Synchronous anal HPV Infection in Patients with HPV-Related Gynaecological Diseases: A Prospective Study.

Why carry out this study? Patients with human papillomavirus-related gynaecological diseases (HPV-RGD) are at risk of synchronous HPV infections in other areas, such as the anal canal. The general risk of developing synchronous HPV infection in the anal canal, as well as the exact distribution of HPV types in this area in patients with HPV-RGD, is unknown. What was learned from the study? This study shows that two-thirds of patients with HPV-RGD have a synchronous anal HPV infection (mostly high-risk types), while in the general population, it is one in ten (mostly low-risk types). Together with the available evidence, the results of this study add insights into the research on stratifying the risk of anal cancer and clinical holistic management (screening and vaccination) of patients treated for HPV-RGD.

This was a prospective, cross-sectional, single-institution study performed in the Department of Gynaecological Oncology in Pomeranian Hospitals in Gdynia, Poland, between May 2023 and May 2025. We examined two groups—research and control. The research group included patients with histologically confirmed HPV-related gynaecological cancers: cervical (CC), vulvar (VC), vaginal (VaC) and their precancers—high-grade intraepithelial lesions (-IN): cervical [HSIL(CIN2/3)], vulvar [HSIL(VIN2/3)], vaginal [HSIL(VaIN2/3)]. In the case of VC and VaC, only patients with histologically confirmed HPV-related disease (p16 positive) were included. The control group included patients with gynaecological diseases that were neither HPV-related (endometrial polyps, uterine fibroid, abnormal vaginal bleedings, or endometrial hyperplasia) nor had a medical history of HPV infections (no history of abnormal cervical screening results, and without other signs or suspicions of HPV infection). We took two swabs in the research group: one from the anal canal and the other from the currently treated gynaecological organ. In the case of CC and CIN, the swab was taken from the cervix, in the case of VaC and VaIN, it was taken from the vagina, and in the case of VC and VIN, it was taken from the vulva [ 19 – 21 ]. The collection of material was performed just before the operation or diagnostic procedure for gynaecologic disease and after induction of general anaesthesia (if needed for the planned treatment). The swab collection was performed in the same manner by two physicians (M.B. and M.S.), thereby reducing the risk of bias associated with different swab collection techniques. The swabs were collected using the clinically validated cytological brush. The tip of the brush was inserted to a depth of 1–2 cm into the anal canal to collect material directly from the anal transformation zone—the region between the dentate line and anorectal junction. Subsequently, the material was fixed in a preservative fluid in the BD SurePath collection vial [ 22 ]. The genotyping test for 18 oncogenic HPV types: 13 types of high oncogenic risk (hrHPV) (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and five types of low oncogenic risk (lrHPV) (6/11, 42, 43, 44, 66)—Anyplex II HPV HR Detection test (Multiplex real-time PCR) was performed from the submitted material [ 23 , 24 ]. Due to the nature of the tests used, it was assumed that the presence of HPV in a given region indicates the infection [ 25 ]. Each participant was screened for the presence of HIV by detecting anti-HIV-1/2 antibodies and antigen p24 in the blood serum. A second confirmation test (real-time PCR test) was performed in case of a positive screening test. HIV-positive patients were not included in the study to reduce the additional impact of lower immunity on the risk of HPV infection. The results were compared between the research and the control groups. If a given participant had both hrHPV and lrHPV types detected, she was included in the hrHPV group. Only cases where lrHPV was solely responsible for the infection were included in the lrHPV group. The total concordance was defined as the same set of specific hrHPV types detected both in the anal canal and in the currently treated gynaecological organ. The partial concordance means that at least one type of hrHPV was present in both the anal canal and in the presently treated gynaecological organ. Discordance was defined as different types detected in the gynaecological organ and in the anal canal. The data were compared using Pearson’s chi-squared test or Fisher’s exact test, depending on the number of results in each group. The level of statistical significance was set at 5% ( α  = 0.05). To obtain meaningful results, the sample size was estimated at 100 patients. It was performed under the following conditions: type I error probability of 0.05, test power of 90%, and hrHPV test sensitivity of 90%. This research was funded by the Medical University of Gdansk (Grant number DB 71-01415/0004620) and partially by the National Science Centre, Poland (Grant number 2023/49/N/NZ5/01125). The Bioethics Committee of the Medical Chamber of Gdańsk approved the study protocol (39/21). Before entering the study, each patient signed an informed consent form for participation in the study and publication of data. This study was conducted in accordance with the Helsinki Declaration of 1964 and its subsequent amendments.

