the vagina, labia, and clitoris (184.0–184.8);Penile cancers .Among procedures, the following codes were also considered:Cervix conization (67.2);Cervical lesion cauterization (67.32);Cervical lesion cryosurgery (67.33).In order to identify all HPV-related hospitalizations, the following codes were considered, according to the ICD-9-CM  and prioIf a patient experienced multiple admissions for the same diseases, it was counted only for the first time. Codes 233.3  and 184.9  were referred to lesions in an unspecified site and, in order to avoid the overestimation of the incidence of HPV-related hospitalization, they were not included.p-value less than 0.05 was considered significant. The statistical analysis was performed with STATA v14.2 software .Annual and total hospitalization rates for each HPV-related disease  were calculated per 100,000 inhabitants using the related attributable fractions, according to most recent literature ,16: cervDuring the study period (2015–2021), a total of 5493 hospitalizations attributable to HPV infection occurred in the Abruzzo region, Italy. The most frequent admissions were related to cervical cancer (3386 cases) and genital warts (638 cases), as reported in Considering the first year of the COVID-19 pandemic (year 2020), a decrease in the standardized incidence of a large part of the diseases considered was reported, particularly among cervical cancer (PC −10.8 from 2019 to 2020), genital warts (PC −30.9 from 2019 to 2020), oropharyngeal cancer (PC −4.7 from 2019 to 2020), and anal cancer (PC −30.3 from 2019 to 2020). On the other hand, a strong increase was reported for all these conditions in the year 2021. Only penile cancer and genito-urinary tract cancers reported an increase between the years 2019 and 2020, with PCs of 50.0 and 91.3, respectively. Oropharyngeal cancer admissions also decreased in the years 2020 and 2021, as reported in Among diseases concerning both genders, the highest incidence was observed for oropharyngeal cancers, particularly among older classes of male subjects. Gender differences in the incidence of anal cancer were observed during first two years of the study, but this difference was reduced during study period, as reported in Oropharyngeal cancer showed a higher incidence among older men (from 1.3 to 1.6/1000 inhabitants) compared to women (from 0.6 to 0.5/1000 inhabitants).The present study reported the trends of hospitalization for HPV-related diseases from 2015 to 2021 in the Abruzzo region, Italy. Cervical cancer demonstrated the highest incidence rate, with a yearly average rate of 36.6 cases/100,000. Despite this, the hospitalization rate appears lower in the Abruzzo region compared to other Italian regions and to Italian data overall ,16. OverDespite these results, Abruzzo has low vaccination coverage against HPV compared to the threshold required by the Italian Ministry of Health ,33. It iPenile cancer was the only affliction that was reported to have an increasing trend, despite the fact that the reported incidence rates remained lower than nation data . The greRegarding the pandemic period, COVID-19 heavily impacted healthcare systems, causing a reduction in hospital admissions for many diseases . Data reFinally, the overall decrease in admission trends for all included diagnosis confirmed the national trend  in most Overall, the results reported by this study were similar to national data recently reported by Restivo et al. . The decThe strength of this study is the large sample analyzed and the long study period considered. This is the first study to have been conducted in the Abruzzo region considering all the hospital admissions related to HPV. In addition, this the first study conducted in Italy that evaluates the most recent years (2019–2021).However, the results of this study should be considered in the light of the following limitations. Firstly, the reported diagnoses in HDR were based on ICD-9-CM codes that did not consider the severity of the condition and the HPV genotype. Secondly, in HDR, some diagnostic codes could have been under-reported or incorrectly reported, causing underestimates of the rate. Thirdly, these results were referred to a small Italian region. Finally, this study evaluated only hospital admissions, and it could not consider procedures performed by private practitioners or ambulatory care facilities, especially for genital warts. Thus, this study cannot be used to evaluate the real incidence of HPV-related illnesses.HPV-related hospitalizations decreased in Abruzzo during the study period. These results could be useful to LHAs and policy-makers in improving vaccination coverage and screening adherence. A decreasing trend in hospitalizations due to the extension of HPV vaccination to male subjects is expected in the coming years. In parallel, trends should also be analyzed in the future to evaluate the impact of the COVID-19 pandemic on the decrease in vaccination and screening adherence.


