HPV vaccination is expected to be eventually included in the nationwide immunization program if the demonstration project is successful. However, introduction and implementation of such a vaccination program raises various ethical concerns. This review paper illustrates a step by step assessment of the ethical concerns surrounding the HPV vaccination implementation in Bangladesh considering specific elements in administering and conducting the program as well as the intended results. Policy-makers, vaccine implementers, vaccine recipients, and an ethics specialist in Bangladesh were interviewed. Electronic database and websites have also been reviewed for relevant published literature and government statementsThis program imparted inadequate knowledge about HPV and cervical cancer to the recipients and participants. There was lack of autonomous and informed choice of the girls and their parents about taking the vaccine. The program did not have any follow-up plan for the adverse effects in the long run. The impact of a female-only strategy in the larger societal context was overlooked. There was lack of awareness among the implementers about safeguarding the ethical issues pertaining to HPV vaccination.Adolescent health education imparted in the scope of the vaccination program should contain adequate information about HPV, its mode of transmission, risk factors along with the importance of secondary prevention despite primary prevention. Adolescent boys should be given HPV related health education as well. The right of making informed choice should be appreciated and respected. More ethical discussion and debate should be done among the public health professionals of Bangladesh in order to increase awareness about ethical issues related to human health. Institutionalized vaccination programs have become part of routine health care services for the Bangladeshi masses. The success of polio and smallpox vaccination programs has been a boon for the population of this country and has greatly increased trust in the system to protect the population from debilitating or deadly diseases. The trust in vaccination as an almost guaranteed protection from the disease for which the vaccine is administered is ironclad. Against this backdrop, in 2016, the human papillomavirus (HPV) vaccine was introduced in Bangladesh through the implementation of a demonstration project in Gazipur district, which is located close to the capital city of Dhaka. This demonstration project is governed by the Ministry of Health and Family Welfare of Bangladesh through the Expanded Program on Immunization (EPI), with financial support from the Global Alliance for Vaccines and Immunization . Eventually HPV vaccination is expected to be included in the nationwide immunization program if the demonstration project is deemed successful.HPV infection is now a global concern. According to the HPV Information Centre, more than a half-million cancers—including cervical, vulvar, anal, penile, and oropharyngeal cancer—and over 250,000 deaths are attributed annually to HPV infection . GloballHPV is a sexually transmitted viral infection (STI) and is associated with various social stigmas and misconceptions , 8. AlsoThe government of Bangladesh has initiated a two-year demonstration program of HPV vaccination in partnership with Gavi, which provides funding for the vaccine, and the United Nations Children’s Fund (UNICEF), which oversees the procurement of the vaccine. Cervarix, the bivalent preparation of the HPV vaccine, is administered in two doses, six months apart, based on the recommendation of the World Health Organization (WHO) . The proMost HPV vaccine recipients, through the health education they received prior to being vaccinated, were made aware about the vaccine and its importance in preventing cervical cancer. However, information about HPV infection, its route of transmission, and the cofactors related to its spread, along with other cervical cancer prevention strategies, were left out of this health education.HPV is the aetiological cause of cervical cancer. HPV 16 and 18 serotypes, especially, are responsible for about 70% of cervical cancer worldwide . HoweverFirst, the fact that sexual contact is the main route of transmission of the vaccine-preventable HPV serotypes is seemingly not openly disclosed to the vaccine recipients, their parents/guardians, or teachers. Not revealing the nature of transmission of the virus against which the girls are being vaccinated raises ethical concerns. There may be scenarios where incomplete information is provided to avoid sociocultural controversies for the greater good. Regarding vaccination, which essentially has a utilitarian benefit, it could be argued that full information might hinder vaccine uptake. Experiences from several low- and middle-income countries (LMICs) have suggested that highlighting the anti-cancer role of the HPV vaccine to the public without mentioning its true role in preventing sexually-transmitted HPV infection is associated with high vaccination coverage . AchieviThe alternative to providing framed partial information is providing full information to enable well-informed choices. Some studies suggest there is no significant effect of information framing on HPV vaccine acceptance . HoweverSecond, not providing information about other cervical cancer prevention strategies, particularly cervical screening, poses ethical questions. Vaccination is not a substitute for screening. The vaccine only prevents 70% of cervical cancers, so surveillance via screening must continue . It has Third, there is an ethical dilemma regarding the actual efficacy of the vaccine . CommuniObtaining consent from adolescents or their parents/guardians before giving vaccination is one of the most challenging tasks in the HPV vaccination program. Adolescence is a distinctive life stage, and hence any health intervention at this age requires special attention. WHO stresses the particular need to obtain informed consent when administering vaccination for this age group . Many HPIn Bangladesh, there is no written guideline or procedure on ensuring the obtaining of parental/guardian consent or the assent of the adolescents for vaccination. In usual practice, it is expected that adolescents are accompanied by their parents/guardians when undergoing any health intervention at health service centres. However, there is no recommendation regarding non-accompanied adolescents receiving vaccines. This raises ethical questions around whether adolescent vaccine recipients can provide their own consent and how vaccination can be ensured while balancing parental/guardian consent with the assent of recipients. At school, adolescent girls are vaccinated without the presence of their parents/guardians based on implied parental/guardian consent. The assent of the girls, which is considered important for vaccination according to WHO , is not In the HPV vaccination demonstration program of Bangladesh, the social mobilization carried out in the vaccination areas has been very poor, both in quality and quantity. Many parents/guardians are unaware of the vaccine their daughters/wards have received at school. One positive feature of the program is that the girls are taught about the HPV vaccine and cervical cancer through a health education session before receiving the vaccine. Although partial, this education helps girls to know something about the vaccine they are going to receive. However, given the regimented way in which the vaccination is administered in schools, all selected girls feel obliged to accept it, either due to peer pressure or to please school teachers. They have no opportunity to take an informed and independent decision regarding the matter. This creates an ethical dilemma, given that no choice, in its truest sense, is available to the recipients or their parents/guardians when deciding whether to take the vaccine. For adolescent girls not attending school, coming to the EPI centres for vaccination is considered as giving implied consent. In reality, however, they are lured into taking the vaccine by hearing it referred to as the ‘cervical cancer vaccine’ during community mobilization, and complete information about the vaccine and its limitations is not disclosed.Immunization of all eligible girls to create herd immunity may be rationalized by the theory of utilitarianism, which considers an action’s rightness or wrongness based on its consequences . HoweverFurthermore, an HPV-vaccination card is maintained for every vaccine recipient, but these are not given to the girls; rather, they are deposited with the relevant school authorities until the two doses are completed. This raises another dimension of ethical concern. Although the reason provided, at the implementer level, for not giving the vaccination cards to the girls is to protect the cards from being lost, it is still a matter of concern that there is no written documentation for the parents/guardians to know about the vaccine administered to their daughters/wards at school. This puts parents/guardians in a blind situation, making them solely dependent on the school teachers’ judgment about the well-being of their daughters/wards and depriving them of being able to make their own decisions.Studies suggest that effective and thorough social mobilization with accurate and complete knowledge about HPV ultimately results in successful vaccination programs in the long run . Also, vInterviews with policy-makers revealed that the HPV vaccine and all the EPI vaccines are used in Bangladesh with WHO approval but without any country-specific trial or licensure by any local drug administration authority. Hence post-marketing vaccine surveillance is poor. This is a concern because, whereas most EPI vaccines’ adverse effects are immediately identified, there may long-term adverse effects associated with cases of HPV vaccinations. Consequently, not performing due diligence in the context of Bangladesh raises ethical questions, given the risks associated with HPV vaccinations, which for the most part is still unknown.Any adverse effect following immunization (AEFI) with HPV vaccine is recorded using an existing EPI form. The EPI form is adequate for EPI listed vaccines, as it captures immediate and well-established adverse effects following immunization. On the other hand, HPV vaccinations may not necessarily result in short-term adverse effects, but in long-term ones yet to be evaluated, which cannot be recorded on the current EPI form. The pharmaceutical manufacturers have reported “local pain” as the most common side-effect of the HPV vaccine , 31. HowThere is still a lack of data on how long the vaccine will give immunity or on the need for any booster dose , 26. As The HPV vaccination program targets only the young female population, whereas the male population, who are in effect a major source of transmission, are left out of the program. Globally the HPV vaccination program targets women to minimize the overall cost of the program , althougThe gender split of the Bangladeshi population is approximately 50–50 . Even thThis female-only approach may also increase sexual irresponsibility in the male partner, especially given that, in Bangladesh, females generally have less of a say regarding choices in sex. Women’s significant progress in matters such as education and financial independence has already produced examples, in Bangladesh, of insecurity on the part of their male partners that has, at times, led to deadly violence. The asymmetrical way in which vaccination is being conducted globally, targeting only females for purported cost–efficiency reasons, will gradually build a misconception that HPV infection is a female-only matter, which may have negative consequences on the larger social psyche that already marginalizes women.Vaccination is a core delivery of public health services. In Bangladesh, the immunization program still holds the public’s trust and is regarded as the most successful public health program in the country. Hence, to preserve ethical standards, adding any new vaccine to the existing program requires thorough investigation of its compatibility, necessity, and fit-for-purpose. To alleviate the concerns raised in this paper, we offer the following recommendations. Adolescent health education imparted in the scope of vaccination should contain adequate information about HPV infection and cervical cancer, as well as other measures to prevent cervical cancer, such as safe personal practices, including use of the barrier method, delayed sexual debut, smoking cessation, regular cervical screening, etc. Any misleading or partial information should be identified and prohibited. The procedure for obtaining parental/guardian consent while ensuring adolescents’ autonomy and assent to vaccination should be established and well-circulated among health service providers. The vaccinated cohort should be kept under long-term follow-up. Adolescent boys should also be given health education regarding HPV-related infection. More ethical discussion and debate are needed among public health professionals in Bangladesh to increase awareness about ethical issues related to human health.