We recruited 134 patients for the research and 100 for the control group. Of all those invited, two women refused to participate. There were two cases of HIV-positive patients, who were excluded. Finally, there were 130 participants in the research group. There were no statistically significant differences between the two groups regarding the participants’ age (mean 48 in the research and 53 in the control groups, p  > 0.05). The mean age for individual subgroups was: 41 for CIN2/3, 51 for CC, 67 for VIN2/3, 62 for VC, 57 for VaIN2/3, and 74 for VaC. Results are summarized in Table  1 . Table S1 (in Supplementary materials) contains detailed information about each case in the research group. Table 1 Total number and percentage of anal HPV infections in various analysed subgroups Number of anal HPV infection (all types) n (%) Number of anal hrHPV infection n (%) p c The mean age Number of total concordance* n (%) b Number of partial concordance a n (%) b Control group N  = 100 11 (11%) 8 (8%) – 53 – – Research group (all) N  = 130 84 (64.62%) 78 (60%)  < 0.0001 48 46 (54.76%) 71 (84.52%) HSIL(CIN2/3) N  = 63 39 (61.9%) 36 (57.14%)  < 0.0001 41 21 (53.85%) 34 (87.18%) CC N  = 50 32 (64%) 30 (60%)  < 0.0001 51 15 (46.88%) 24 (75%) HSIL(VIN2/3) N  = 1 0 (0%) 0 (0%) – 67 – – VC N  = 7 7 (100%) 6 (83.33%)  < 0.0001 62 5 (71.43%) 7 (100%) HSIL(VaIN2/3) N  = 6 6 (100%) 6 (100%)  < 0.0001 57 5 (83.33%) 6 (100%) VaC N  = 3 0 (0%) 0 (0%) – 74 – – Statistical significance ( p ) was calculated in comparison to the control group CC cervical cancer, CIN2/3 cervical intraepithelial neoplasm grade 2 or 3, HPV human papillomavirus, hrHPV high-risk human papillomavirus, HSIL high-grade intraepithelial lesion, VaC vaginal cancer, VaIN2/3 vaginal intraepithelial neoplasm grade 2 or 3, VC vulvar cancer, VIN2/3 vulvar intraepithelial neoplasm grade 2 or 3 * Total concordance, the same set of specific hrHPV types was detected both in the anal canal and the currently treated gynaecological organ a Partial concordance, at least one type of hrHPV was present in both the anal canal and in the currently treated gynaecological organ b Percentage of all positive results c Statistical significance was calculated using Pearson’s chi-squared test. In VC and HSIL(VaIN2/3), statistical significance was calculated using Fisher’s exact test due to the small number of results Total number and percentage of anal HPV infections in various analysed subgroups Control group N  = 100 Research group (all) N  = 130 HSIL(CIN2/3) N  = 63 CC N  = 50 HSIL(VIN2/3) N  = 1 VC N  = 7 HSIL(VaIN2/3) N  = 6 VaC N  = 3 Statistical significance ( p ) was calculated in comparison to the control group CC cervical cancer, CIN2/3 cervical intraepithelial neoplasm grade 2 or 3, HPV human papillomavirus, hrHPV high-risk human papillomavirus, HSIL high-grade intraepithelial lesion, VaC vaginal cancer, VaIN2/3 vaginal intraepithelial neoplasm grade 2 or 3, VC vulvar cancer, VIN2/3 vulvar intraepithelial neoplasm grade 2 or 3 * Total concordance, the same set of specific hrHPV types was detected both in the anal canal and the currently treated gynaecological organ a Partial concordance, at least one type of hrHPV was present in both the anal canal and in the currently treated gynaecological organ b Percentage of all positive results c Statistical significance was calculated using Pearson’s chi-squared test. In VC and HSIL(VaIN2/3), statistical significance was calculated using Fisher’s exact test due to the small number of results The research group included 63 patients with HSIL(CIN2/3), 50 CC, six