In the context of cervical cancer prevention, where human papillomavirus (HPV) infection is pivotal, HPV testing is replacing Pap Smear in primary screening. This transition offers an opportunity for integrating self-sampling to enhance coverage. We evaluated the accuracy of HPV testing using self-collected urine and vaginal samples, comparing them to physician-collected cervical swabs. From a cohort of 245 women with abnormal cytology, we collected self-sampled vaginal, urine, and clinician-administered cervical specimens. Employing Anyplex™II HPV28 assay, outcomes revealed HPV positivity rates of 75.1% , 78.4% , and 77.1% (urine). Significant, hr-HPV detection concordance was observed between self-taken cervical samples and clinical counterparts . This study extends beyond accuracy, highlighting self-collected sample efficacy in detecting high-grade cervical lesions. The insight underscores self-sampling’s role in bolstering participation and aligns with WHO’s goal to eliminate cervical cancer by 2030. Cervical cancer (CC) is one of the most common cancers in women and ranks fourth among female malignancies  according to GLOBOCAN 2020 data [Analogous to its relevance in other neoplastic conditions, screening has emerged as a pre-eminent strategy for mitigating the impact of cervical carcinoma on the populace. Since the inception of the Pap test in the 1950s, the conspicuous effectiveness of preventive screening has rapidly manifested in the confrontation against cervical cancer. However, the outcomes of cytological examination are contingent upon the expertise of medical pathologists for interpretation.Human papillomavirus (HPV), an established DNA virus, assumes a pivotal role as a requisite factor in the genesis of cervical cancer. Consequently, the emergence of methodologies proficient in detecting the presence of viral DNA specific to high-risk HPV (hr-HPV) variants has furnished supplementary tools to amplify the efficiency of screening initiatives, engendering heightened clinical sensitivity compared to the Pap test ,5. The IThere are several different commercially available HPV tests already validated following the Meijer or VALGENT protocols ,7,8. AllOne of the major obstacles in implementing efficient prevention programs is to ensure the regular participation of women at the highest risk for cervical cancer development (age range of 25–65 years). In the context of cervical cancer screening in Italy, the data derived from surveillance efforts reveals that several emerging factors contribute to non-adherence to screening protocols. These factors encompass the lack of comprehensive information, a disposition towards indolence, and constraints imposed by limited time availability. Moreover, women who abstain from screening have communicated additional concerns, including feelings of embarrassment and apprehension linked to the prospect of physician examination . The proThe collection of vaginal specimens allows HPV detection because most of detached cervical cells are released with mucous in the vagina. Vaginal self-collected sampling using a vaginal swab is easy to execute and could be performed at home. Another sample that could be used for self-sampling is first-void urine (FVU), the collection of which is non-invasive and has showed similar results in HPV testing compared to cervical specimens ,17. DuriTherefore, the aim of this study is to evaluate the accuracy of HPV DNA testing conducted on urine and vaginal self-samples compared to clinician-collected cervical samples and to investigate the distribution of HPV genotypes (both high- and low-risk) in different sample types.The study protocol was approved by the Ethics Committee of University of Milano-Bicocca (protocol numbers 0037320/2017 and 0086409/2018). All subjects provided written informed consent to participate in the study.®, Novosanis, Wijnegem, Belgium) and a vaginal self-sample  before colposcopy examination. An information brochure of the study and instructions on how to use the devices were given to all participants.A cohort comprising 245 women with a recent diagnosis of cervical dysplasia were enrolled at the Colposcopy Clinic of IRCSS San Gerardo dei Tintori, Monza, Italy, during the period spanning from May 2017 to January 2021. All women were adequately informed about the study by Colposcopy Clinic staff and were asked to autonomously collect a urine specimen (20 mL)  and the Pap smear test was repeated if it had been performed over 6 months before the date of the colposcopy. A cytological diagnosis of cervical lesions was performed at the hospital, and findings were assessed according to the 2001 Bethesda System for cervical cytological reporting. Cytological findings were classified as follow: HSIL ; ASC-H ; LSIL ; ASC-US ; AGC ; and NILM . Women underwent biopsy examination or conization treatment depending on the colposcopy results. Histological findings were classified as cervical intraepithelial neoplasia grade 1, 2, or 3 ; squamous cell carcinoma; adenocarcinoma; or adenosquamous carcinoma. The classification of histological findings was conducted according to the WHO histological classification of tumors [During the colposcopy examination, the gynecologist collected a cervical sample using an L-shaped Endocervical/Esocervical FLOQSwabf tumors . All physician-collected cervical, vaginal, and urine samples were tested at the Clinical Microbiology Laboratory of the Department of Medicine and Surgery, University of Milano-Bicocca, Italy.® and transported in a tube with 20 mL of ThinPrep® PreservCyt® Solution . All samples were well shaken using vortex for 30 s and 1.5 mL aliquots were made. One aliquot was used for DNA extraction purposes.Cervical samples were collected using the L-shaped Endocervical/Esocervical FLOQSwab® PreservCyt® Solution. Five aliquots were made (one was used for DNA extraction and the others were stored at −20 °C).Vaginal self-samples were transported dry to the laboratory and immediately suspended in 5mL of ThinPrepAfter arriving to the laboratory, urine specimens were shaken for 30 s using the vortex and 1.5 mL aliquots were made. One aliquot was used for nucleic acid extraction, while the others were stored at −20 °C.