Infection with human papillomaviruses is associated with a series of benign and malignant hyperproliferative diseases that impose a heavy burden on human populations. A subgroup of mucosal human papillomavirus types are associated with the majority of cervical cancers and a relevant fraction of vulvar, vaginal, anal, penile and head and neck carcinomas. Human papillomaviruses mediate cell transformation by the expression of two pleiotropic oncoproteins that alter major cellular regulatory pathways. However, these viruses are not complete carcinogens, and further alterations within the infected cells and in their microenvironment are necessary for tumor establishment and progression. Alterations in components of the extracellular matrix for instance, matrix metalloproteinases and some of their regulators such as tissue inhibitors of metalloproteinases, have been consistently reported in human papillomaviruses-associated diseases. Matrix metalloproteinases function by remodeling the extracellular matrix and alterations in their expression levels and/or activity are associated with pathological processes and clinical variables including local tumor invasion, metastasis, tumor relapse and overall patient prognosis and survival. In this review we present a summarized discussion on the current data concerning the impact of human papillomavirus infection on the activity and expression of extracellular matrix components. We further comment on the possibility of targeting extracellular matrix molecules in experimental treatment protocols. Human papillomaviruses (HPVs) are small (∼50 nm), nonenveloped viruses with marked tropism for the stratified epithelia of mucosa and the skin. The viral cycle is strictly dependent on the epithelial differentiation program since the availability of cellular factors expressed in different strata of the epithelium play a role on viral gene expression and genome amplification (see below). The genome of these viruses is composed of a circular molecule of double-stranded DNA of approximately 8 kbp packaged with cellular histones. The viral DNA can be divided into three functional regions. The long control region (LCR) or upstream-regulatory region (URR) is a noncoding stretch of DNA that contains the viral origin of replication and sites for binding viral and cellular factors. The combination of viral and cellular proteins that bind to this region regulates both viral genome replication and gene transcription. In addition, HPVs exhibit two coding regions. The early region (E) codifies the early genes that regulate viral DNA replication and gene expression E1, E2, E7), viral persistence , immune evasion , inhibition of apoptosis (E6 and E7) and virion maturation and release (E4). The late region (L) contains the genes that codify the major (L1) and minor (L2) capsid proteins , E2, E7,HPV infections are ubiquitous and are associated with a series of hyperproliferative pathologies of epithelia and mucosa as well as most cases of cervical cancer and genital warts in vivoHigh-risk HPV types express two oncogenes, E6 and E7, that collaborate to immortalize normal human keratinocytes in cell culture systems and are essential to maintain the transformed phenotype Despite the high impact of the expression of HPV proteins on cellular homeostasis, these viruses are incomplete carcinogens. Therefore, further alterations in the cell as well as in its microenvironment are necessary for tumor establishment and progression. This process includes dysregulation of the extracellular matrix (ECM). In some cases, alterations in the levels and activity of defined ECM components have been experimentally associated with the expression of specific HPV proteins, suggesting the direct involvement of the virus in the deregulation of these factors. Other studies, mainly those conducted using clinical samples, have identified alterations in the levels of ECM molecules during the progression of HPV-related diseases. However, in most situations the direct involvement of viral proteins on these alterations has not been addressed. In this review we discuss the current state of knowledge concerning the impact of HPV infection on the expression and function of components of the ECM.in situ [CIS]) represents a heterogeneous disease, and it can be considered a precursor lesion to a more advanced, invasive cervical cancer The natural history of cervical cancer development arises from precursor lesions, called cervical intraepithelial neoplasias (CINs). CIN 1 and a subgroup of CIN 2 lesions are classified as productive lesions, in which the viral cycle is complete. On the other hand, a subgroup of CIN 2 and all CIN 3 lesions are cervical cancer precursors. The development of these lesions is mainly caused by persistent infection with oncogenic HPV types. Productive lesions present low to moderate histological alterations and spontaneously regress in the period of 1 to 2 years. In the event of high-risk HPV persistent infections, high-grade precancerous lesions  may develop within 3 to 5 years. Morphologically, CIN 3  expression and activity. Additionally, alterations in different molecules of the ECM can be observed in HPV-associated CINs. The main alterations in ECM components in CIN are summarized in MMP-2 and its regulator tissue inhibitor of metalloproteinases type 2 (TIMP-2) are important determinants in the behavior of invasive tumors and in their metastatic potential. A previous study conducted with clinical samples from patients with CIN or invasive squamous cell cervical carcinoma (CC) showed that MMP-2 expression levels presented a positive correlation with CIN grade, and was further increased after progression to invasive cancer. However, TIMP-2 expression presented no alterations when normal tissues were compared to CIN 3 tissues In an early study, Davidson et al. Recently, Valdivia et al. Alterations in other ECM components have also been explored in the cervical tissue transformation context. Branca et al. Moreover, analysis of the expression of sindecan-1 and claudins (CLDNs) in CIN 1, 2, and 3 and CIS lesions demonstrated that sindecan-1 and CLDNs 1, 4 and 7 expression levels are similar among CIN 2 and 3, CIS and normal epithelium. However, CLDN3 was not detectable in the squamous epithelium HPV infection has also been linked to a relevant percentage of lesions in other epithelia of the anogenital tract including the vulva, vagina and anus. Vulvar, vaginal and anal cancer precursor lesions are also denominated as vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VAIN) and anal intraepithelial neoplasia (AIN), respectively Finally, a retrospective study analyzed the expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 by IHC in VIN 1, 2 and 3 samples and invasive vulvar carcinoma. The results from this study suggested that overexpression of MMP-2, MMP-9 and TIMP-2 proteins may be related with the progression of VIN to invasive carcinoma The carcinogenic process naturally culminates in the overcoming of multiple ECM regulatory mechanisms in order to promote tissue invasion of established tumors Disruption of ECM remodeling regulation through imbalanced proteolysis has a major role in loss-of-tissue homeostasis and pathological processes such as cancer. In cancer, this event can impact (I) tissue tension and (II) the release of chemotactic fragments of ECM components that influence the local microenvironment, as it favors (III) cell migration and recruitment of stromal, endothelial and immune cells to the tumor vicinity. The heterotypical association of cancer cells and other elements observed in the tumor microenvironment, such as inflammatory infiltrates, endothelial cells and tumor-associated fibroblasts, must also be explored in order to fully understand the alterations in ECM remodeling resulting from this intricate crosstalk Historically, the most investigated proteases present in this crossroad are the MMPs. The activity of specific MMP types such as MMP-2, MMP-9 and membrane type 1 (MT1)-MMP is upregulated in most human cancers. These MMPs play a central role in basement membrane breakdown and cell invasion, as well as in neoangiogenesis and metastasis The crucial role of specific MMPs in the process of carcinogenesis has set an objective task for researchers in the field to explore the potential of MMPs as therapeutic targets Several authors have studied ECM alterations in both structural and remodeling molecules in invasive cervical cancer. More specifically, alterations in the expression/activity of galectins, collagens, proteoglycans, laminins, fibronectins, integrins and proteases and their regulators were observed in both cervical cancer samples and derived cell lines. Considering the current review, we aim to assemble a more complete view of the spectrum of ECM alterations in HPV-associated tumors, mainly regarding cervical cancer.HPV16 seems to be associated with higher expression/activity of specific MMPs during cervical cancer progression Several authors have reported the upregulation of specific MMPs in samples of cervical carcinoma when compared to control healthy tissue, including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14 (MT1-MMP) and MMP-15 Even though MMP proteolytic network alterations are common during the progression of cervical cancer, only upregulation of the expression/activity of MMP-2 (gelatinase A) and/or MMP-9 (gelatinase B) seems to be indicative of a poor prognosis in cervical cancer patients Branca et al. Moreover, another study analyzed both protein levels and proteolytic activity of 9 MMPs  and TIMP-1, TIMP-2, and TIMP-3 in normal cervical tissue and LSIL, HSIL and cervical cancer clinical samples using IHC assays and gelatin zymography. The authors observed an increase in the protein levels of MMP-2 and MMP-9 in 13%, 80% and 90% of LSIL, HSIL and cervical cancer samples, respectively The differential expression of specific modulators of ECM protease activity plays a central role in tumor microenvironment remodeling. These positive and negative modulators present an intricate interplay that impacts both their expression and function in HPV-associated tumors Indeed, the protein levels of a disintegrin and metalloproteinase 17 (ADAM17), amphiregulin (AREG), ECM metalloproteinase inducer (EMMPRIN), and MMP were increased in cervical carcinoma clinical samples when compared with normal adjacent cervical tissues. Moreover, strong ADAM17, AREG, and EMMPRIN protein expression was associated with several poor prognosis parameters, including the presence of lymph node metastasis and advanced tumor stage COL17A1 gene was hypomethylated in cervical cancer clinical samples Different types of collagens, including collagen types I, II, III, V, and IX may show increased deposition in the tumor microenvironment Syndecan 1, a transmembrane heparan sulfate cell surface proteoglycan that is highly expressed in the normal cervical epithelium , presents a marked downregulation in invasive cervical carcinomas when compared with CIN samples CLDNs and occludins are families of proteins associated with the establishment of tight junctions and epithelial cell polarity and intercellular permeability. Expression levels of CLDN type 1, 2, 4 and 7 proteins were upregulated in HSIL lesions and in invasive cervical tumors when compared with normal cervical tissues The expression of the 67-kd high-affinity laminin�binding protein (67LR) was increased in both CIN and cervical cancer samples when compared with normal cervical tissues in situ hybridization. The expression of high levels of versican in tumor stromal myofibroblasts was associated with (I) a lower frequency of tumor-infiltrating CD8-positive T cells, (II) increased tumor parametrial invasion and infiltration depth (III) and no change in cervical cancer survival Versican, an ECM proteoglycan, was evaluated in cervical cancer samples by IHC and In 2015, a new study presented more results on ECM changes in cervical cancer clinical samples when compared with normal cervical tissues obtained from hysterectomy The data discussed above show that alterations in the ECM composition and function are common in HPV-associated lesions and cancers. Altogether, these alterations highlight the complex molecular pathways that lead from initial infection to disease. For instance, the analysis of the impact of HPV on components of the MMP family that have been addressed by studies from different groups around the world have produced a plethora of data that could be used for disease diagnosis and for the identification of targets for therapy. The data summarized here also show that in regard to the mechanisms by which HPV modulates MMP expression and activity, there is still much to learn. Finally, ECM alterations may impact the HPV-infected tissue microenvironment, affecting the recruitment of inflammatory infiltrates, altering the fate of different cell populations present in the tumor and, ultimately, determining the disease progression and prognosis. Therefore, further studies are necessary to understand how HPV proteins affect the dynamic ECM balance in associated pathologies. This will help us to better understand the genesis of the disease and define more suitable clinical interventions. Importantly, a great majority of the ECM alterations described in this review have also been observed in HPV-independent tumors. Therefore, understanding the virus-mediated molecular events leading to ECM disturbance may prove of value for understanding the basic mechanisms of carcinogenesis and the development of more general antitumor approaches.Herbster S prepared the text describing ECM alterations in HPV-associated tumors. Paladino A prepared the text describing the general characteristics of HPV and the effect of the virus on ECM alterations present in precursor lesions and prepared the illustration in Figure 1. Freitas S assisted in the preparation of text describing the general characteristics of HPV and the effect of the virus on ECM alterations present in precursor lesions. Boccardo E prepared, revised and corrected all the text.