HSIL(VaIN2/3), three VaC, one HSIL(VIN2/3), and seven VC. The total prevalence of anal HPV infection in the research group was significantly higher than in the control group and was 64.62% and 11%, respectively ( p  < 0.05). When considering only hrHPV infections, the prevalence was 60% and 8%, respectively ( p  < 0.05). The prevalence of anal HPV in the cohort of patients diagnosed with CC was 64% for all HPV types and 60% for hrHPV ( p  < 0.05). Among patients with HSIL(CIN2/3), the prevalence of anal HPV (all types) was 61.9% and 57.14% for hrHPV ( p  < 0.05). Anal HPV was detected in all cases of VC ( n  = 7) and VaIN ( n  = 6). None of the patients with VaC ( n  = 3) and VIN ( n  = 1) had HPV detected in the anal canal. The research group’s overall total concordance (the same hrHPV types in the anal canal and the gynaecological organ) was 54.76%, the highest in the VaIN subgroup (83.33%), followed by VC (71.43%) and HSIL(CIN2/3) (53.85%). The research group’s overall partial concordance (at least one HPV type the same in the anal canal and the gynaecological organ) was 84.52%, the highest in the VC HSIL(VaIN2/3) subgroups (100%), followed by HSIL(CIN2/3) (87.18%), and CC (75%). The most common HPV type detected in the anal canal in the research group was 16, present in 53.6% of cases. The prevalence of the other HPV types was 17.9% for type 31, 14.3% for type 51, and 11.9% for type 33. The exact prevalence of each of the detected HPV types in the research and in the control groups is presented in Table  2 . Table S2 (in Supplementary materials) contains detailed data regarding anal and gynaecological HPV prevalence divided into specific cohorts. Figures S1 and S2 (in Supplementary materials) contain the percentage distribution of HPV types in the anal canal and gynaecological organs in the research group. Table 2 Distribution of anal and gynaecological HPV genotypes among all HPV infections in the research and control groups. (Note: multiple genotypes detected in the same sample were counted separately for calculation of the number of infections; percentages may not yield 100) HPV type Anal infection Gynaecological infection Research group n (% of all positive infections) Control group n Research group n (% of all positive infections) Control group n hrHPV 16 45 (53.6%) 0 53 (44.4%) – 18 8 (9.5%) 1 16 (13.4%) – 31 15 (17.9%) 1 12 (10.1%) – 33 10 (11.9%) 1 10 (8.4%) – 35 1 (1.2%) 1 1 (0.8%) – 39 8 (9.5%) 1 4 (3.4%) – 45 1 (1.2%) 1 5 (4.2%) – 51 12 (14.3%) 3 6 (5%) – 52 8 (9.5%) 2 5 (4.2%) – 56 4 (4.8%) 0 4 (3.4%) – 58 8 (9.5%) 1 4 (3.4%) – 59 3 (3.6%) 0 4 (3.4%) – 68 6 (7.1%) 3 3 (2.5%) – lrHPV 6/11 3 (3.6%) 0 0 (0%) – 42 7 (8.3%) 4 6 (5%) – 43 3 (3.6%) 1 4 (3.4%) – 44 5 (6%) 0 3 (2.5%) – 66 7 (8.3%) 1 8 (6.7%) – HPV human papillomavirus, hrHPV high-risk human papillomavirus, lrHPV low-risk human papillomavirus Distribution of anal and gynaecological HPV genotypes among all HPV infections in the research and control groups. (Note: multiple genotypes detected in the same sample were counted separately for calculation of the number of infections; percentages may not yield 100) HPV human papillomavirus, hrHPV high-risk human papillomavirus, lrHPV low-risk human papillomavirus In the control group, no specific HPV type was more prevalent than others. The most common type of HPV detected in the anal canal was low-risk 42, present in 4 out of 11 positive cases (4% of all cases), followed by 51 and 68, both present in 3 out of 11 positive cases (3% of all cases). There was no case of type 16 detected in the anal canal in the control group.

