Prevalence of human papillomavirus and subtype distribution in male partners of women with cervical intraepithelial neoplasia (CIN): a systematic review

Background Human Papillomavirus (HPV) infection is estimated to be the most common sexually transmitted infection. The present systematic review summarizes data regarding the prevalence of HPV and the distribution of subtypes in heterosexual male partners of women, who were diagnosed with any grade of cervical intraepithelial neoplasia (CIN). Methods We conducted a systematic review of the literature by Medline and Google Scholar databases using the terms “Human Papillomavirus” or “HPV” plus “men” or “male partners” or “women with CIN”. We included original published English-language articles published from 1/1/2000 until 1/1/2018 that had screened male partners of women with CIN using HPV DNA testing. We excluded studies that they overlapped with other included studies or were unrelated to the study subject. Results We included a total of 12 publications, which reported the prevalence of HPV in free-clinical signs male partners of women with CIN. The largest proportion of the studies were from South America (seven studies), and the rest from Europe. The mean age of participants was 35.18 + − 3.47 years. HPV prevalence ranged from 12.9 to 86%; the total HPV prevalence among the studies was 49.1%, while ten out twelve studies (83.3%) demonstrated prevalence > 20%. Between the studies, the distribution of HPV subtypes varied on the basis of the method used, on the population and on the geographic region. A great variety of subtypes were detected, including 6, 11, 16, 18, 31, 33, 40, 42, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 66, 68, 81 and 83. In six studies the HPV 16 was the most frequent, while in two others the HPV 6 and HPV 83. Conclusions Until now, there are not precise screening or surveillance guidelines for the management of partners of women with CIN. This population is frequently colonized by various HPV subtypes and therefore need to be screened in an effort to reduce the infection in both sexes. The screening test could include detection/identification of HPV subtypes by a molecular assay, followed by peniscopy only in the positive cases.


Background
Human Papillomavirus infection (HPV) is estimated to be among the most common sexual transmitted infections. Most HPV infections are asymptomatic or subclinical and become undetectable over time. It is well known that some HPV subtypes can cause anogenital warts, dysplastic and/or neoplastic lesions in women and men (heterosexual and men who have sex with men), generating a considerable economic distress within societies [1][2][3].
There are more than 150 HPV subtypes, which have been grouped according to their oncogenic capacity into High-Risk (HR) and Low-Risk (LR) [4]. Epidemiological studies show that HR are associated in women with invasive cervical cancer and its precursor lesion, the cervical intraepithelial neoplasia (CIN), whereas in men HR subtypes can cause head and neck squamous carcinoma and penile cancer [5,6].
CIN is a potentially premalignant transformation and dysplasia of squamous cells of the cervix, caused mainly by the HR-HPV types 16 and 18 [7]. Usually, CIN is eliminated by the host's immune system without any intervention, but, in some cases, when it left untreated, CIN can progress to cervical cancer [8]. Even though there is a vast bibliography regarding the management of the women diagnosed with CIN, there is very limited number of studies focused on the measures that must be applied in male sexual partners of women with CIN. A positive result for HPV infection usually stress women, who are worried about disclosing the result to others and the fact that there is not a clear management of their sexual partners make the disclosing even more difficult [9]. Previous studies have demonstrated that partners of women with CIN can be infected by the virus, while, the risk of developing cancer seems to be higher in men's second wives when their first wives died from cervical cancer [10].
The present study summarizes data regarding the global prevalence of HPV and the distribution of subtypes in heterosexual male partners of women, who were diagnosed with any grade of CIN. The scope of this review primarily focuses on the characteristics and the results of the selected studies. Assessing the prevalence of HPV infection of male partners is the first step in order to understand the natural history of HPV in couples with women with CIN, and to finally clarify the management of the male partners.

