Prevalence of the human papillomavirus (HPV) expression of the inner prepuce in asymptomatic boys and men
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- Klinglmair, G., Pichler, R., Zelger, B. et al. World J Urol (2013) 31: 1389. doi:10.1007/s00345-012-0997-8
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To investigate the prevalence of high-risk (HR) and low-risk (LR) human papillomavirus (HPV) in prepuces of boys and men without any HPV related lesions.
Patients and methods
Between 2009 and 2011, a total collective of 250 boys and men were investigated in this prospective study. The samples were subdivided into 3 groups regarding their age, consisting of 125 (50 %) children (0–10 years), 38 (15.2 %) adolescents (11–20 years) and 87 (34.8 %) adults (>20 years). In situ hybridization (ISH) was performed to detect HR and LR virus types within the epithelium, followed by microscopic interpretation and determination between episomal and integrative signal pattern.
Our results revealed high levels of HPV concentration (HR and LR) in all age groups: HR versus LR positivity was seen in 45.5 versus 35 % (children), 60.6 versus 63.6 % (adolescents) and 58.3 versus 48.6 % (adults). The topmost rate of HR (59.8 %)- and LR (50.6 %)-positive probes was found in the group with high estimated sexual activity (>14 years).
Concerning the high prevalence of male HPV infection even in children, indicating non-sexual transmission pathways, inclusion of boys in the vaccination program seems to be required reducing their burden of HPV related disease.
Human papillomavirus (HPV) is a common sexually transmitted infection and accounts for diseases like invasive cervical cancer, genital intraepithelial neoplasias or condylomata acuminata. High-risk oncogenic HPV types (16, 18, etc.) are responsible for about 70 % of cervical cancer, whereas low-risk HPV (6, 11) causes about 90 % of anogenital warts . Women suffering from cervical carcinoma or CIN frequently have partners with a penile intraepithelial neoplasia. Although a connection between an intact prepuce and cervical carcinoma has been suspected for a long time, it was first confirmed in 2002 by a large multicenter study . Although there has been a lot of research on HPV infection in women, there is only little data on the male HPV prevalence.
It is presumed that in uncircumcised men the frail, easily infectable inner prepuce is exposed to the vaginal secretion of an infectious partner, when retracted during erection. After infection and inoculation, there is a high risk of transmitting the virus during intercourse. Regarding this theory, the aim of the study was to evaluate the association between the foreskin and HPV infection in asymptomatic male individuals.
Materials and methods
After positive approval from local ethical committee (application number 20090609-68), we analyzed prepuces of 250 male individuals (without HPV related lesions) after circumcision due to congenital (children, adolescents) or acquired (adults) phimosis. Specimens were collected from 3 different Austrian institutions. The samples were labelled anonymously with ongoing numbers and the patient’s age. Therefore, anonymity was preserved, and tracking of patient data was not possible. We configured three groups regarding age: children (0–10 years), adolescents (11–20 years) and adults (>20 years), and estimated sexual activity relating to the epidemiological study on sexual habits (start of sexual activity) by Boccalini et al. (2012) : group 1 (low frequency 1–14 years) and group 2 (high frequency >14 years). The frequency of HPV infection was evaluated in comparison with the different groups. In situ hybridization was performed to detect high-risk (HR) (16, 18) and low-risk (LR) (6, 11) virus types within the epithelium of the easily infectable inner prepuce, followed by microscopic interpretation and determination between episomal and integrative signal pattern. Some slides could not be evaluated since their epithelium had been wrenched off or the morphological structure had been severely damaged during processing. Those were excluded. A case was considered positive when we found at least one tissue area showing a specific HPV positive signal.
Statistical analysis was performed using the SPSS program. The number of positive and negative probes was calculated from our original data. A probe was considered invalid if it was not possible to accurately evaluate the slide in microscopic interpretation. Mann–Whitney U test was performed to show that there is no statistical difference between HPV positive and -negative patients regarding the age groups. Chi-square test was done to investigate the statistical difference in the distribution of the HPV status regarding the age groups. A statistical significance was assumed at p value <0.05.