Two-thirds of patients with HPV-RGD had a synchronous anal HPV infection (mostly hrHPV), while in the control group, it was one in ten (mostly lrHPV). The highest prevalence of synchronous anal HPV infection was among patients with VC and VaIN, followed by CC and HSIL(CIN2/3). There was a higher prevalence of anal HPV infection in patients with CC (64%) compared to those with HSIL(CIN2/3) (61.9%). The duration of the infection may be important here. It has been shown that HPV infection must persist for about 5–20 years to develop CC [ 26 ]. In the case of HSIL(CIN2/3), the lesion may occur after several months of chronic infection [ 26 ]. At least one HPV type was the same in both the anal canal and the gynaecological organ in 84.5% of patients. It appears that a longer presence of HPV in the gynaecological organ increases the risk of spreading to the entire anogenital zone, including the anal canal. We were unable to recruit enough patients with vulvar and vaginal cancers and corresponding precancerous conditions to determine statistical significance and draw meaningful conclusions. However, the fact that every case of VC involved a synchronous HPV anal infection aligns with reports from other studies, where patients with VC are included in the group with the highest risk of developing AIN/AC [ 4 ]. Interestingly, in our study, all patients with VaIN2/3 also had synchronous anal HPV infections. To our knowledge, there is currently no available research investigating the prevalence of anal HPV infections in the subgroup of women with VaIN2/3. A larger database of patients with VC and VaIN2/3 is needed to accurately determine the rate of synchronous anal infection in these patients. The fact that two-thirds of patients with HPV-RGD had synchronous anal HPV infection (and in 53.6% of cases caused by type 16) warrants considering the clinical implications of this finding. There is evidence that active HPV infection of the anal canal, similar to HPV infection of the cervix, may induce neoplastic changes, which, over time, can lead to the development of anal precancer and cancer [ 27 ]. As many as 90% of anal cancers are related to HPV infection [ 1 ]. For this reason, the latest IANS guidelines recommend that each positive test for hrHPV (and/or abnormal anal cytology) should be an indication to perform a high-resolution anoscopy (HRA), which is the gold standard for detecting AIN/AC [ 17 ]. However, performing an anal swab in every patient with HPV-RGD would mean referring about two-thirds of them for HRA. Unfortunately, there are not enough health resources to provide HRA triage for such a large population. Considering the above, it seems reasonable to select a subgroup of patients at the highest risk of developing AIN/AC. This was expressed in the meta-analysis [ 4 ] and the recent IANS international guidelines for AC screening [ 17 ]. Accordingly, the HRA is considered a method for verification of positive screening tests for the AIN [ 28 ], while if the HRA is not available, the assessment of symptoms and digital anorectal exam (DARE) (not HPV or cytology tests) is considered a method for screening for AC [ 29 ]. In general, most HPV infections are transient, with nearly 80% being undetectable within 36 months in the case of cervical infections [ 30 ]. The approximate time from HPV acquisition to cervical cancer detection is 5 to 20 years [ 26 ]. There are many suggestions that the model of anal carcinogenesis is similar to cervical carcinogenesis. Accordingly, the process takes three stages. Persistent HPV infection may lead to the viral genome integration into nuclear DNA and, as a result, induce the expression of viral oncogenes E6 and E7. This can lead to the development of a precancerous condition of the anus (AIN), which, without treatment, can progress to AC [ 31 ]. Little data is available regarding this process; however, some authors suggest that the transition from AIN to AC may take longer and be less common compared to CIN [ 13 ]. Another important issue that should be addressed is the risk of developing AIN/AC for different hrHPV types. Hoots et al. conducted a systematic review and presented on the group of 991 patients with AC that the most frequently detected HPV type in the anal canal in women was 16 (73.7%), followed by 18 (7.6%) and 33 (2.1%). A similar situation occurred with AIN: the most frequently detected types were 16 (68.4%), 18 (7%), and 33 (5.3%) [ 32 ]. Relationship between anal HPV type, and anal disease severity (defined in four categories as: normal, including normal cytology only; low-grade cytology or histology diagnoses; high-grade cytology or histology diagnoses; anal cancer) has been analysed by Lin et. al. They found that in women, HPV16 positivity increased with lesion severity, whereas HPV18 positivity was relatively stable and HPV31/33/45/52/58 positivity fell in anal cancer from intermediate diagnoses [ 33 ]. Thus, the