® easyMag® , an automated system used for isolating nucleic acid from clinical samples. The protocol chosen was “specific B”, characterized by a higher final elution temperature (about 70 °C), and silica beads (diluted in a ratio of 1:2) were used, in accordance with the manufacturer’s instructions. The nucleic acids extracted from cervical and vaginal samples were eluted in 100 μL of NucliSENS® easyMag® elution buffer , and the ones from urine were eluted in 40 μL.For 180 patients, DNA isolation was performed starting from 1 mL of each specimen using the NucliSENSSamples from the remaining 65 women were extracted using the STARMag 96 × 4 Universal Cartridge Kit  on Microlab Nimbus, a totally automated platform for nucleic acid extraction and PCR plate preparation. The starting volume used for the extraction was 200 µL, and the elution volume was 100 µL for all the three different sample’s types.Cervical, vaginal, and urine samples obtained from 50 women were analyzed using both nucleic acid extraction methods to evaluate the concordance of the results obtained following different protocols.Cervical, vaginal, and urine specimens underwent HPV typing using a commercial kit known as Anyplex™II HPV28 . This kit is proficient in identifying 19 high-risk HPV types  and 9 low-risk HPV types  with two different real-time PCR reactions performed on CFX96  as reported by the manufacturer’s instructions. DNA detection was performed with 5 μL of DNA in each of the two 20 μL reaction mixtures with two different primer sets. Data recording and interpretation were performed with Seegene Viewer software, in accordance with the manufacturer’s instructions.Qualitative and quantitative variables were summarized with absolute (relative) frequencies and medians , respectively.The HPV typing analysis was performed considering as high-risk types: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 and low-risk types: HPV 6, 11, 26, 40, 42, 43, 44, 53, 54, 61, 69, 70, 73, and 82.http://graphpad.com/quickcalcs). Agreement was interpreted as slight (k < 0.200), fair (0.200 < k < 0.401), moderate (0.400 < k < 0.601), good (0.600 < k < 0.801), very good (0.800 < k < 1.000), and perfect (k = 1.000) [The agreement of HPV typing results between paired cases was evaluated with Cohen’s kappa (κ) statistics and their uneven distribution was evaluated with McNemar’s test using GraphPad QuickCalcs software  .Cervical, vaginal, and urine samples collected from 245 women were analyzed. The average age of the patients at the time of enrollment in the study was 38.6 years (range: 17–67 years), while the median was 38 years (IQR: 31–46 years). Abnormal Pap smears at the enrolment were detected in 92.6% (227/245) of the study participants with low-grade lesions, with ASC-US  and LSIL  being the most frequently reported. Data regarding cytological lesions are reported in ® easyMag® extraction protocol, we observed hr-HPV positivity rates of 80% (40/50), 78% (39/50), and 76% (38/50) in cervical, vaginal, and urine samples, respectively. We obtained the following results using the platform MicroLab Nimbus: 72% (36/50), 80% (40/50), and 70% (35/50) in cervical, vaginal, and urine samples, respectively. A good concordance in hr-HPV detection was observed using the two different extraction protocols by comparing the results obtained from all three types of samples analyzed, as reported in Samples collected from 50 women were randomly chosen and analyzed to compare results obtained using the two different nucleic acid extraction protocols. The concordance was evaluated considering just the positivity for hr-HPV genotypes. A very high percentage of hr-HPV positivity was obtained among all types of samples analyzed and using both extraction methods. In particular, using the NucliSENSOverall, 75.1% (184/245), 78.4% (192/245), and 77.1% (189/245) of cervical, vaginal, and urine samples, respectively, were positive for at least 1 of the 28 HPV genotypes detected using the Anyplex™ II HPV28 Detection Kit. In particular, 161 women (65.7%) were positive for at least one hr-HPV both in cervical and urine samples, while 170 women (69.4%) were positive in vaginal self-samples. lr-HPV infection was detected in 40.0% (98/245) of the cervical samples, 48.6% (119/245) of the vaginal self-samples, and 50.2% (123/245) of the urine samples. HPV16 and HPV31 were the most frequently reported hr-HPV types , while HAs shown in Almost half of hr-HPV-positive samples were coinfected by more than one hr-HPV genotype, with urine and vaginal self-samples showing more multiple cervical sample infections: 49.7% (80/161), 48.2% (82/170), and 42.2% (68/161), respectively .Considering the positivity for at least one of hr-HPV, the observed agreement between cervical and vaginal samples was 95.5% with k = 0.898. HPV16 and HPV18, which are the main responsible for cervical cancer, showed a very good agreement with k = 0.945 and k = 0.940, respectively. The concordance in lr-HPV detection was 88.2% with k = 0.762. Concordance in the hr-HPV test results between cervical and vaginal specimens is shown in The concordance rates between cervical and urine specimens were 90.6% (with k = 0.792) for hr-HPV and 85.7% (with k = 0.715) for lr-HPV. In particular, a very good concordance rate in HPV16 (k= 0.874) and HPV18 (k = 0.866) results was found. Genotype-specific HPV agreement rates are reported in ® easyMag® and Seegene Microlab Nimbus platforms, implemented with the STARMag 96X4 Universal Cartridge Kit. A good analytical concordance was observed ; however, slightly higher hr-HPV positivity was observed among aliquots extracted using NucliSENS® easyMag®, probably in part resulting from the different starting volumes: 1 mL for NucliSENS® easyMag® compared to 200 µL for the STARMag 96X4 