Overexpression of miR-203 and miR-200c suppressed cancer stemness in TNFα-treated HPV16-immortalized cells. Overall, our study suggests that chronic inflammation promotes cancer stemness in HPV-infected cells, thereby promoting HPV-associated oral carcinogenesis.High-risk human papillomaviruses (HPVs) are involved in the development of several human cancers, including oropharyngeal squamous cell carcinomas. However, many studies have demonstrated that HPV alone is not sufficient for the oncogenic transformation of normal human epithelial cells, indicating that additional cofactors are required for the oncogenic conversion of HPV-infected cells. Inasmuch as chronic inflammation is also closely associated with carcinogenesis, we investigated the effect of chronic exposure to tumor necrosis factor α (TNFα), the major proinflammatory cytokine, on oncogenesis in two immortalized oral keratinocyte cell lines, namely, HPV16-immortalized and human telomerase reverse transcriptase (hTERT)-immortalized cells. TNFα treatment led to the acquisition of malignant growth properties in HPV16-immortalized cells, such as (1) calcium resistance, (2) anchorage independence, and (3) increased cell proliferation in vivo. Moreover, TNFα increased the cancer stem cell-like population and stemness phenotype in HPV16-immortalized cells. However, such transforming effects were not observed in hTERT-immortalized cells, suggesting an HPV-specific role in TNFα-promoted oncogenesis. We also generated hTERT-immortalized cells that express HPV16 E6 and E7. Chronic TNFα exposure successfully induced the malignant growth and stemness phenotype in the E6-expressing cells but not in the control and E7-expressing cells. We further demonstrated that HPV16 E6 played a key role in TNFα-induced cancer stemness  The ubiquity of HPV infections, the regression of most HPV-induced dysplasias, and the long incubation period between initial infection and development of cancer indicate that HPV infection by itself may not be sufficient for neoplastic conversion of normal epithelial cells.12High-risk human papillomaviruses (HPVs) infection is closely associated with the development of female genital epithelial cancers. Over 90% of cervical cancer biopsies contain high-risk HPV DNA, such as HPV-16 and HPV-18 DNA.13 Although the molecular and cellular mechanisms linking chronic inflammation to tumourigenesis have not been fully understood, tumor necrosis factor α, TNFα, a major mediator of inflammation, plays a crucial role in inflammation-associated cancer development. Disruption of the TNFα signaling pathway could significantly inhibit chemical-induced carcinogenesis in the skin.15 Many studies have suggested that TNFα promotes inflammation-associated tumourigenesis by activating nuclear factor-κB signaling,17 which inhibits the death of precancerous or transformed cells during the development of inflammation-associated cancers.20 In addition, TNFα has been shown to be a potential mutagen that causes DNA damage through the induction of reactive oxygen species.21There is increasing evidence of chronic inflammation-associated tumourigenesis.23 However, the precise mechanism by which chronic inflammation might contribute to HPV-associated tumourigenesis is unclear. TNFα has been shown to promote HPV gene expression in HPV-immortalized keratinocytes. This suggests that TNFα contributes to HPV persistence and subsequent neoplastic progression by increasing viral gene expression.25 TNFα increases the proliferation of HPV-immortalized and HPV-infected cervical cancer cell lines by upregulating amphiregulin, an epidermal growth factor receptor ligand.26 These data suggest that TNFα and HPV may act in concert to induce neoplastic cell growth within the squamous epithelium infected with HPV, leading to the development of secondary molecular and genetic events, which eventually result in malignant conversion.21 However, most of the molecular events underlying the co-carcinogenic role of TNFα in HPV-associated carcinogenesis remain elusive and warrant further study.28Chronic inflammation has been identified as a cofactor for HPV-associated cervical carcinogenesis.29 CSCs share many molecular similarities to embryonic and normal adult stem cells. Many molecular determinants of normal stem cells, such as self-renewal ability and multi-lineage differentiation capacity, are retained in CSCs.29 CSCs have been isolated from various primary tumors and established cancer cell lines via cell surface markers, and they typically have the following properties: high tumourigenicity upon injection in immunodeficient mice, the ability to grow as tumor spheres in undifferentiating medium and resistance to cancer therapeutic agents.29 Therefore, CSCs drive the perpetuity of the disease while producing cellular heterogeneity of cancer tissues and are becoming new targets of anticancer therapies.29 The phenotypes of CSCs have been reported to be maintained by several endogenous signaling pathways, including Notch, Hedgehog, and Wnt.30 In addition to the endogenous pathways, CSCs could be enriched by exogenous carcinogenic factors. For instance, nicotine has been shown to increase the aldehyde dehydrogenase (ALDH)-positive CSC population in human breast cancer via a Notch-dependent pathway.31 Furthermore, recent studies have demonstrated that the proinflammatory cytokines TGFβ and TNFα generate CSCs in human cancer.34 In the present study, we investigated the effect of chronic inflammation on HPV-associated oral carcinogenesis by treating HPV-immortalized and non-tumourigenic human oral keratinocytes with TNFα for extended periods and studied the phenotypic and molecular biological changes.Recent studies have uncovered and validated the pathophysiologic role of self-renewing cells, namely, cancer stem cells , in long-term maintenance of cancers.Chronic TNFα exposure induces calcium resistance in HPV-immortalized cells but not in non-HPV-immortalized cells.2+ (0.15 mmol·L−1) keratinocyte growth medium (KGM) but not in high-Ca2+ (1.5 mmol·L−1) DMEM containing 10% serum. Proliferation capacity at the physiological calcium level (1.5 mmol·L−1), also known as calcium resistance, is a transformed phenotype of keratinocytes.35 To investigate the effect of inflammation on HPV-associated carcinogenesis, we first examined the effect of short-term proinflammatory cytokine TNFα exposure (2–10 days) on the proliferation of HPV-positive HOK-16B and HPV-negative OKF6/tert cells in low-Ca2+ medium , KRT10, and involucrin (INV), in HOK-16B but not in 16B/TNF cells  were used in this study. Keratinocytes normally proliferate in low-Caium Fig. . The shoTNF Fig. . HoweverTNF Fig. . Moreovells Fig. . Our datlls Fig. . E6 and Chronic TNFα exposure induces malignant growth properties in HPV-immortalized cells but not in non-HPV-immortalized cells.36 16B/TNF cells formed an irregular epithelial sheath showing increased epithelial thickness and invasion into the subepithelial layer  were consistently increased in 16B/TNF compared to control HOK-16B cells , high motility, and resistance to cancer therapeutic agents.29 There was a significant increase in the CD44high/CD24low CSC population in 16B/TNF compared to HOK-16B cells . The assay revealed that stemness-related miRNAs were frequently dysregulated in 16B/TNF cells (data not shown). As validated by qPCR . The expression of NICD was significantly elevated in 16B/TNF compared to HOK-16B cells  anchorage-independent growth ability, (3) increased cell proliferation in vivo, and (4) self-renewal capacity. Moreover, TNFα increased the CSC population and properties in HPV-immortalized keratinocytes. Importantly, such TNFα effects were not observed in non-HPV-immortalized keratinocytes. Subsequently, our study revealed that HPV16 E6 played a key role in the TNFα-induced CSC phenotype via suppression of stemness-inhibiting miR-203 and miR-200c. Restoration of miR-200c and miR-203 reversed the TNFα-induced CSC properties in HPV-immortalized cells. Our findings provide evidence of the role of chronic inflammation in HPV-associated carcinogenesis and a mechanism through which chronic inflammation promotes HPV-associated carcinogenesis.In this study, we propose that chronic inflammation promotes HPV-associated carcinogenesis by increasing cancer stemness. Chronic exposure of TNFα led to the acquisition of a malignant growth phenotype in HPV-immortalized keratinocytes, namely, (1) proliferation capacity at the physiological level of Ca9 Our prior work suggests that HPV infection plays an important role in the early stage of oral carcinogenesis. However, the possible role of chronic inflammation in the malignant progression of HPV-immortalized cells, i.e., HOK-16B cells, has not been well documented.43 Many studies have reported the tumor-promoting effects of TNFα, a major mediator of inflammation. TNFα increased the malignant behavior of tumor cell lines.45 TNFα knock-out mice are resistant to chemically induced skin carcinogenesis.46 TNFα increased chromosomal instability by virtue of its ability to induce ROS.47 Similarly, our study clearly demonstrated that chronic exposure to TNFα induced a malignant growth phenotype in HPV-immortalized keratinocytes but not in non-HPV-immortalized keratinocytes, i.e., OKF6/tert cells. Moreover, chronic TNFα exposure induced greater chromosomal instability in HPV-immortalized keratinocytes than in non-HPV-immortalized keratinocytes (unpublished data). Previously, we reported that HPV-immortalized keratinocytes displayed impaired DNA repair activities.49 More specifically, the E6 and E7 oncoproteins of HPV16 interfered with the DNA repair process.51 Thus, we speculate that chronic TNFα may