HPV type should be taken into account when planning the screening for AC in women. Another factor, specifically for women who were diagnosed with HPV-RGD, seems to be the time since the gynaecological diagnosis. It was partially addressed in the IANS guidelines [ 17 ]. However, data showed that the peak age of AC diagnosis in white women was 65–69 [ 34 ], and CC was 35–44 [ 35 ]. The issue of when to begin screening for AC/AIN seems even more complicated when thinking about AC/AIN risk as a synchronous or metachronous disease to the HPV-RGD. In our research, among HIV-negative women, anal HPV16 positivity was detected in 53.6% of patients with HPV-RGD at the time of the treatment of gynaecological disease, with other hrHPV types prevalence of 1–18%. It was impossible to establish the time of the infection in our research, nor is it a regular clinical management of patients with HPV-related diseases. Other important risk factors for the development of AC/AIN that should be addressed are immunodeficiency (primary or secondary e.g. to organ transplant) and its duration, autoimmune diseases, receptive anal intercourse, high-risk sexual behaviours, and smoking. Information regarding sexual behaviour and smoking status was not collected from patients participating in our study. None of the study participants had autoimmune diseases (rheumatoid arthritis, lupus, Crohn’s, or ulcerative colitis) or were receiving systemic steroid therapy. The factor that should reduce the risk of AIN is vaccination against HPV. Vaccine type and age at vaccination play a crucial role in determining the level of protection against HPV infections. Studies show that the 9-valent vaccine administered before the age of 18 provides the best protection against the development of HPV-RGD in the future [ 36 ]. Currently, vaccinations are the best form of primary protection against HPV-related diseases [ 37 ]. Although taking swabs from the anal canal may seem to be an embarrassing procedure for patients, in our study, only two participants in the research group did not agree to join the study after carefully explaining the goals and benefits they could obtain. This refusal percentage is surprisingly low and may result from the fact that this was a group of patients with an HPV-RGD already diagnosed (patients were just before entering the appropriate treatment process). This situation may have a crucial impact on the increased awareness regarding HPV-related morbidity. Thoroughly explaining to the patients that they constitute a subgroup at increased risk of developing another HPV-related disease also contributed to their willingness to participate in the study. The strength of our research was the prospective design of the study with a specifically defined target population and a control group. This allowed us to precisely determine the differences in the risk of anal HPV infections between patients with HPV-RGD and those without a risk of HPV gynaecological infection. Screening each participant for HIV also made it possible to eliminate the bias related to the potential impact of the presence of this virus (and the associated reduced immunity) on the study results. Another advantage of our study was the fact that we collected a sufficient number of patients with CC and HSIL(CIN2/3) to directly compare these two subgroups. The limitation of our study was the fact that we did not obtain information from the patients regarding whether they had been previously vaccinated against the HPV virus, and if so, at what age the first vaccination took place, what kind of vaccination they received, and how many doses were administered. However, the mean age of patients in our cohort was 48 years. Thus, the majority did not have an opportunity to participate in any vaccination against HPV in their childhood. Moreover, Poland is a country with a relatively low vaccination rate. Approximately 10% of the target population was estimated to receive vaccination, mainly younger individuals [ 38 ]. Considering this, it can be assumed that most of the participants in our study did not receive the HPV vaccination. Another limitation was that we obtained the prevalence of anal HPV infection, but without its clinical significance (at the time of test positivity). Further research is required to determine the clinical implication of synchronous anal HPV infection in patients with HPV-RGD, HIV-negative. A longitudinal, observational study on the real incidence of AIN/AC and a long follow-up would allow us to determine which patients are at the highest risk of developing AC and who would benefit the most from introducing routine screening for AC. The vaccination status and the age at vaccination will be variables to consider in a multivariate analysis, as there are data supporting and recommending vaccination against HPV, also in patients treated for HPV-RGD, e.g., after cervical conization for HSIL(CIN2/3) [ 39 ].