Universal Cartridge Kit protocol on Microlab Nimbus. The clinical relevance of these differences in HPV detection is still not fully understood due to the low number of samples analyzed.According to the World Health Organization, without any additional actions, the annual number of new cases of cervical cancer will rise from 570,000 to 700,000 between 2018 and 2030. In order to eliminate cervical cancer as a public health problem, one of the targets to reach by 2030 is to screen 70% of women with a high-performance test by 35 years of age and again by 45 years of age . Over thIn this study, we also compared the accuracy of the HPV test on vaginal self-samples and urine compared to cervical samples (gold-standard) in women referred to colposcopy for an abnormal cervical cytology. At the time of enrolment, a very high rate (75.1%) of women were positive for at least 1 of the 28 HPV genotypes in cervical sample, as expected in a colposcopy setting. This HPV positivity rate is similar to other percentages reported in previous studies conducted on a population with the same characteristics ,31,32. HPositivity for at least one of the hr-HPV genotypes was found in 65.7% of women, with the most common genotypes being HPV16 and HPV31. Analyzing self-taken specimens provided similar results, with a percentage of 69.4% for HPV-positive women and 65.7% in vaginal and urine samples, and the agreement with cervical swab results was 95.5% (k = 0.898) for vaginal samples and 91.4% (k = 0.764) for urine samples. These promising results are in accordance with previous studies that have evaluated self-collected samples using molecular methods for HPV detection ,34,35,36Moreover, this study demonstrated that the hr-HPV testing of self-collected samples using Anyplex™II HPV28  is not inferior to the gold standard for the detection of cervical intraepithelial lesions that are potentially at risk of progression. Relative sensitivity rates for the detection of CIN2+ were 1.00 and 0.95 for vaginal self-samples and urine samples, respectively. The relative specificity was 0.89 for self-collected vaginal samples and 0.95 for urine samples based on the total population. From the analysis of women with histological outcomes, the relative specificity was 1.00 for both types of self-collected samples. A good clinical sensitivity rate for the detection of CIN2+ lesions was obtained using the three sample types; even the analysis was restricted to the seven hr-HPV samples described as mainly responsible for cervical cancer development  ,40, undeThe high HPV positivity rate in this population and the presence of transient HPV infections that spontaneously regress are responsible for the low specificity rates for the detection of CIN2+ lesions for the three sample types which were demonstrated to be higher when the population examined was constituted by women undergoing screening .Indeed, a limitation of this study is that, in order to achieve statistical power, it was conducted in a population of women referred to colposcopy due to previous cytological abnormalities, as indicated by the VALHUDES protocol . Such a Another important consideration concerns the choice of HPV test to be used on self-samples. In the present study, we selected the Anyplex™ II HPV28 Detection  Kit in order to evaluate the distribution of both lr-HPV and hr-HPV in the different anatomical sites. However, as previously recommended, cervical cancer screening should not include testing for low-risk HPV types . MoreoveA specific study design, the VALHUDES protocol, has been proposed for the combined validation of the HPV test and the self-collection device to establish the proper assay cut-offs and achieve clinical accuracy in providing non-inferior self-collection respect to the cervical specimen . Finally, present triage guidelines for hr-HPV-positive women are based on cervical cytology. As cervical cytology cannot be performed on self-collected samples, it is important to consider that in a screening program based on self-sampling, hrHPV-positive women need to be recalled in order to collect an additional cervical sample to perform cytology triage. This introduces an inherent risk of loss associated with the follow-up of HPV-screen-positive women, which may be further exacerbated among non-attendant individuals, a group already predisposed to challenges in adherence. In the future, it would be important to identify new molecular biomarkers suitable for the triage of hr-HPV-positive women, such as methylation- and genotype-specific viral loads, which would allow triage to be performed directly on self-collected samples.These results confirmed that performing Anyplex™II HPV28  on urine and self-collected vaginal samples for the detection of hr-HPV in women with CIN2+ is comparable with that of clinician-taken cervical samples. These findings support initiatives to implement self-sampling as an option to improve participation of non-attending women in screening programs.


A T3 transformation zone had a protective effect, reducing the likelihood of significant pathology. Infection with HPV high risk (non 16/18) or HPV high risk (16/18), on the contrary, increased the likelihood of CIN2+ four times and seven times, respectively, compared to the lower-risk category. Conclusion: A differential approach in the workup of ASC-H cytology is advisable. Younger, premenopausal patients with positive HPV high-risk findings are at a significantly higher risk for CIN2+ lesions than older postmenopausal women with negative high-risk HPV test results. As the risk increases from HPV high-risk non 16/18 to HPV 16/18 infections, using HPV PCR testing is advisable.Background: This study aims to investigate whether specific characteristics of the patient group with ASC-H (PAP III-p) findings increase the likelihood of clinically significant disease (CIN2+), offering implications for risk-adapted clinical management. Methods: 225 patients with an ASC-H smear presenting to our colposcopy unit between 