In situ hybridization (ISH)
Sample staining was carried out on Discovery XT® automated slide processing system (Ventana Medical Systems, Strasbourg, France). Unless stated otherwise all products were used from Roche/Ventana Medical Systems, Strasbourg, France. First step of processing included dewaxing by heat with EZ-prep®. Next, a pre-fixation was performed with RiboPrep, followed by clearing with RiboClear®. Cell conditioning with CC2 (citrate-based buffer) was performed. DNA targets were retrieved by applying a combination of heat treatment with Reaction Buffer® and digestion with Protease 3®.
INFORM HPV 3® probes (oncogenic types) and INFORM HPV 2® probes (non-oncogenic) were diluted in RiboHybe® buffer.
Probes and target DNA were denatured, and hybridization was conducted. Stringent washing steps were carried out using RiboWash® followed by fixation with RiboFix®. DNP hapten detection was performed using primary rabbit anti-DNP antibody (Antibody 110®), followed by treatment with a goat anti-rabbit antibody (Universal secondary Antibody®). The Blue Map Kit® was used for visualization. Counterstaining with Nuclear-Fast-Red-counterstain® was performed. Finally, specimens were dehydrated and mounted onto slides . Each run was performed with a positive and negative control. A condyloma sample was used as positive control, whereas one with the probe cocktail omitted served as negative control.
For microscopic analysis, the Olympus BX 50 F4 microscope (Olympus Optical Co., Ltd.) was used. We differentiated between episomal and integrative signal pattern according to the interpretation guide for Ventana INFORM® HPV II probes . Interpretation was sometimes difficult since the virus load is very low, and in many cases, we found a faint integrative pattern. Unspecific findings of a signal on top of the mucosa and in the subepithelial stroma were evaluated as invalid.
The preputial prevalence of HR and LR–HPV was investigated in 125 (50 %) infants, 38 (15.2 %) adolescents and in 87 (34.8 %) adults.
Overall HR- and LR positivity was very high with 49.8 % (115/231 patients) and 41.9 % (99/236 patients). Regarding the different age groups, there was no significant statistical difference in HR (p = 0.323) and LR (p = 0.323) positive status: HR positivity was seen in 55 (45.5 %) children, in 20 (60.6 %) adolescents and in 42 (58.3 %) adults. LR positivity was very high in all age groups too: Adolescents demonstrate the highest amount of LR positive probes with 63.6 % (21/31), in children and adults, a positive LR status was detected in 35 % (43/123) and 48.6 % (36/74).
Regarding the results of Boccalini et al.  (mean and median age of the first sexual intercourse was 15.4 ± 1.4 and 15 years), we configured age groups estimating the frequency of sexual intercourse with group 1 (low sexual activity, 1–14 years) and group 2 (high sexual activity, >14 years). The frequency of HR-positive probes showed its peak in the group of most sexual active patients (group 2): 59.8 and 50.6 % of the probes within this age group were evaluated as HR and LR positive. The group of low sexual activity noticed a HR and LR positivity status in 47.1 and 39.4 %.