potentiate its mutagenic effect in HPV-infected cells with defective DNA repair capacity. Collectively, our data suggest that the chronic inflammatory microenvironment is an important additional factor for HPV-associated oral carcinogenesis.Previously, we reported successful immortalization of normal human oral keratinocytes by transfection with a high-risk HPV whole genome , but this strategy failed to yield neoplastic conversion of normal cells.24 reported that TNFα increased the expression of HPV16 E6/E7 in HPV16-immortalized cervical keratinocytes. We demonstrated that acute and chronic TNFα exposure had no effect on viral gene expression in HPV16-immortalized oral keratinocytes, indicating different effects of TNFα on HPV expression in a different cellular context. Our study suggests that the acquired calcium resistance, malignant growth and stemness phenotype in 16B/TNF cells is independent of the overexpression of E6/E7 induced by TNFα in HPV16-immortalized oral keratinocytes.Gaiotti et al.52 CSCs are considered the seed of cancer for their crucial roles in the malignant behavior of cancer cells, i.e., metastasis, drug resistance, and tumourigenicity. We demonstrated that chronic TNFα exposure endowed HPV-immortalized cells with self-renewal capacity, a key feature of CSCs, and concomitantly increased the expression of self-renewal transcription factors. TNFα also led to a robust increase in the CD44high/CD24low CSC population. CD44high/CD24low cells have been identified as CSCs in different types of cancer, including OSCC.53 CD44high/CD24low cancer cells displayed higher self-renewal capacity and important CSC properties, such as migration, chemo-radioresistance, and tumourigenic potential compared to other subpopulations.53 TNFα also increases the migration ability and chemo-radioresistance of HPV-immortalized cells. Furthermore, we documented that chronic TNFα treatment results in activation of the Notch1 signaling pathway, a critical CSC maintenance pathway.42 Therefore, the increased CSC population and properties induced by TNFα in HPV-immortalized cells is also an important observation supporting the role of chronic inflammation in HPV-associated carcinogenesis and cancer progression. This observation suggests that chronic inflammation further promotes the malignant progression of HPV-immortalized oral keratinocytes by increasing cancer stemness. It is interesting to note that epithelial-mesenchymal transition (EMT) is considered the critical biological change for cytokine-induced CSCs.32 We observed increased EMT phenotypes, such as downregulation of genes involved in cell junctions (data not shown) and enhanced migration ability in TNFα-treated HPV-immortalized keratinocytes.Recent studies demonstrated that proinflammatory cytokines, including TNFα, increased the CSC population and properties in human cancer, suggesting a possible link between CSCs and inflammation.38 Many studies have identified various miRNAs affecting the CSC phenotype. For instance, miR-203 and the miR-200 family  were downregulated in CSCs isolated from various cancer types, and their regulation could alter CSC markers and properties, indicating that they are CSC-inhibiting miRNAs.54 miR-203 suppresses CSCs by targeting Bmi-1.39 miR-200 members inhibit CSC self-renewal and properties by suppressing Notch signaling.55 Activation of Notch signaling has been implicated in CSCs in mammalian cancer.42 We found that miR-203 and miR-200 members were substantially decreased by chronic TNFα treatment in HPV-immortalized cells. Further analysis revealed that miR-203 and miR-200c were suppressed by TNFα in HPV16 E6-expressing but not in HPV16 E7-expressing OKF6/tert cells, indicating the requirement of E6 for TNFα-induced suppression. In our study, overexpression (restoration) of miR-203 and 200c resulted in suppression of self-renewal capacity, migration and chemoresistance in TNFα-treated HPV-immortalized cells, supporting their functional role in the TNFα-induced CSC phenotype. Furthermore, miR-203 and 200c inhibited Notch signaling in TNFα-treated HPV-immortalized cells. Therefore, we hypothesize that the exposure of HPV-infected cells to chronic inflammation generates a CSC population and allows CSCs to subvert immune-mediated elimination,56 thereby permitting long-term survival, which would promote the further oncogenic transformation of the cells.There is increasing evidence of the importance of miRNAs in the genesis and maintenance of CSCs.57 Our study showed that chronic TNFα exposure activated the Notch pathway in HOK-16B cells. Together with the fact that E6 and E7 expression were not altered in 16B/TNF cells, our findings suggest that activation of the Notch pathway in 16B/TNF cells is independent of the E6/p53 axis. Moreover, we showed that ectopic expression of miR-203 and miR-200c suppressed the Notch pathway in 16B/TNF cells. Therefore, our findings indicate a novel activation mode of the Notch pathway via the TNFα/miR-203/miR-200c axis in HPV-immortalized oral keratinocytes.It is interesting to note that E6 is known to inhibit the Notch pathway by downregulating Notch expression via inhibition of p53.Our findings also have strong implications for whether an HPV-infected keratinocyte promotes amplification of the HPV genome and propagation of the virus or blocks propagation of the virus, stabilizes the HPV genome and leads to cancer. This decision for the HPV-infected cell to yield virus or form cancer has not been adequately addressed or understood in the field. The relevant observations are the induced CSC properties and inability to differentiate when cells with the HPV genome are treated with TNFα. Keratinocytes must differentiate in order to amplify the HPV genome and ultimately yield viral particles. Therefore, it is possible that keratinocytes infected with HPV in the host will be shunted towards cancer formation instead of viral production if the cells persist in a strong inflammatory state.In conclusion, this study provides novel information on the role of chronic inflammation in HPV-associated oral carcinogenesis. Our findings provide evidence for a novel synergism between chronic inflammation and HPV infection and a plausible mechanism underlying the cooperative effect of both critical cancer factors in carcinogenesis.58 The HOK-16B cell line was established by transfecting normal human oral keratinocytes (NHOK) with the HPV-16 whole genome.8 The OKF6/tert cell line was established by transducing NHOK with hTERT.58 Both cell lines were treated with 5 ng·mL−1 TNFα (Sigma-Aldrich) for extended periods. SCC4, a human tongue squamous cell carcinoma cell line, was cultured in DMEM/F12 medium (Invitrogen), as described in our publication.59Two non-tumourigenic immortalized human oral keratinocyte cell lines (HOK-16B and OKF6/tert2) were cultured in Keratinocyte Growth Medium (KGM) (Lonza), as described previously.4 cells were plated in culture medium containing 0.3% agarose over a base layer of serum-free medium containing 0.5% agarose. The assay was performed as described previously.59To determine colony-forming efficiency in semi-solid medium, 1 × 10nu/nu, Charles River Laboratories). The animal study was performed as described previously.59Ten million cells were subcutaneously injected into the flanks of immunocompromised mice , as described in our previous study.59For tumor sphere formation, 4 000 cells were grown in 4 mL DMEM/F12 medium with 1:50 B27 (Invitrogen), 20 ng·mL59 The primer sequences were obtained from the Universal Probe Library (Roche), and the sequences are available upon request.cDNA was synthesized by the SuperScript first-strand synthesis system (Invitrogen). The cDNA pool of miRNAs was synthesized by the QuantiMir cDNA Kit (System Biosciences) according to the manufacturer′s protocol. PCR amplification was performed with SYBR Green I Master Mix (Roche) on LightCycler 480 (Roche), as described previously.Cell surface expression of CD44 was determined by flow cytometry analysis on a FACScan (Becton Dickinson). Anti-CD44 FITC (BD Pharmingen) and secondary antibody FITC goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) were used.34Cell migration was measured using transwell chambers with polycarbonate membranes (Corning) according to the method described in the manufacturer’s protocol and in our previous publication.34 For the radioresistance assay, the cells were exposed to ionizing radiation (IR) at varying doses using the Mark I-30 Cesium-137 irradiator  with a delivery rate of 4.86 Gy ng·min−1 as described in our previous study.34Cisplatin and methotrexate were purchased from Sigma-Aldrich. Chemoresistance of cells was determined by measuring cell viability using the tetrazolium salt (MTT) cell proliferation assay kit (ATCC) as described elsewhere.51 Retroviruses expressing the empty vector or a viral gene were harvested from the transfected PA317 cells. Forty to 60 percent confluent OKF6/tert cells were infected with the retroviruses. After the infection, the cells were cultured in medium containing 100 μg·mL−1 G418 (Invitrogen). Then, G418-resistant clones were collected and used for the experiments.Retroviral vectors capable of expressing HPV16 E6 or E7 were constructed from pLXSN plasmids as described in our previous publication.59 We used the following primary antibodies for this study: anti-p53 (Oncogene Science), anti-NICD , anti-actin (Santa Cruz Biotech) and anti-GAPDH (Santa Cruz Biotech).Western blotting was performed as described previously.Cells were transfected with pre-miR negative control , pre-miR-200c (Ambion), or pre-miR-203 (Ambion) at a final concentration of 50 pmol using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.