2014 and 2021 were identified and examined retrospectively. A total of 203 patients were included in the cross-sectional study using multivariate logistic regression. Results: Of the 12 variables that entered the regression model, transformation zone ( Infections with high-risk human papillomaviruses (HPVs) are the leading risk factor for the development of cervical cancer and its precursor lesions, cervical intraepithelial neoplasia (CIN) ,2,3,4. TIn July 2014, the Munich Nomenclature III for gynecological cytodiagnosis of the cervix became effective in Germany . For theThe German screening guidelines for abnormal findings demand a colposcopy within three months for all patients with an ASC-H screening result, regardless of the age group and HPV test result . HoweverASC-H findings display a heterogeneous spectrum of underlying entities, ranging from benign findings to CIN3 and cervical cancer ,17. At tFrom a total of 3791 patients examined at the dysplasia unit of the Department of Obstetrics and Gynecology at the University Hospital Aachen from July 2014 to August 2021, the 3218 patients presenting with cytological abnormalities were screened for a history of ASC-H or an ASC-H result at the time of examination. Patients who were presenting for vulvar or vaginal disorders were not considered for screening. Subsequently, all 225 identified patients with a documented ASC-H smear were examined retrospectively. Afterwards, patients who had a total hysterectomy with an ASC-H smear of the vaginal stump were excluded. Additional exclusion criteria were pelvic radiotherapy in temporal relation to an ASC-H PAP smear collection, a presentation at our dysplasia unit more than 12 months after an ASC-H result, and missing outcome data. Finally, 203 patients were included in the cross-sectional study .During the time of investigation, 2374 high-grade lesions were diagnosed at our center of colposcopy. Of these, 1330 were excised.The standardized colposcopic examination was performed in the DGK-certified colposcopy unit of the University Hospital Aachen by experienced and highly qualified AG CPC-certified personnel. It was executed with a Leisegang 3MCV colposcope. Every colposcopy included the systematic collection of a conventional PAP smear (control cytology). At the beginning of the study period, a test for human papillomavirus (HPV) was only taken if no external HPV test result was available or if it was older than six months at presentation. The collection of an HPV test at every colposcopy was standardized at a later stage during the study. The Seegene Anyplex II HPV 28 detection kit, an HPV DNA PCR test, was utilized throughout the study. This test kit identifies 19 high- and intermediate-risk HPV genotypes and 9 low-risk types.In some cases, an additional swab was taken for microbiological examination at the examiner’s discretion. Indications were evidence of genital infection in the examination, such as an abnormal PH level; conspicuous fluor vaginalis; reddening of the vaginal wall; or reported clinical symptoms, such as itching, burning, or suspicious genital discharge. The colposcopic findings were classified according to the terminology of the International Federation for Cervical Pathology and Colposcopy (IFCPC) of 2011 for the cervix uteri . The traThe patient’s baseline characteristics  were rec“Significant pathology” was defined as the presence of CIN2, CIN3, adenocarcinoma in situ (AIS), or cervical cancer, respectively, CIN2+, in the histopathological findings of the biopsy, LEEP, or TLH. The category “non-significant pathology” subsumes CIN1 and benign findings of the categories “healthy”, “inflammation”, and “atrophy”. If the biopsy displayed a higher-grade CIN than the subsequent LEEP or TLH finding, it was assumed that the higher-grade lesion had already been completely removed during the biopsy. The results were considered consistent and classified according to the higher-grade finding. All data on the patients’ histopathological findings, as well as the classification into significant and non-significant pathology, are based on original diagnoses; no review of a histological specimen was performed. One patient was classified in the “non-significant pathology” group based on the visual diagnosis of extensive atrophy in the colposcopy; no histopathological sample was taken.The patient’s menopausal status was gathered from previous medical findings or classified according to the German S3 Guideline “Peri- and Postmenopause- Diagnosis and Interventions” , based oAll statistical analyses were performed using IBM SPSS Statistics 27. Continuous variables are expressed as median ± interquartile range (IQR) and mean ± standard deviation (SD). The continuous variables were checked for normal distribution graphically utilizing a histogram with a normal distribution curve. Categorical variables are given as absolute frequencies and percentages.Significant pathology (CIN2+) is regarded as the primary endpoint for the primary analysis. A frequency analysis of the histopathological outcome was conducted. Binomial logistic regression was performed to identify predictor variables of significant cervical pathology in patients with ASC-H smears. A crosstab between the categorical predictors and the outcome variable was created to identify empty or small cells with very few cases. Accordingly, categories of the variables “HPV infection” and “contraceptive method” were combined to prevent the logistic regression model from becoming unstable . Since ip < 0.2 were considered as candidates for the multivariate logistic regression model. The first category was chosen as the reference category for all categorical predictor variables in the dummy coding. For the final multivariate logistic regression model   .2 (15) = 65.597, p < 0.001, resulting in a good amount of explained variance, as shown by Nagelkerke’s R2 = 0.427 > 0.26 [The final binomial logistic regression model  was stat7 > 0.26 .p = 0.045) and HPV infection (p = 