Methods
In this systematic review we conducted a systematic search in two online databases, Medline and Google Scholar, searching for studies published from 1/1/2000 until 1/1/2018. For our research we use the terms "HPV" OR "Human Papillomavirus", plus "men", "male partners" and "women with CIN". References cited in retrieved articles were also assessed. Eligible studies had to: 1) screen a population of heterosexual male partners of women with CIN, 2) include HPV DNA testing, and 3) be written in English language; all of which were including criteria. The evaluation of articles was performed based on their relevance of the title, abstract and manuscript review. In order to minimize the risk of bias, the evaluation of the articles was performed by two reviewers, independently. We performed a qualitative synthesis of the data for the prevalence and subtype distribution, since the articles varied significantly based on the study design, on the participants characteristics and on the molecular assays used. The prevalence of HPV in the selected studies was calculated by dividing the number of HPV-positive male partners by the total number of male partners of women with CIN. Unfortunately, there were no data for male partners of women without CIN in order to conclude if male partners of women with CIN have higher risk for HPV infection. Hence, no further statistical analysis was possible to be done. All the computations were calculated by R program (RStudio Team (2015). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL http://www.rstudio.com/).
Τhe articles were classified based on the time period when the samples were collected rather than the year of publication (Table 1). Five studies originated from Brazil, two from Spain, one from Netherlands, one from Italy, one from Mexico, one from Colombia and one from Czech Republic. There were differences regarding the time of the samples' collection: in eight before 2010, in three after 2010. Only one study did not mention the period of the collection of specimens. This study was published in 2012, took the ethical approval in 2009 and the collection of samples would have been rationally done after 2009 and before 2012; so, it was placed between Afonso (collection 2000-2010) and Rob (2013Rob ( -2015. In these studies, a variety of molecular assays were used for the detection of HPV in samples, obtained from men, who were sexual partners of women with CIN (Table 3). Information regarding the characterization of HPV subtypes (both HR and LR) were given in nine studies; the remaining three studies assessed HPV detection without subtyping (Table 3). In these three studies the methods used (PCR with universal primers followed by restriction or hybridization) had only the capacity to detect the virus and not to identify subtypes.  Overlap with a previous study by the same group, which was included (Benevolo et al. 2008). In addition, the men were stable partners of women who had been HPV/CIN positive in the past 3 years, but whose pathological data at enrolment were not available so it was not possible to distinguish the participants with HPV + partners from participants with CiN partners. Regarding the collection of specimens, the majority of the studies describe similar anatomical sites (the penile groove area, the glans penis, penile body and procure) for sampling by brushing (Table 3). Only one study used self-obtained samples as previously described by Weaver et al. [23]. Apart from the HPV DNA test, half of the studies used peniscopy as an additional diagnostic tool. Table 4 describes the characteristics of the couples. Four articles included only monogamous couples, while the remaining eight articles either did not mention whether couples were monogamous or if they included both monogamous and non-monogamous couples. In addition, differences in the time of relationship were also observed; four studies included couples with minimum duration of 6 months, two studies at least 1 year, one study at least 2 years and five studies did not mention the duration of the relationship.
Regarding the circumcised participants, four studies included circumcised male partners, one study excluded them; the rest of the studies did not mention if their male partners were or were not circumcised. On the other hand, differences in the use of condoms have also been observed; six studies mentioned the percentage of participants which used condoms, one excluded couples who used condoms and the remaining five articles did not mention the percentage of condom use. Finally, only five studies described the proportion of women with CIN I/ II/ III, whose partners participated in the studies.
Finally, only five out of 12 studies have studied the concordance of HPV-subtyping between the couples [15,17,19,21,22]. Benevolo et al, reported that 42.8% of the couples, that were HPV-positive, harbored at least one identical subtype, including HPV 16, 51, 52, 53, 56 and 58 [15]. This percentage is lower than that described by Lopez-Diez et al, who has demonstrated that 62% of infected couples had at least one subtype in common [22]. In both studies, HPV 16 was detected in a high proportion of infected couples. Vargas et al also showed that 28% of the sexual partners shared at least one viral subtype (16, 51, 52, 54, 68, 73 and 81) [21], while Afonso et al demonstrated that 53.3% of the couples had the same subtype (HPV 16) [18]. de Lima-Rocha et al found that 56.5% of the couples had at least one common subtype (HPV 6, 11, 16, 18, 31), whereas, absolute concordance was observed in only one case (4.3%) [19]. Furthermore, in the same study, the male partner had the same HR sub-types as their female sexual partner (HPV 16, 18, 31).