Human papillomavirus as a sexually transmitted infection (STI) causes cervical and penile cancer, other types of cancer, or genital warts . Prophylactic vaccination against HPV types 6/11, 16/18 among female adolescents results in a rapid reduction in genital warts and cervical cytological abnormalities. Additionally, diagnostic and therapeutic procedures with substantial reduction in cervical, vulvar and vaginal cancer are well established nowadays . The aim of this study was to point out the high HPV prevalence of non-lesional preputial mucosa in asymptomatic men among all age groups. Approximately 40 % of invasive penile carcinomas are attributable to oncogenic HPV types. In addition to HPV infection, the risk factors most strongly associated with penile cancer are lack of neonatal circumcision, phimosis and anogenital warts . Two systematic reviews investigating the association between circumcision and HPV infection indicated that circumcised men are less likely to have prevalent genital HPV infection than uncircumcised. Thus, circumcision provides additional benefit in reducing HPV prevalence in men [8, 9]. Regarding this fact, Tobian et al.  showed that circumcision does not only reduce HIV acquisition in men, but also HR-HPV prevalence in both HIV-negative and -positive men. Compared to HIV-positive men, circumcision of HIV-negative men also reduces the incidence of HR-HPV infections in their female partners showing that circumcision is most effective prior to coitarche and that the presence of foreskin facilitates HIV and HR-HPV infection in men and their female partners. Wawer et al.  also confirmed this statement and indicated that circumcision should be accepted as an efficacious intervention to reduce the prevalence and incidence of HPV infections in female partners. The exposition of the inner preputial epithelium during intercourse and its low level of keratinization enhance the possibility of infection with epitheliotropic viruses like HPV. Therefore, circumcision and keratinization of the surgical scar are able to hamper the infection . Comparing various studies on Kenyan men, Smith et al.  analyzed 2.705 sexually active, uncircumcised, HIV seronegative men. The prevalence of virus-infected cells was 51.1 % in penile cells from the glans/coronal sulcus compared with 19.1 % in the penile shaft. HR-HPV was detected in 31.2 % of glans and 12.3 % of the shaft samples. Backes et al.  evaluated the association of circumcision with the prevalence of HPV associated penile lesions in Kenyan men (151 circumcised, 124 uncircumcised). Flat penile lesions (= increased risk of HPV transmission) were much more frequent in uncircumcised than in circumcised men (26 vs. 0.7 %) and associated with higher prevalence of HPV and higher viral loads.
Considering these studies and the data from our study with a high HR and LR–HPV positivity in juvenile and adolescent patients, circumcision of boys and adolescents may reduce the prevalence of HPV related disease. Consequently, the rate of the sexual transmission would be likely to decrease. Globally, even more relevant is protection from cervical cancer, which is 10 times more common, and much higher in women with uncircumcised partners, so circumcision provides indirect protection against infections in women .
As we expected, the highest percentage of HPV positivity was detected in the sexually most active group (>14a) with HR positivity in 59.8 % and LR positivity in 50.6 %. Remarkably, we also found a great amount of HPV positive specimens in the group with low sexual activity (1–14a) with a low-risk and high-risk HPV signal pattern in 39.4 and 47.1 %. This may indicate a high frequency of non-sexual transmitted HPV infection. Apart from sexual abuse, other modes of HPV transmission are more likely in children, including perinatal transmission, autoinoculation, heteroinoculation and indirect infection via fomites [19, 20]. Regarding our results with LR–HPV positivity in 35 % (infants) and 63.6 % (adolescents), Tarnoud et al.  confirmed the significant reduction in LR–HPV infection among circumcised young men, increasing the mean number of LR–HPV genotypes with number of lifetime sexual partners, decreasing with education level and consistent use of condoms.
Regarding evaluation, the biggest challenge we had to face was a very small virus load since we only included patients without any clinically detectable HPV infection. A staining protocol originally developed for routine diagnostic of cervical lesions was modified to set a higher sensitivity in staining. The virus load in lesional specimens like condyloma compared to a sample from the present study is many times higher than in non-lesional mucosa. We only found a few probes with episomal pattern, many cases with a discrete integrative pattern and quite a number of specimens with a dark blue band covering the apical epithelial layer, probably representing drying associated artifacts. The latter reaction pattern cannot be interpreted as a positive signal. We can only speculate that it might be true viral DNA, therefore, we marked and dissected areas with that signal as well as clear episomal and integrative signals in order to perform real time PCR studies to confirm our preliminary results in a further study. ISH has one big advantage: In contrast to a smear or a urine test for HPV , ISH conserves the histomorphology and gives insight into each single cell of epithelium instead of just investigating the apical cell layers. This might be the reason why we found such a high percentage of HPV positive specimens (49.8 % HR and 41.9 % LR).