0.005) contributed significantly to predicting CIN2+. Compared to the reference category of patients with a T1 transformation zone, T3 transformation zones had a protective effect, reducing the likelihood of significant pathology, OR = 0.244 . Infection with HPV high risk (non 16/18) or HPV high risk (16/18), on the contrary, increased the likelihood of CIN2+ compared to the lower-risk category, OR = 4.053  and OR = 7.410 . The variable menopausal status (p = 0.050) showed a low significant effect. The model included 170 cases; 33 subjects were excluded from the analysis due to missing data.The overall percentage of accuracy in classification was 74.1%, with a sensitivity of 77.3% and a specificity of 70.7%. Of the 12 variables that entered the regression model, transformation zone  could not be included in the multivariate model due to a too high number of missing values. The variable “age” was excluded from the final multivariate regression model due to a lack of significance in the multivariate regression and the known correlation with the variables “menopausal status” and “transformation zone”.The univariate significant variable “HPV vaccination” (2 (4) = 58.478, p < 0.001, resulting in a good amount of explained variance, as shown by Nagelkerke’s R2 = 0.358 > 0.26 [A stepwise forward selection  was conducted. The established selection model  was stat8 > 0.26 .p-values of F, using a p-value < 0.05 as the default entry criterion. The overall percentage of accuracy in classification was 74.3%, with a sensitivity of 77.9% and a specificity of 70.7%. A total of 16 subjects have been excluded from the analysis due to missing data; the final selection model included 187 cases.The final selection model consisted of the variables transformation zone, entered in step one, and HPV infection, entered in step two. At each step, variables were chosen based on In the present study, we were able to show that high-risk human papillomavirus infection is a predictor of significant underlying pathology, a CIN2+ lesion, in ASC-H patients . The likAccording to the German screening algorithm for abnormal findings in cytology, a colposcopic examination is indicated within three months for all patients with ASC-H findings, regardless of age and HPV status . Howeverp = 0.045) and the selection model (p < 0.001). In this context, a type 3 transformation zone was a protective factor, reducing the risk of significant pathology by a factor of 0.244  and should only be understood as a trend. Perimenopausal status reduced the likelihood of CIN2+ in comparison to premenopausal status (p = 0.014), OR = 0.177 . Future studies with larger sample sizes might demonstrate corresponding effects. Nevertheless, we observed that CIN2+ was the most common outcome in the premenopausal patient group; premenopausal women were more likely to have significant pathology than those with peri- or postmenopausal status.Considering the association between age and menopausal status, premenopausal women with ASC-H are more likely to show significant pathology than postmenopausal women ,38,39,40In this study, there appeared to be mixed results concerning age, menopausal status, and T3 transformation zone. However, the results suggest that the biological age of patients, expressed by the T3 transformation zone, plays a role in the likelihood of underlying significant pathology in ASC-H patients.In our opinion, a differential approach in managing patients with ASC-H would be advisable. Younger, premenopausal patients with positive HPV high-risk findings could be evaluated by colposcopy. In older, postmenopausal patients with negative HPV high-risk findings, a wait-and-see approach seems reasonable. In this group of ASC-H patients, local estrogenization could be performed to prevent over-interpretation in cytomorphology due to atrophic changes, followed by a repeat smear . SubsequWhen looking at the mere diagnostic accuracy in predicting significant pathology, of course much higher sensitivity and specificity values can be achieved when including the result of the colposcopy in the model. However, in the presented analysis, we sought intrinsic patient characteristics that are not examiner dependent for a better characterization of the patient collective. Thus, our results can help triaging patients for immediate colposcopy versus a wait-and-see strategy based on their intrinsic characteristics.This is a retrospective study design with a specific patient population and a limited number of cases. Due to the retrospective approach, some limitations must be addressed. At the beginning of the study period, no new HPV smear was taken during the colposcopy if the external HPV smear was not older than six months at the time of presentation. Furthermore, the pathologist’s evaluation of the PAP smear could have been biased because the histological sample of the respective patient was simultaneously available for diagnosis. In addition, in some cases, not all anamnestic factors were documented, which led to missing data in the analysis. During the study period, the patient’s HPV vaccination status was not collected by default. However, ASC-H is a rare subcategory, and this study collected a variety of sociodemographic and clinical risk factors and included a wide range of patients. Previous research on predictors of clinically significant disease in ASC-H patients has frequently drawn on fewer cases or only looked at a small number of putative predictors ,38. The Our data show that HPV PCR testing with discrimination of HPV 16/18 and non-16/18 HR infections and consideration of biological age is significant for the risk assessment of underlying clinically significant disease (CIN2+) in patients with ASC-H findings. Younger, premenopausal patients with positive HPV high-risk findings are at a significantly higher risk for CIN2+ lesions than older postmenopausal women with negative high-risk HPV test results. Therefore, a differential approach in the workup of ASC-H cytology is advisable.