Discussion
HPV infection is common in asymptomatic men. In a systematic review of the literature, Dunne et al have shown that the prevalence of HPV infection in asymptomatic men ranges from 1.3-72.9% [24], while Smith et al have demonstrated that HPV prevalence among high-risk men (such as sexually transmitted infection clinic attendees, human immunodeficiency virus-positive males, Unsuccessful PCR analysis in some specimens in the selected studies male partners of women with HPV infection or abnormal cytology and men who have sex with men) was from 2 to 93% versus 1-84% in low-risk men [25][26][27]. In addition, the Centers of Disease Control and Prevention (CDC) reported that, between 2013 and 2014, in the USA, the prevalence of genital HPV for men aged from 18 to 59 years old was 45%, while, 25% of men had HR genital HPV infection [28]. A recent meta-analysis revealed a prevalence of 49% of any type of HPV and 35% of HR-subtypes in men [29]. However, the real incidence and prevalence of HPV infection in asymptomatic men is difficult to estimate, due mainly to the silent behavior of this virus not only in men but also in women.
According to data from studies conducted in North and Latin America, genital HPV prevalence is indicated to be higher in men than in women [30][31][32]. A meta-analysis by de Sanjose et al has demonstrated that the overall HPV prevalence in women with normal cervical cytology was 10.4%; the highest percentages were observed in Africa (22.1%), Central America and Mexico (20.4%), Northern America (11.3%), Europe (8.1%), and Asia (8.0%) [33]. On the basis of these estimates, approximately 291 million women worldwide are carriers of HPV DNA, of whom 32% are infected with HPV 16 or 18, or both.
In the present systematic review, we have found that the mean prevalence of HPV infection, in male partners of women with CIN was 49.1%. Although HPV 16 was the most common subtype, many other subtypes (6,11,16,18,31,33,40,42,45,51,52,53,54,56,57,58,59,61,62,66,68,81 and 83) were also detected. An interesting finding was that none of the participants had any clinical signs indicating that this target group was a reservoir for the dissemination of the virus. Apart from the HPV detection, the subtyping is very important not only for epidemiological purposes but, also, for evaluating the oncogenic potential of new subtypes in order to establish an effective and safe vaccination program.
Despite the variety of methods for the diagnosis of HPV in men, investigation into the presence of the virus has not been consensual [34]. Unfortunately, the identification of the presence of HPV in men is far more difficult than in women, due to the smaller quantity of plane squamous non-keratinized mucosa of the male genital organ in relation to that of the female [35]. Diagnostic tools are peniscopy, biopsy and HPV DNA testing. Recent studies have demonstrated that even when carried out by experienced professionals, peniscopy has very low specificity, leading to unnecessary biopsies [36]. Today, the identification of HPV has been carried by molecular assays using polymerase chain reaction (PCR) or hybrid capture; these methods are rapid, sensitive and easy to be performed [37][38][39][40]. Several of them are commercial, having the capacity not only to detect infection presence but also to characterize the different subtypes. Clinical specimens obtained using brushes from different anatomical sites can be directly tested for the presence of HPV DNA. Giuliano et al have shown that the optimal anatomical sites for detection of HPV are the penile shaft, the glans penis/coronal sulcus and the scrotum; specimens obtained from urethra and semen seem to have the lowest sensitivity [32]. However, sometimes, the detection of genital HPV is technically more complicated in men than in women because cells are more difficult to harvest from skin than from moist mucosal surface. Specimens, such as urine, are more easy to be obtained and to be tested for HPV presence. Neha Pathak et al, have demonstrated that HPV DNA testing of urine may be an alternative and an easier approach [41,42].
According to the data of the studies included in this review, the use of condoms was limited. In addition, only a minority of participants were circumcised. Previous studies have demonstrated that constant condom use is associated with reduced prevalence of HPV [43]. On the other hand, circumcision seems to minimize the risk for HPV penile infection and in the case of men with multiple sexual partners, circumcision reduce the risk of cervical cancer in their sexual partners [44].
There is a question how to manage the male partners of women diagnosed with CIN. It is well known than men who are found positive for HPV, could be HPV negative after 12 months. This could be explained by the fact that the epithelial cells of the penile skin are more resistant to HPV infection than the cervical epithelium, the clearance rates differ by gender and the duration of HPV infection is shorter in men than in women [45]. Morales et al have shown that the median clearance time for any HPV subtype was 5.1 months (3.5-7.7), while the duration of the colonization was similar for oncogenic and nononcogenic HPV subtypes [46]. Also, Guiliano et al stated that the median clearance rate of any HPV subtype was 5.9 months, with no observed difference in clearance time between oncogenic and nononcogenic HPV subtypes; 75% of participants were negative for any HPV subtype after 12 months [47].
Summarizing the results of the studies included, healthy sexual male partners of women with CIN may be HR HPV-positive, maintaining the risk of viral transmission and consequently the risk of recontamination of their female partners. Therefore, when the male partners were found to be positive by a penile HPV test, they should be advised to undergo a clinical follow up as previously reported by Gupta et al [48]. In addition, the introduction of the 9-valent HPV vaccine, that includes the subtypes 6,11,16,18,31,33,45,52 and 58, most of which were detected in the partners of women with CIN, combined with education regarding the prevention should limit the spread of the virus within couples [49,50].

Limitations
The main limitations of this review are the differences between the characteristics among the participants and the different HPV DNA testing assays. Some studies have included regular and monogamous sexual partners, but others just regular partners. Some studies have included men who have been circumcised, which may alter the results since circumcision seems to protect men from HPV infection. Among the studies, the proportion of women with CIN I, CIN II and CIN III differed significantly, while sometimes the proportion was not mentioned. So, searching for correlation between HPV infection of male partner and the grade of CIN was not possible. Also, the studies have been held in different countries, where the prevalence of HPV infection varies in the general population. Finally, among the studies, various HPV DNA tests with different specificity were used for the detection of the virus, in addition, each study characterized specific subtypes.

Conclusion
Until now, there are not precise screening or surveillance guidelines for the management of partners of women with CIN. This population is frequently colonized by various HPV subtypes and therefore need to be screened in an effort to reduce the infection in both sexes. The screening test could include detection/identification of HPV subtypes by a molecular assay, followed by peniscopy only in the positive cases.
Given that the virus is associated with neoplastic lesions, these men are also at risk for HPV-related tumors (penile cancer etc). The introduction of vaccines could play an important role to the prevention and therapy. Prophylactic HPV vaccination (B-cell-mediated immunity) provides lifelong protection against subtypes included in the vaccine; therefore, a vaccination program for children of both sexes is counted to the primary prevention strategies and might reduce the HPV prevalence. On the other hand, therapeutic vaccines, based on an antigen-specific T-cell immunity are promising approaches for the treatment of already existing intracellular HPV infections and are under investigation.