Patients with HPV-related gynaecological diseases, who are also HIV-negative, constitute a group with a significantly increased risk of synchronous anal HPV infection, with type 16 being the most common. Further research is warranted to define the clinical significance of the synchronous anal HPV-positive test in patients treated for HPV-related gynaecological diseases to guide holistic management—screening for anal cancer and HPV vaccination.

Human papillomavirus (HPV) is responsible for the development of almost all cases of cervical cancer (CC) and can be detected in about 74–78% and 24.9% of vaginal (VaC) and vulvar cancers (VC), respectively. Moreover, it is associated with almost 90% of anal cancers (AC) [ 1 , 2 ]. In the transformation zone of the cervix, where the squamocolumnar junction is located, the virus may directly infect a single layer of cuboidal epithelial cells [ 3 ]. A similar situation can be observed in the anal canal, where the squamous epithelium of the anal transition zone borders the columnar epithelium. Due to the relative proximity of the cervix, vagina, and anus, and the ease of spreading the HPV throughout the anogenital area, patients with HPV-related gynaecological disease (HPV-RGD) may be at risk of secondary HPV infections in the anal canal. This connection has been demonstrated in clinical observations [ 4 ]. HPV types 16 and 18 are the virulent high-risk genotypes, responsible for about 70% of all invasive CC worldwide [ 5 ]. In the case of cervical precancers, type 16 is also the most common. However, the prevalence of type 18 is decreasing in favour of types 31/33/45/52/58 [ 6 ]. Additionally, HPV type distribution in CC appears to depend on age—the older the individual, the less frequent type 16 is (in a population of ≥ 65 years of age, type 16 was detected in 42.7% of CC), and the more frequent other types are [ 7 ]. In AC, the prevalence of type 16 is much higher than in CC and is approximately 85%. HPV 18 is the second most detected type, but its prevalence is minimal and comparable to other types [ 8 ]. In the general population, AC is considered a rare malignancy. In 2020, there were 50,865 new cases confirmed worldwide. The rate of new cases was 1.9 per 100,000 (men and women) per year. However, the incidence and mortality of AC have been increasing over the past decades by 2.7% annually [ 9 – 11 ]. In fact, in the US, AC incidence recently surpassed CC among white women older than 65 (8.6 vs. 8.2 per 100,000 in 2015) [ 12 ]. Specific subgroups, where the incidence of AC or anal intraepithelial neoplasia (AIN) is significantly higher, can be identified. These include: immunosuppressed patients (especially HIV-positive people or transplant recipients), men who have sex with men (MSM), and patients with HPV-RGD [ 4 , 13 – 16 ]. It is reflected in the recent International Anal Neoplasia Society (IANS) guidelines for anal cancer screening. The authors defined two risk groups and outlined the clinical use of different tests in screening for anal high-grade squamous intraepithelial lesion [HSIL(AIN)] and AC. While publishing such guidelines marks a breakthrough in providing appropriate care for at-risk groups, the authors acknowledge a lack of comprehensive literature data to fill numerous knowledge gaps. We still cannot determine the harms and benefits of various screening algorithms, such as the ideal age to initiate or terminate screening and the optimal screening intervals. Furthermore, most data mainly concern HIV-positive patients or MSM. There is little data regarding other risk groups, e.g., patients with HPV-RGD [ 17 ]. The actual incidence of AIN and AC after a previous diagnosis of HPV-RGD is unknown. Available data rely on historic national registries, which generally did not include AIN incidence [ 18 ]. This study aimed to determine the risk of synchronous anal HPV infection in patients treated for HPV-RGD. The secondary goals were to test HPV type distribution and risk of anal canal infection depending on the localization of the HPV-RGD.

Below is the link to the electronic supplementary material. Supplementary file1 (PDF 375 KB) Supplementary file1 (PDF 375 KB)