Precancerous lesions of the uterine cervix, due to HPV infections, are still today a great medical challenge. This clinical case highlighted the effectiveness of epigallocatechin gallate (EGCG), vitamin B12, folic acid, and hyaluronic acid (HA) in counteracting HPV lesions in a 39-year-old patient with a long history of viral persistence, cervical lesions of various degree, and several unsuccessful surgical approaches. After eight weeks of treatment, both the histological and cytological analyses revealed only a chronic cervicitis without any malignant lesions or cellular dysplasia, thus reducing the urgency of an invasive surgery, a total hysterectomy. The Human Papilloma Virus (HPV) is the etiologic agent of cervical cancer, the second most frequent tumor in women between 15 and 45 years of age . Over 10Depending on the oncogenic potential, papilloma viruses may be divided into low-risk (LR) and high-risk (HR). The former may cause benign external genital lesions (warts and condylomas), while the latter are associated with almost all types of cervical and anogenital cancers ,4. In about 80% of cases, the infection naturally reverts within one year due to the clearance activity by the immune system. However, in the remaining 20% of patients, the infection can persist over one year , thus faQuite often, the infection of HPV correlates with affections of cervical tissue, determining the occurrence of cervical intraepithelial lesions of various degrees, depending on the severity and the risk of tumoral progression. In detail, premalignant changes represent histological abnormalities ranging from Atypical Squamous Cells of Undetermined Significance (ASCUS) and mild dysplasia (CIN1) to moderate dysplasia (CIN2) and to severe dysplasia or carcinoma in situ (CIN3). Depending on the histological diagnosis and the degree of the severity, the treatment of the lesions is different. In the case of ASCUS/CIN1, physicians recommend no treatments at the time of diagnosis due to the high rate of spontaneous regression, while in the case of CIN2 or CIN3 they recommend surgical procedures. These latter include ablative methods that destroy the affected cervical tissue , as well as excisional methods, such as loop electrosurgical excision procedures (LEEPs) . NeverthIn light of this, current treatments only target the clinical signs of the infection, such as condylomas or cervical lesions, but no specific therapies are available to eradicate the virus and its persistence. As of today, vaccines and screening programs are the only available tools to prevent HPV infection , reducinMoreover, a randomized clinical study from Ahn and colleagues investigated the efficacy of green tea extracts on 88 HPV positive women with squamous intraepithelial lesions of various degrees. The results revealed a significant improvement in the degree of the lesions after an 8/12-week treatment with 200 mg/day of oral EGCG . Vitamin B12 and folic acid are crucial micronutrients whose deficiency may correlate with different pathological conditions, including cancer . Both thHyaluronic acid (HA) is one of the most intriguing and versatile natural molecules. Despite its simple structure, HA has several functions according to its molecular weight. In particular, the reparative action of very low molecular weight HA (<5 kDa)  plays a Despite several advances in scientific research and screening programs, HPV infection remains a global burden. Therefore, developing new therapeutical strategies to manage this infection and its persistence represents a great medical challenge. The aim of this clinical case was to describe the effect of the treatment with EGCG, vitamin B12, folic acid, and HA on the cervical lesions of a 39-year-old patient with indication for hysterectomy due to the recurrence of HPV infection.A 39-year-old patient with a 9-year clinical status of HPV persistence received a medical indication for hysterectomy. As it is an irreversible procedure and considering the fertile age of the patient, she decided to ask for another medical opinion, becoming the subject of this case report study. Regarding the background of the patient, her personal history reported she was a former smoker since 2016, while the obstetric and gynecological history revealed she had one physiological pregnancy with a cesarean delivery. In addition, she had no immunological diseases and she didn’t assume any immunosuppressive treatments. In 2013, a biopsy of the uterine cervix revealed a severe cervical intraepithelial neoplasia (CIN3), and she underwent loop electrosurgical procedure (LEEP). The histological analysis revealed a high-grade intraepithelial lesion (CIN2/3) with free margins followed by a persistent condition of low-grade squamous intraepithelial lesion (LSIL). In 2017, the HPV DNA test diagnosed the positivity for the high-risk HPV16. The next year, the Papanicolaou test (Pap-test) revealed a high-grade squamous intraepithelial lesion (HSIL). Later, the endocervical curettage and the relative tissue microscopical analysis revealed a condition of mild cervical intraepithelial neoplasia (LSIL/CIN1). Despite the clinical guidelines for LSILs, the patient underwent a second LEEP. Postsurgical analysis revealed no squamous intraepithelial lesions. Five months later, the Pap-test evidenced another HSIL, which was confirmed after 3 months. Postsurgical analysis after a third LEEP revealed a LSIL (CIN1) lesion. About 4 months later, another Pap-test revealed the presence of abnormal squamous cells in the tissue (ASCH) with a persistent positivity for HPV16, which was present for more than two years. Three months later, the HPV DNA test was still positive for HPV16. In 2020, seven years later from the initial diagnosis of HSIL (CIN3) and despite all the medical procedures, the patient still presented HSILs. Seven months later, the HPV positivity was still present, and the patient underwent cervicoscopy and hysteroscopy. The analysis revealed the presence of proliferating endometrial polypus and squamous intraepithelial lesions, which were consistent with a condition of HSIL (CIN3) with the margins of the resection free from lesion. The HPV DNA test was still positive for HPV16 type. Following these results, the patient underwent laser conization to remove the high-grade lesion. Five months later, the HPV16 positivity was still present, and the Pap-test revealed another HSIL. One month later, the patient repeated the colposcopy with biopsy. The objective gynecological exam revealed a regular vagina, without any alterations. The colposcopy reported that performing the exam was very difficult due to the anatomical condition of the uterus; the squamocolumnar junction (SCJ) was visible only back, and the transformation zone (TZ) grading was 1 back to the SCJ. So, physicians performed a biopsy that revealed again cytological atypia of low and high grade. At that time, following the troubled clinical history of the patient and considering her previous histological results and her anatomical condition, which did not allow further conservative interventions, the physicians proposed a surgery of total hysterectomy as the only solution. Therefore, following the request of the patient, we evaluated the effects of four natural molecules to explore alternative solutions to this invasive surgical procedure or at least to delay the need for this surgery.