Guo et al. compared in situ hybridization to rtPCR on cervical tissue with CIN and carcinoma and found that the results they gathered from ISH are concordant to those from PCR. They also compared the Inform HPV III probe—used in the present study—to precursor probes and observed significantly higher HPV positive rates using Inform HPV III, indicating an increased sensitivity of this probe. They also observed that interpretation becomes difficult on specimens with weak and sporadic integrative signal patterns. Another considerable aspect is that especially in lesions with low virus load an ISH positive/PCR negative result was reported frequently. The main reason seems to be the heterogeneous distribution of the signal pattern resulting in the target molecule missing and thus leading to false-negative results in PCR .
Artifacts we had to face in this study are explainable by drying, overdigestion or leukocyte association. Overdigestion is a result of prolonged treatment or an inadequate strong amount of protease and leads to loss of tissue integrity. Non-specific staining of leukocytes is very common and resembles an unspecific reaction of cytoplasmatic components with the secondary antibody resulting in cytoplasmatic staining of leukocytes. The same type of unspecific reaction pattern is seen in specimens with acute inflammation. Drying artifacts emerge when liquid reactants drip off the slides which can result in a focal accumulation of chromogen after drying. A pale blue “fog or cloud-like” artifact can be seen either all over the whole specimen or only on a focal area. This type of artifact can also appear as a blue band-like stain over a group of cells making interpretation difficult and requiring further investigation as mentioned above .
The concept of precautionary HPV vaccination is the insertion of antibodies to prevent infection of the mucosa or the skin. Currently, the quadrivalent vaccine Gardasil® (Sanofi Pasteur MSD GmbH, Austria) and the bivalent Cevarix® (GlaxoSmith-Kline Pharma GmbH, Austria) are available in Austria. Both of them take effect against the HPV types 16 and 18, whereas the quadrivalent vaccine is effective against HPV 6 and 11 as well. Clinical studies have shown that both are safe and are well tolerated in women from 9 to 26 years, and the immunogenicity is provided for at least 5 years. The main target of vaccination programs against HPV is girls prior to coitarche [19, 20, 23–25]. Austria’s department of health (BMGV) recommends HPV vaccination for girls before sexual activity, since they benefit most from the vaccine. According to the department of health, it is basically reasonable to also vaccinate boys and male adolescents, regarding the possibility of virus transmission. Using a quadrivalent vaccine is considered useful on male individuals since it provides protection against condylomata. Regardless of vaccination, PAP smears and prerequisite treatment against HPV infection have to be continued .
The expenses for HPV vaccination are not covered by Austrian insurances at the present day. The price for the vaccine is about € 200 per vaccination, adding up to a total of about € 600 for the complete three partite vaccinations .
Regarding the data on HPV prevalence in children (LR positive 35 %, HR positive 45.5 %), adolescents (LR positive 63.6 %, HR positive 60.6 %) and in the age group with the low sexual activity (LR positive 39.4 %, HR positive 47.1 %), it would be rational to downgrade the ideal age for vaccination to maybe 6–10 years also in boys. General recommendation of HPV vaccination for boys would be a reasonable step too. Most significantly the cost coverage for the vaccine should be provided by the public health system. If the quadrivalent vaccine is successfully disseminated to large segments of young males in further multicenter studies, there is potential for substantial reduction in HPV related disease in men. A limitation of this study is that the number of adolescents compared to the other two groups was relatively small. A comparison of LR and HR viral-infected cells between patients with and without phimosis was not addressed in this study, so we proved only the prevalence of HPV associated lesions/HPV infection in asymptomatic, uncircumcised men.
In conclusion, we confirmed an overall high prevalence of LR and HR-HPV infections in asymptomatic boys and men with phimosis. Considering this fact vaccination programs would be beneficial to both men and women in reducing the HPV related health burden. Further studies using real time PCR of the specimens are necessary to confirm the preliminary results.
We want to thank Gabriel Djedovic, Erich Brenner, Elisabeth Richter, Irma Sottsas, Linda Lovdok and Stefan Reininghaus for their support and their contribution to this work.
Conflict of interest
The authors declare that they have no conflict of interest.