Self-sampling to test for high risk human papilloma virus (HPV) is becoming an increasingly important component of cervical cancer screening. The aim of this observational study is to examine how women treated for high-grade cervical intraepithelial neoplasia (CIN) view HPV self-sampling.Invited to participate in the present study were patients who had undergone treatment of high-grade CIN (grade 2 or higher) and were followed-up at 6-months at the Karolinska University Hospital, Stockholm. The participants were instructed as to how to perform HPV self-sampling. Thereafter, the participants completed a questionnaire about HPV self-sampling and other cervical cancer screening methods, as well as about self-perceived risk of cervical cancer without regular gynecologic follow-up and about specific knowledge regarding HPV, CIN and cervical cancer.Altogether 479 women enrolled in this study. The participation rate was 96.6%. Nearly 75% of the participants stated they would consider performing the HPV self-sampling prior to their next gynecologic follow-up. Confidence in HPV self-sampling was a significant independent predictor of willingness to perform HPV self-sampling. However, confidence in HPV self-sampling was significantly lower than confidence in Papanicolaou smears and in HPV testing with samples collected by health professionals. Higher specific knowledge about HPV, CIN and cervical cancer was also a significant independent predictor of willingness to perform HPV self-sampling, as was having travelled longer distance to attend gynecologic follow-up. Participants with lower income and without completed university education expressed significantly higher confidence in HPV self-sampling and lower confidence in Papanicolaou smears than the other women.To the best of our knowledge, this is the first study to examine the views of women treated for high-grade CIN vis-à-vis HPV self-sampling. The latter is an acceptable option for the vast majority of this cohort of women. Cervical cancer is the fourth most common cause of cancer mortality among women worldwide . In 2012th lowest in the EU [Screening programs and subsequent treatment of cervical dysplasia have successfully reduced cervical cancer mortality –11. Sincn the EU , 14. Desn the EU , 16.Persistent infection with HPV has been identified as the key etiologic factor for cervical cancer. This finding is having a major impact upon cervical cancer screening and prevention strategies. Although frequently less specific than cytology-based examination, testing for HPV is reportedly more sensitive for detection of high-grade cervical intraepithelial neoplasia (CIN) , 9. OverIn addition to HPV testing on samples collected by health professionals, patients themselves can collect the samples for HPV testing. Self-sampling for HPV is one proposed approach for expanding access to cervical cancer screening. A meta-analytical comparison through mid-2013 of self-sampling versus clinician-collected samples indicated that the latter are more accurate . In partNotwithstanding the earlier described limitations, self-collection of samples for HPV testing is becoming an increasingly important and accepted option for cervical cancer screening , 25–31. For women in whom high-grade CIN has been detected and treated, these issues are of special importance. This group of women needs more intense follow-up than the general population, but evidence-based guidelines for the most appropriate screening protocols are lacking , 37–39. On the other hand, a negative HPV test plus negative cytology at six months were found to be a reliable test of cure after three-year follow-up among 330 women with cervical dysplasia treated with the loop electrical excision procedure (LEEP) . Our groAs noted, self-sampling to test for HPV has been quite extensively studied in a number of settings throughout the world. This modality could be a helpful option for women with high grade CIN. However, to the best of our knowledge, there have not been any published papers examining self-sampling to test for HPV among women diagnosed and treated for high-grade CIN. In the present study we focus upon HPV self-sampling among this cohort of women, examining this in relation to other diagnostic modalities, self-perceived risk of cervical cancer and knowledge about HPV, CIN and cervical cancer. Sub-groups within this cohort for whom this option might be particularly helpful will be examined in more depth. The wider aim of these efforts is to optimize and tailor follow-up for the needs of these women at increased risk for developing cervical cancer. Vital to these efforts is to ensure adequate safety against recurrent/residual disease.As described in Ref. , to be eA printed invitation letter was sent by postal mail to each eligible patient about 5 months after treatment. Therein, a short summary of key facts about HPV and cervical cancer were included, together with information about how to contact the Attending Gynecologist and Principal Investigator (SA) and the Research Coordinator (EÖ). The study was presented as including a self-collection of a sample for HPV testing and completion of a questionnaire. The stated aim of the study was to better prevent cervical cancer. Full assurance was given of confidentiality and freedom to withdraw from the study at any time without any adverse consequences whatsoever.Major efforts were made by the clinical research team to arrange convenient scheduling of the 6-month follow-up appointment for each patient. Thereby, all the patients who had been treated for high-grade CIN came to the follow-up examination.The Research Coordinator (EÖ) met with each patient to explain the study procedure at the 6-month follow-up visit. An informed consent form was signed by all the invited patients. The options were agreement or declining to participate in the study. The Karolinska Ethics Committee  approved the study protocol.Since the questionnaire was in Swedish, Swedish language proficiency was a requirement for participation in the questionnaire portion of the study. There were 480 patients who were eligible to participate in the questionnaire portion of the study. Altogether, 479 of these patients signed the informed consent agreeing to participate and thereby were included in this study. All but six of the patients included in this study were treated for CIN for the first time.There were also sixteen patients who were treated for high-grade CIN and attended 6-month follow-up and who would otherwise have been eligible to participate. However, these sixteen patients were not fluent in Swedish, and thus did not receive the questionnaire. Including the latter sixteen non-participating patients, the participation rate was 96.6%. These seventeen non-participants were of a mean age of 42 years (standard deviation 7.8 years).At the clinical care site, and before the gynecologic examination, each study participant was given a written description of how to use the self-sampling kit  device for collection of the vaginal fluid specimens, as well as instructions for taking urine samples (with first void urine in a urine cup). The self-collection was performed at the restroom reserved for patients at the clinical care site, with the urine sample collected first. The samples were analysed for HPV with the Abbott RealTime high-risk HPV assay . After completing self-collection, the patient gave the samples to the Research Coordinator (EÖ) for further handling, according to the study protocol. The gynecological examination including colposcopy was performed by gynecologists with special expertise in colposcopy  with cervical scrape samples collected from each woman. All samples collected were analyzed for HPV using a clinically validated real-time polymerase chain reaction (PCR)-based test for the detection of HPV DNA. Close agreement of HPV results between self-collected vaginal and urine samples in comparison to physician-collected cervical reference material was found .After the self-sampling in the restroom at the care site and before completion of the gynecological examination, each participant was given a printed questionnaire (S1). This was completed at the clinical care site, with full anonymity guaranteed, as per the consent form. Socio-demographic queries were posed in the first part of the questionnaire. Inquiries about incurred expenses, travel time and arrangements needed to attend the gynecologic examination were posed in part 2. Part 3 surveyed knowledge about CIN, HPV, and attendant risk of cervical cancer and other malignancies. These results were analysed in detail in Ref. . These kHuman papilloma virus (HPV) is sexually transmitted,HPV can be asymptomatic,HPV can persist leading to cell changes in the cervix,Cell changes in the cervix over a long period of time can lead to cervical cancer,Vaccination can protect against cell changes in the cervix and cervical cancer,Gynecologic screening is important even if vaccinated against HPV.Included in part 4 was a rating scale from 10 to 1 , on how the participant views her own risk of developing cervical cancer if she does not receive regular gynecologic follow-up.The final portion of the questionnaire concerned the HPV self-sampling. The patient was asked whether the self-sampling was easy for her to carry out, and if not, to state the reason(s) why not, as an open-ended question, whether the self-sampling instructions were clear, and if not, what was missing, as an open-ended question. The participant was then asked to rate on a scale from 10 to 1, (10 highest and 1 lowest), her confidence that cervical cell changes would be found, such that she would be protected from developing cervical cancer. The same query and rating scale were then presented for HPV tests from self-collected samples, and for Pap smear performed by health professionals. Thereafter, the participant was asked whether she could see herself performing HPV self-sampling at home before coming to the next gynecologic follow-up. Open queries ended the questionnaire: reasons for or against carrying out the self-sampling.Univariate analysis was thoroughly performed on available data from the study (S2). (Note questions F101 (age in years) and F102  are not included in the data set to protect the patients’ confidentiality). Therein, the distribution of all continuous and semi-continuous variables was evaluated by inspection, as well as by examining skewness and kurtosis. When the continuous or semi-continuous variables had skewness as well as kurtosis less than one, and appeared to be normally distributed, parametric bivariate analysis was performed. If not, the bivariate analysis was done non-parametrically. Yates chi-squared analysis was employed for bivariate analysis of dichotomous variables.The open-ended queries concerning the use of the HPV-self sampling were subjected to content analysis by two independent observers (SA and EÖ). These two observers came to consensus as to the items that should be included.Using binomial logistic regression, we computed odds ratios (OR) and 95% confidence intervals (CI) for unadjusted and adjusted models, with the outcome variable being: Could see oneself collecting a sample for HPV testing before the next gynecologic follow-up. Statistica 64 software was used to perform the statistical analysis.As reported in Ref. , more thThe univariate data concerning the HPV self-collected samples, other means of follow-up, as well as specific knowledge and risk assessment show that nearly three fourths of the women could see themselves performing the HPV self-sampling at home prior to their next gynecologic examination . The mosOver 80% of the participants gave high ratings (≥ 7) regarding their confidence in the HPV test collected and performed by health professionals. However, only 54.3% rated the self-collection of samples at that high level. Nearly 75% expressed the high confidence (≥ 7) in the Pap smear.As also reported in Ref. , about 7Just over 15% of the women considered that without gynecologic follow-up their risk of developing cervical cancer was maximally high (score of 10). Nearly half considered their risk to be high but not maximally (7 to 9), whereas almost 30% viewed their risk as moderate to low.The total distance travelled had a very wide range, up to 254 kilometers . The totThere was no significant association