®  in a regimen of two tabs/day for eight weeks. Each tablet contains 200 mg of EGCG, 50 mg of HA, 1 mg of vitamin B12, and 400 mcg of folic acid. The patient underwent a treatment with the food supplement named PervistopThe exam of cervical–vaginal cytology is primarily a cervical cancer screening test. This test consists of a procedure that gently removes cells from the surface of the cervix and the surrounding area, and then perform the microscopical analysis. The histological analysis was performed by using the hematoxylin and eosin (H&E) staining. The hematoxylin stains cell nuclei purplish blue, while the eosin stains the cytoplasm and extracellular matrix pink. The pattern of the coloration shows the general layout and distributions of cells providing a general overview of the tissue structure. After the eight-week supplementation, the young patient underwent a Pap-test and cervical biopsy for cytologic and histological analysis, respectively. The cervical–vaginal analysis reported an adequate picked-up sample, with the presence of endocervical tissue component. The medical response indicated a strong inflammation, but it was strikingly negative for intraepithelial or malignant lesions. In addition, the histological analysis of the cervical biopsy reported chronic cervicitis as the unique diagnosis, confirming the absence of atypia in the laminae of squamous epithelium. Before the treatment, the cervical biopsy revealed the presence of both LSIL and HSIL . Koilocytosis and nuclear atypia, associated with an ongoing viral infection, can be observed in the LSIL region A, while Eight weeks after the oral assumption, the histological analysis highlighted parakeratosis and focal cellular alterations of koilocytosis attributable only to chronic cervicitis, without dysplasia A,B.The analysis of the collected cervical scraping from the Pap-test showed a squamous epithelium with focal perinuclear halos and hyperchromatic nuclei A,B. The Later, considering the complexity of the clinical history of the patient, she underwent a six-month follow up to monitor the HPV infection and the progression of cervical lesions. In particular, she underwent a colposcopy, Pap-test, and an HPV DNA test, and all the exams reported negative results. The objective gynecological exam reported regular external genitals and vagina, without alterations as regards the internal genital apparatus with linear endometrium and no effusion. The colposcopy did not observe any cervical lesions, and the Pap-test revealed no more cervical intraepithelial or malignant lesions. Finally, the HPV DNA test was also negative, thus confirming the improvement in the clinical conditions of the patient.In this clinical case, we demonstrated for the first time the effectiveness of a combination of four natural molecules—EGCG, vitamin B12, folic acid, and HA—in reducing the urgency of an invasive surgery, a total hysterectomy, in a young fertile patient. So far, clinical practice has had no tools for counteracting the persistence of the virus, since an effective treatment against the clearance of HPV is still lacking . In the In the case of this 39-year-old patient, the HPV infection had persisted for nine years. She underwent surgical procedures, more or less invasive, but none of them was effective in eradicating the HPV infection, with the occurrence of new lesions. Therefore, physicians proposed the surgical remotion of the uterus as the only irreversible solution. The eight-week oral administration of EGCG, folic acid, vitamin B12, and HA improved her pathological condition; in fact, both the cytological and the histological analysis revealed no more squamous intraepithelial malignant lesions for the first time after nine years. The exams only reported a chronic cervicitis, without dysplasia or malignant events. The presence of some cellular morphological alterations indicated a strong inflammatory ongoing process, but no signs of malignant lesions appeared from the analysis, confirming the positive effects of the treatment. Both in vitro and in vivo pieces of evidence have already demonstrated the protective role of these natural molecules by blocking the progression of squamous intraepithelial lesions. Studies on the individual molecules demonstrated the efficacy of (i) EGCG as an antiproliferative and proapoptotic factor ,15,16,17Here, we demonstrated that the association of four natural molecules had a protective effect in this complex HPV-pathological picture. They improved the existing cervical lesions, and they delayed the need for a hysterectomy, considered the only available solution for this patient. Subsequent exams, performed as a six-month follow up of the oral treatment, confirmed the initial beneficial results. The colposcopy, Pap-test, and HPV DNA test confirmed the effectiveness of the treatment, indicating that the oral assumption of these combined natural molecules may improve cervical intraepithelial lesions and contribute to a condition of HPV negativity. Although they are preliminary results reported at eight weeks and six months, this clinical case may pave the way for an innovative approach in the management of HPV cervical lesions and viral infection. Along with this, a recently published pilot clinical study also supports such positive effects. A small group of 20 women with persistent HPV infections and cervical lesions showed no cytological or histological evidence of lesions following the treatment with the same dosages of these four natural molecules .In the meantime, the patient is still taking the therapy, without experiencing any adverse events, and longer monitoring will be necessary to assess the long-term beneficial effect of such treatment. The management of HPV infections remains a critical topic among physicians, especially considering the persistence of the virus with its evolutionary success. To date, current strategies only act on preventing HPV infections through vaccines and screening programs or on targeting the clinical manifestations, such as cervical lesions and condylomas. Specific treatments that counteract the viral persistence, eradicating the virus, are still lacking. In this clinical case, the eight-week oral supplementation, containing EGCG, vitamin B12, folic acid, and HA, restored the high-grade cervical lesions, thus preventing or delaying the surgery of hysterectomy in a fertile patient. In addition, the six-month follow up after the beginning of the oral assumption, corroborated the initial beneficial outcomes. The negativity of the HPV DNA test and the improved colposcopy examination, along with the objective gynecological exam, open the possibility of considering such results not casual but relative to the treatment.Overall, in light of these results and considering (i) the antiproliferative and proapoptotic activities of EGCG, (ii) the preventive effect of vitamin B12 and (iii) folic acid against viral integration, and (iv) the re-epithelizing property of HA, the combination of these molecules provides a promising tool in the management of HPV infection.