between readiness to do the self-sampling with age, income or educational level (2-sample “t” test) nor with any of the civil status categories  (Yates chi-squared).Confidence in HPV tests collected and performed by health professionals and confidence in the HPV test from self-collected samples were correlated . Confidence in the Pap smear and confidence in the HPV test collected and performed by health professionals were also correlated . However, there was no correlation whatsoever between confidence in the self-sampled HPV test and confidence in Pap smear . The confidence in the three tests differed significantly  chi-squared = 394, p < 0.0001), with the lowest rank being for confidence in the HPV self-collection.There was no significant association between readiness to do the self-collection and refraining from another activity or needing another person’s help to attend the exam (Yates chi-squared). Neither total costs nor total time expended were associated with readiness to do the HPV self-sampling (Mann-Whitney test). However, greater total distance traveled was associated with readiness to do the HPV self-sampling .The variable: “Could you see yourself collecting a sample for HPV testing at home before your next gynecologic follow-up” was the outcome measure in the logistic regression analysis. The dichotomization was endorsement versus non-endorsement .In the unadjusted logistic regression model, total distance travelled dichotomized at >35 kilometers, as the independent variable, showed a significant association with readiness to do the HPV self-collection . This moThe adjusted binomial logistic regression models include age, annual income and education as covariates. Adjusting for these 3 covariates, the Specific Knowledge score retained a significant OR. This multi-variate model included nineteen fewer patients, due to the missing data for age, income and education. Total distance travelled, confidence in the HPV self-collected samples, and specific knowledge score were all in that adjusted model, which included only 390 of the participants.We selected two subgroups for further analysis. The first of these is the women who rated their risk as the highest of developing cervical cancer without gynecologic follow-up. The other group includes the women with lower income and education compared to the cohort as a whole. We focus on how these two subgroups of patients view the HPV self-sampling . For refAs noted, there were 74 women who indicated that without appropriate gynecologic follow-up they considered their risk to be maximum (10 of 10) of developing cervical cancer. Their mean Specific Knowledge score was higher (5.15 ± 1.2) than for the other participants . Their confidence in HPV testing by health professionals and in the HPV self-collection was very similar to that of the rest of the participants. However, their mean confidence score for the Pap smear was higher (9.5 ± 1.1) .Altogether, 56 of the 74 women who rated their risk to be 10 (75.7%) endorsed that they could see themselves doing the HPV self-sampling, as indicated in the middle column of Altogether, sixty-two of the participants had an annual personal income below 260 000 Swedish kronor (~ 28 000 USD) and had not completed university education. This subgroup of women had lower mean Specific Knowledge  compared to the other participants. Their mean perceived risk of developing cervical cancer without gynecologic follow-up was also lower . Their mean confidence in HPV testing by health professionals was similar that of the other women. However, their mean level of confidence in the HPV self-collection was higher . On the other hand, their mean level of confidence in the Pap smears was lower .Over 75% of the women in this subgroup were ready to do the HPV self-sampling, as seen in the right-most column of The present study provides clear and consistent evidence that the HPV self-sampling is an acceptable option among the vast majority of this cohort of women who have been treated for high grade CIN. Nearly three fourths of the women stated that they would consider performing the self-collection before their next follow-up examination. An even larger percentage of the women considered the procedure easy to perform. The level of confidence which these women indicated in the self-collection, albeit significantly lower than the examinations performed entirely by health professionals (HPV test and Pap smear), was still quite high. Namely, the majority of women ranked their confidence in the HPV self-sampling to be well above the midline. Moreover, confidence in the self-collected samples was the strongest multivariate predictor of readiness to perform the HPV self-collection. Concordantly, the most common reason for not being ready to perform the self-collection was concern about its reliability. This concern is consistent with the somewhat earlier data from the literature . As the In the present study, the HPV self-sampling was introduced to the participants as a new way to help prevent cervical cancer. It is possible that this positive presentation encouraged the participants to respond in what they perceived as a socially-desirable manner, notwithstanding the guarantees of confidentiality. However, the finding that confidence in self-sampling was significantly lower than for samples collected by health professionals as well as for Pap smear, suggests that bias due to social desirability is likely to be fairly minimal.The two other significant multi-variate associations with readiness to perform the HPV self-collection are also noteworthy. First was that the women with higher specific knowledge about HPV, CIN and cervical cancer are those who are the most ready to perform the self-collection. On the other hand, we can identify a group of women with high-grade CIN for whom coming to the clinic exam is likely to be more difficult. Namely, it was those who were obliged to travel longer distances. These women also appear to be more amenable to performing the self-collection, according to the results of the present study. Along these lines, a study of barriers and facilitators to cervical cancer screening among women in rural Ontario, Canada  indicateOur findings that HPV self-collection was well-accepted among the subgroup of women with lower income and lower educational levels are also broadly consistent with the findings that self-collection is a viable, and even preferred option among women who are underscreened –35. As mAnother sub-group of patients upon whom we focused attention was those who assessed their own risk of developing cervical cancer without gynecologic follow up as the highest. Although direct data about anxiety were not ascertained in the present study, it can be surmised that these women are the most anxious about their risk of developing cervical cancer. Data from other studies indicate that colposcopy and subsequent HPV testing , 48 are To the best of our knowledge, there have not yet been any published studies explicitly examining the relationship between HPV self-sampling and empowerment. However, a qualitative interview study of young women undergoing HPV DNA and cytological testing indicates that empowerment through the knowledge of results and the possibility to prevent future disease was a key outcome of their participation in the screening program . MoreoveA key component of empowerment which motivates participation in cancer screening is that, in addition to potentially saving one’s life, control is put back into one’s own hands . ConcordA major strength of the present study is the very high participation rate. The efforts of the investigative team in personally explaining the study to all the eligible women seem to have contributed to creating an atmosphere of trust. It has been clearly demonstrated that personal contact encourages women to participate in studies of early cancer detection and prevention , 57.Although the age-adjusted incidence of cervical cancer in Sweden is reportedly similar among women born outside Sweden and those born in the country , the canIt is not known whether the high level of acceptability of self-sampling found among the participants in the present study also holds true for women with high-grade disease who are non-adherent to gynecologic follow-up. Particularly in light of the savings in time and costs, this latter group of patients could certainly benefit from the option of collecting samples at home for HPV testing. Whether they would agree to do so warrants investigation.Nevertheless, the findings of the present study have broad implications. Notably, even among women who participate in cervical cancer screening programs in Sweden, on-time screening is frequently low , 60. ComThe wider aim of this study effort was to optimize follow-up management of patients treated for high-grade CIN. Currently, in Sweden, these patients are referred for double testing (HPV and cytology examination) 6 months after treatment within the framework of the organized screening programme. The results from this study show a high level of acceptability of HPV self-sampling among women after treatment for high-grade CIN who adhere to follow-up. Insofar as further research confirms the reliability of HPV self-sampling in comparison to clinician-collected samples, recommendations could be modified. Namely, recommendations could then include the option of self-sampling for women after treatment for high-grade CIN, due to the need for lifelong surveillance of these women related to their increased risk for cervical cancer. Special attention is needed to investigate the acceptability of HPV self-sampling among women treated for high-grade cervical disease who have not adhered to follow-up recommendations.S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Attendees of sexually transmitted diseases (STD) clinics could also benefit from HPV vaccination. Healthcare providers’ recommendation is the most effective published method in motivating HPV vaccination initiation. This study was to investigate practice of recommending HPV vaccination to attendees among healthcare providers (doctors and nurses) working in public STD clinics in Hong Kong, China.Participants were medical doctors, registered nurses and enrolled nurses working in all eight public STD clinics in Hong Kong. All of them (29 doctors and 82 nurses) were approached by telephone. A self-administered questionnaire was mailed to them. A total of 98 completed questionnaires were received (28 doctors and 70 nurses). The study was conducted during January to May, 2018. Using recommendation of HPV vaccination to any attendees in the last year as the dependent variable, univariate and multiple logistic regression models were fitted.In the last 12 months, 16.3% and 36.7% of the participants had recommended HPV vaccination to any male attendees and to any female attendees of their clinics; 41.8% had recommended it to either male or female attendees. Adjusting for significant background variables (professions and years of working experience in the clinic), three constructs of the Theory of Planned Behavior were significantly associated with the dependent variable in expected directions. They were: 1) the Positive Attitude Scale , 2) the Negative Attitude Scale , and 3) the Perceived Behavioral Control Scale .STD clinics are ideal settings that allow healthcare providers to access individuals who are at high-risk of HPV infection and promote HPV vaccination. Health promotion targeting these healthcare providers is warranted to enhance their perceived importance of recommending HPV vaccination. Such promotion should modify their attitudes and perceived behavioral control related to recommending HPV vaccination to attendees. Human papillomavirus (HPV) vaccination is highly effective in preventing vaccine-type general warts and cancers among both males and females , 2. GrowBivalent, 4-valent and 9-valent HPV vaccines are available in Hong Kong where the study was conducted. These vaccines are provided by private physicians to males and females age ≥9 years. The cost to complete the 3-dose course ranges from HK$2,500 to 4,000 (US$ 321–514) . There aHealthcare providers’ recommendation is regarded as the most effective method in motivating HPV vaccination initiation –16. ThirIn this study, we surveyed doctors and nurses working in all eight public STD clinics in Hong Kong, China. Their practices of recommending HPV vaccination to the clinic attendees in the last year were investigated. Associated factors were also investigated. These factors included their background characteristics, knowledge related to HPV vaccination and perceptions related to recommending HPV vaccination based on the TPB.An anonymous cross-sectional survey was conducted among healthcare providers working in public STD clinics in Hong Kong during January to May, 2018. Participants were medical doctors, registered nurses and enrolled nurses working in public STD clinics in Hong Kong. There are 29 doctors and 82 nurses currently working in eight public STD clinics in Hong Kong.The questionnaire was piloted among three healthcare providers  working in public STD clinics. Based on the feedback, the questionnaire was modified and finalized by the authors . Data obThrough telephone, the first author approached all medical doctors and nursing officers in these clinics, briefed them about the study, and invited them to participate. She also invited the nursing officers to pass the study information and invitation to all their nursing colleagues. Oral informed consent was obtained before a self-administered questionnaire was mailed to each prospective participant. A statement was printed on the questionnaire indicating that completion of the survey implied consent to join this study. Return envelops with stamps were enclosed for participants to send back the completed questionnaires. No incentive was given to the participants. A total of 98 completed questionnaires were received (28 doctors and 70 nurses). Ethics approvals were obtained from both the Survey and Behavioral Research Ethics Committee of the Chinese University of Hong Kong and the Department of Health, Hong Kong SAR.Information collected included professions, gender, age, marital status, religion, education level and years of working experience in STD clinics.Participants were asked whether they had recommended HPV vaccination to any male and female attendees of their clinics in the last 12 months. A composite variable was created representing recommendation of HPV vaccination to any attendees  in the last 12 months. This composite variable was used the dependent variable in subsequent logistic regression analysis. Number of male and female attendees that they have recommended HPV vaccination to was also recorded.Seven items were used to assess knowledge related to HPV vaccination . A composite variable was constructed by counting the number of correct responses to knowledge items with the range from 0 to 7.Four scales were constructed to assess participants’ perceptions related to recommending HPV vaccination. They were based on the TPB.Positive attitudes toward recommending HPV vaccination to STD clinic attendees were measured by four items . The Positive Attitude Scale was formed by summing up individual item scores. Higher score on the scale indicated more positive attitude towards recommending HPV vaccination to attendees of their clinics.Negative attitudes toward recommending HPV vaccination to STD clinic attendees were measured by seven items . The Negative Attitude Scale was formed by summing up individual item scores. Higher score on the scale indicated more negative attitude towards recommending HPV vaccination to attendees of their clinics.Two items were used to measure participants’ perceived support from their significant others (their colleagues and supervisors) for recommending HPV vaccination to STD clinic attendees. The Subjective Norm Scale was formed by summing up individual item scores. Higher score on the scale indicated perceived subjective norm more supportive of giving such recommendations.Another three items were used to measure perceived behavioral control in recommending HPV vaccination to STD clinic attendees . The Perceived Behavioral Control Scale was formed by summing up individual item scores. Higher score on the scale indicated higher behavioral control in giving such recommendations.The response categories of all items ranged from 1 (strongly disagree) to 5 (strongly agree). Cronbach’s alpha of these four scales ranged from 0.57 to 0.89.Descriptive statistics of all studied variables were presented. Using recommendation of HPV vaccination to any attendees of their clinics  in the last 12 months as the dependent variable, univariate odds ratios (ORu) for the associations between background independent variables and the dependent variable were estimated. Those background variables with p<0.10 in the univariate analysis were adjusted for the subsequent multiple logistic regression analysis. Three multiple logistic regression models were constructed in this study. Each model contained three variables, they were: 1) years of working experiences in the STD clinic, 2) professions, and 3) one scale measuring perceptions based on the TPB . Adjusted odds ratios (AOR) and respective 95% confidence interval (CI) were derived from such analyses. SPSS version 24.0 was used for data analysis, with p values <0.05 taken as statistically significant.Over half of the participants were aged >40 years (53.1%), female (52.0%), married (80.6%), had obtained bachelor degree or above (83.7%), and without any religion (70.4%). Majority of them had at least 5 years’ working experience in STD clinics (56.1%), and self-reported seeing at least 100 attendees per week in their STD clinics (70.4%) . There wIn the last 12 months, 16.3% and 36.7% of the participants had recommended HPV vaccination to any male attendees and to any female attendees of their clinics; 41.8% had recommended it to either male or female attendees. Among those who had given such recommendation, 6.2% and 22.2% had recommended to over 10 male and female attendees, respectively .The prevalence of correct response to seven knowledge items related to HPV vaccination ranged from 70.4% to 99.0%. Item responses and means  of the scales measuring perceptions related to recommendation of HPV vaccination to STD clinic attendees were described in As compared to healthcare providers with <5 years working experiences, those having 5–10 years working experience were more likely to have recommended HPV vaccination to any STD clinic attendees in the last 12 months . The difference in prevalence of the dependent variable between doctors and registered nurses was of marginal statistical significant  . AdjustiEmbarrassment to talk about sex-related topics and perceived getting vaccinated for HPV might be seen as a symbol of promiscuous were common barriers for sexually active adults to take up HPV vaccination , 33. STDRelatively few healthcare providers working in the public STD clinics in Hong Kong have recommended HPV vaccination to attendees of their clinics (41.8%). The proportion of the participants who have recommended HPV vaccination to female attendees (36.7%) was lower than general physicians, obstetricians, gynecologists and pediatricians in other countries (47.0–83.8%) , 25, 28.Since the number of healthcare providers in public STD clinics is relatively small, health promotion to improve the current situation should be feasible. The findings of this study provided some insights for developing such health promotion. As compared to doctors, registered nurses had lower likelihood of recommending HPV vaccination to attendees. It is understandable as nurses in Hong Kong are less involved in decision making related to diagnosis, prescription and treatment. Since nurses have the most contact with the patients, they can play important roles in future HPV vaccination promotion. Healthcare providers with longer working experience in STD clinics had higher likelihood of recommending HPV vaccination to attendees, as compared to those having less experience. Since HPV vaccines became available in Hong Kong since 2006, participants having longer working experience might have exposed to more information disseminated by pharmaceutical companies. However for those with more than 10 years’ experience, many of them are supervisors in these clinics. They have fewer number of consultation and hence less chance of giving recommendation to attendees. More attention should be given to registered nurses and healthcare providers with less working experience in the STD clinics in future health promotion.Our participants had good knowledge about HPV vaccination for females, as nearly 95% of them knew that HPV vaccination could protect females. Such proportion was comparable to healthcare providers in Italy , and wasThe TPB is a potential useful framework to guide the design of future health promotion targeting healthcare providers in STD clinics, as three out of four constructs of TPB were significantly associated with recommendation of HPV vaccination in expected directions. It is important to strengthen positive attitudes toward recommending HPV vaccination. Although over two-third of the healthcare providers perceived that HPV vaccination was effective in preventing HPV/HPV-related diseases among sexually active people in general, fewer of them (27.6–43.9%) believed that similar efficacies applied to those with history of HPV and/or HIV infection. Since HPV vaccination research is a rapid developing area, healthcare providers should be regularly updated about the new evidences in this area. Future training for healthcare providers should be conducted. The contents of the training should focus on the fact that both sexually active men and women who have already infected with one or more HPV types could benefit from HPV vaccination.It is also important to remove some negative attitudes, as they were negatively associated with recommendation of HPV vaccination to STD clinic attendees. Over 70% of them indicated that there was lack of guideline or protocol for them to recommend HPV vaccination to attendees, and 26.5% hence perceived it was not their responsibility to do so. Given the recent development of HPV vaccines, the Department of Health should consider update current guideline to include clear instruction about HPV vaccination for sexually active people and high-risk groups. Insufficient time for recommending HPV vaccines during consultation sessions was another major barrier perceived by the participants. It is understandable as healthcare providers working in public sectors in Hong Kong are having heavy workload . DevelopPerceived behavioral control is another construct that was positively associated with recommending HPV vaccination to attendees. Availability of relevant guideline/protocol and sufficient information about the HPV vaccination may be useful to increase their perceived behavioral control.Very few of participants perceived their colleagues or supervisors would support them recommending HPV vaccination to attendees. Perceived subjective norm was not significantly associated with recommending HPV vaccination to attendees. Therefore, building up new norms supporting recommendation of HPV vaccination to attendees among healthcare providers may not be an effective approach in future health promotion. Health promotion should be evidence-based.This study had the strengths of being theory-based and was based on a representative sample. However, it also had some limitations. First of all, recommendation of HPV vaccination to attendees was self-reported, report bias might exist. Second, we did not use items/scales validated by previous studies. Scales constructed for this study demonstrated acceptable internal reliability; however, they have not been externally validated. Third, given the limited number of healthcare providers working in public STD clinics in Hong Kong, the sample size was relatively small as compared to other similar studies –26. HoweSexually active people and some high-risk groups can also benefit from HPV vaccination. STD clinics are ideal settings that allow healthcare providers to access these groups and promote HPV vaccination. Only few of healthcare providers working in public STD clinics in Hong Kong had recommended HPV vaccination to their attendees. Health promotion targeting these healthcare providers is warranted. Such promotion should modify their attitudes and perceived behavioral control related to recommending HPV vaccination to attendees.S1 Survey questionnaire(DOCX)Click here for additional data file.


Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determinants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it’s association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues harboring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetectable pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promotion/maintanence of tumorigenic phenotype. Progression to cervical cancer is a multi-step process etiologically-linked with persistent infection of high-risk human papillomaviruses (HPVs). Though HPV infection is a necessary prerequisite, but it is not sufficient for the initiation of cervical cancer . The phycis-elements in the URR , and 70 cancer tissues qualified for analysis of viral load and the physical state of HPV16 by PCR-based method and pSTAT3 expression using western blotting (n Table 2–39.Table 2). Subsequently, HPV16 positive samples were subjected to comprehensive HPV genotyping by PGMY-Reverse Line Blot, which can detect about 32 HPV types including all high-risk and low-risk types . The The present study specifically focused on HPV16 genotype. Viral load of HPV16 but not the HPV type 18 was demonstrated to express the highest predicted value (96% specificity and 88% sensitivity) in an earlier study . In a siin vivo situation in HPV16-infected cervical precancer and cancer lesions.The present investigation revealed that expression of pSTAT3(Y705) within different stages of the cervical carcinogenesis  individually as well as collectively, was associated with the degree of HPV16 viral load. A recent study has experimentally shown that loss of STAT3 severely impacts episomal maintenance of HPV18 in undifferentiated keratinocytes . The stuA comparative analysis of pSTAT3 expression with HPV16 physical state further demonstrated that in most of the LSIL cases where the HPV16 genome was present in predominantly episomal form, STAT3 activation was weak. On the other hand, tissues of HSIL and cancer lesion harboring either mixed or predominantly integrated form of the HPV16 genome showed an elevated level of active pSTAT3. This correlation sustained in cancer tissues harboring episomal form demonstrated a low level of active STAT3. Our data, therefore, indicated a possible association between STAT3 activation and integration of the viral genome in HPV16 infections. The mechanism by which STAT3 mediates its effect on the process of integration, which is considered as an opportunistic and random event during carcinogenesis, is not known. In such a scenario, an indirect mechanism, such as the induction of genomic instability  by whichTaken together, our study shows for the first time a clinical correlation between pSTAT3, viral copy number, and physical state of the HPV16 genome during cervical disease progression. Independent of its direct role in epithelial carcinogenesis , STAT3 m


HR‐HPV) genotype distribution in cervical intraepithelial neoplasia (CIN) before the widespread using of HPV vaccines in Chinese mainland and to improve risk stratification of HR‐HPV–positive women. Retrospectively, the data of age, cervical HPV genotypes, cytology, and pathology were collected from 1166 patients who received loop electrosurgical excision procedure (LEEP). HPV genotypes were analyzed with Flowcytometry Fluorescence Hybridization Method. And then HPV prevalence, HR‐HPV genotype distribution and the correlation of HR‐HPV genotypes with CIN2+ (CIN2 or severer) were analyzed. The role of multiple HR‐HPV types infection with or without HPV16/18 in the pathogenesis of CIN2+ was also analyzed. The 6 most common HR‐HPV genotypes were HPV16, 58, 52, 33, 18, and 31 in descending order. Compared to HR‐HPV–negative women, HPV16, 33 or 58 positive women had higher risk of CIN2+ . And women who were infected by multiple HR‐HPV types infection with HPV16/18 also had higher risk of CIN2+ . However, multiple HR‐HPV types infection without HPV16/18 did not increase the risk significantly (P = .08). Compare to bivalent Cervarix® and quadrivalent Gardasil®, HPV prophylactic vaccine targeting HPV31, 33, 52, and 58 might provide women more protection from HPV‐induced cervical cancer in China. The women who infected by HPV16, 33, 58, or multiple HR‐HPV types with HPV16/18 have higher risk of CIN2+ and need to be paid more attention in screening processes. And the role of multiple HR‐HPV types infection without HPV16/18 needs be further identified in more studies.To add the growing literature on baseline of high‐risk human papillomavirus ( In addition, to improve risk stratification of HR‐HPV–positive women, the role of specific HR‐HPV genotypes, and multiple HR‐HPV types infection with or without HPV16/18 in the progress of CIN would be shown.22.1Retrospectively, this study included 1166 women who underwent LEEP and were diagnosed with CIN subsequently at the 1st Affiliated Hospital of Wenzhou Medical University between January 2012 and December 2015. These women were the ones who had complete results of cytology, HPV typing, and pathology of cervix including biopsy and LEEP, had no use of vaginal medication or no sexual intercourse in the previous 3 days before sample collection, had not been vaccinated against HPV infection before, had not underwent any medical or invasive treatment for CIN previously, and were not immunosuppressive or immunodeficient. And to ensure the results from same test methods, the part of women using other test methods was excluded. Finally, age, results of screening tests, and final pathologic outcomes of CDB and LEEP were documented from patients' files. This study was approved by the Hospital Ethics Committee of the 1st Affiliated Hospital of Wenzhou Medical University. As a retrospective study, written consents were not required.2.2Cervical samples were collected using a disposable cervical brush  after menstruation, and then the samples were stored and transported in PreservCyt solution for thinprep cytologic test (TCT) using ThinPrep 2000 system (Hologic Inc.). The results were classified into 7 groups: negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASC‐US), low‐grade squamous intraepithelial lesions (LSIL), atypical squamous cells‐cannot exclude high‐grade squamous intraepithelial lesions (ASC‐H), high‐grade squamous intraepithelial lesions (HSIL), atypical glandular cells (AGC), and carcinoma (including squamous cell carcinoma and adenocarcinoma) based on the Bethesda System of 2001,2.3HPV genotypes were analyzed with Flowcytometry Fluorescence Hybridization Method  using Nucleic acid genotyping kit for human papillomavirus according to the manufacturer's instructions. A disposable sterile cervical sampler  was used to collect cervical exfoliated cell. The samples were analyzed within 1 week from collection. Using a suspension bead array method, HPV genotypes were differentiated. The experimental procedure had been described in previous study including DNA extraction, PCR amplification, and hybridization.2.4The referral indications for CDB were mainly based on ASCCP guideline.The referral indications for LEEP were primarily based on the results of CDB. In addition, women were selective to referral for LEEP in following cases: unsatisfactory colposcopic vision combined with suspicion of HSIL, patients' wish for maximum safety after the persistent infection of HR‐HPV for more than 2 years, reduplicated of abnormal cytology and the clinical manifestation, such as recurrence of contact bleeding. And it was performed using a diathermal electrocauterizer with a wire loop which size was determined by colposcopic examination of lesions. The specimen was fixed in formalin and then sent to pathology department.According to the World Health Organization criteria,2.5SD), numbers, or frequencies as appropriate. Chi‐square test was used to evaluate the association between HR‐HPV infection and CIN2+. Student's t test was used to evaluate the association between age and CIN2+. Kruskal‐Wallis test was used to evaluate the association between cytology and CIN2+. Univariate analysis and multivariate logistic regression analysis were used to find the association between HR‐HPV and CIN2+. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated when risks were estimated. P value corresponded to two‐sided tests and P < .05 was considered as statistically significant.Statistical analyses were performed with SPSS software version 19.0 for Windows. Results were presented as means ± standard deviations .The women with CIN1 had the lowest positive rate for HR‐HPV as 89.5% (221/247), followed by CIN2 , and the women with CIN3 had the highest positive rate as 96.1% (344/358). There was significant difference in these 3 groups (χP = .45). And there was significant difference found in groups with different cytologic results (P = .00). To investigate whether different HR‐HPV genotype and infection with multiple HR‐HPV types were risk factors for CIN2+, the patients were classified to 3 types of grouping. In type 1, there were HR‐HPV–positive (72/1161) and HR‐HPV–negative group (1089/1161). In type 2, there were 7 groups: HR‐HPV–negative (72/797) and single type infection with HPV16 (290/797), 18 (36/797), 31 (31/797), 33 (81/797), 52 (107/797), or 58 (180/797). In type 3, there were 4 groups: HR‐HPV–negative (72/675), single type infection with HPV16 or 18 (326/675), coinfection with HPV16/18 (156/675), and coinfection without HPV16/18 (121/675). And significant differences were found among the groups in these 3 types of grouping mentioned above (P = .00).To minimize the bias caused by small sample size when comparing risk factor profiles, we excluded the 5 cases with cytology‐AGC from correlation analysis. Table In multivariate logistic regression models, using CIN2+ compared with CIN1 as the multivariate dependent variable, there were 3 models according to the group types of HR‐HPV Table . As pres4As etiologic cause of cervical cancer, HPV has been studied in many ways including its prevalence and genotype distribution. The results of relative studies were varied because of different regions, races, age groups, and different methodologies or assays used.In concordance with previous researches, our data reported that HR‐HPV prevalence rate was increased with the severity of CIN.Up to now, many researchers had studied the association between CIN2+ and infection with multiple HPV types.This study provides reference data mainly for HR‐HPV genotype distribution in CIN and the association between HR‐HPV and CIN2+. HR‐HPV–positive women remain at an elevated risk for CIN2+. According to the high prevalence of top 6 HR‐HPV genotypes , we recommend these HR‐HPV genotypes have the priority to be included in second‐generation prophylactic vaccines. And women who infected by HPV33 or 58 may have higher risk for CIN2+ and need be heeded. The role of infection with multiple HR‐HPV types showed in the study triggers an intensive study by differentiating patients according to the presence of HPV16/18. And the question whether there is interaction among HR‐HPV genotypes in infection with multiple HPV types needs to be studied further.As a retrospective study, there are several limitations. The patients of this study were mainly from Wenzhou city which could not represent all women in China. And it is necessary to carry multicenter cooperation to study it further.The authors declare that they have no conflict of interest.