**4. Discussion**

This systematic review was performed to evaluate the prevalence of HPV infection in BCa, keeping into consideration confounding demographic, histological, and diagnostic variables.

An overall HPV prevalence of 19% was found in 46 studies, in line with previous meta-analyses which reported a prevalence of 16.88% [10] and 14.3% [61]. Despite the growing number of reported studies, the role of HPV in cancers other than genital, anal, head, and neck cancers is still debated, due to the heterogeneity in the study design, population enrolled, and HPV detection methods [6]. In fact, several systematic reviews, which evaluated the prevalence of infection of DNA-based vs. non-DNA-based methods confirmed the higher specificity and sensitivity of molecular-based methods [62,63]. Moreover, the use of genotype primers designed for shorter DNA sequences reduced the risk of "false negative" results in comparison with broad-spectrum primers (i.e., GP5+/6+), especially for FFPE specimens often undergoing DNA damage [10]. Therefore, the implementation of a standardized procedure for HPV detection could better clarify the impact of HPV in BCa pathogenesis.

A statistically significant difference in the prevalence of infection was found in different histological subtypes, with higher estimates in SCC than those in TCC and UC. The high affinity of HPV to differentiating squamous epithelium, previously demonstrated in cervical, head and neck, and anogenital carcinomas [64], as well as the ability of the virus to evade and inactivate the immune response, could explain the mechanism of carcinogenesis in the bladder epithelium. Since SCC has poor prognosis and is associated with worse outcomes in different sites [65], the confirmation of the role of HPV in SCC could improve the epidemiological burden and the prognosis of potential cases.

The assessment of the role of HPV as causative agent in different histological subtypes could have been affected by the adoption of the new WHO BCa classification [66]. This recent recommendation changed the traditional nomenclature of "transitional cell carcinoma" in "urothelial carcinoma", causing potential misinterpretations of the results. On this basis, the identification of standard accurate procedures for the detection and diagnosis of HPV infection could be helpful to estimate the burden of cancers associated with HPV. However, the available molecular techniques show a high diagnostic accuracy.

Although several studies suggested a relationship between advanced stage (i.e., ≥T2) and HPV prevalence [27,43,44], the poor information collected in the selected studies did not prove this association.

Similarly to other HPV-related cancers, HPV-16 was the most prevalent genotype in BCa, supporting previous findings on the increased risk of BCa in cases of infection caused by high-risk HPV genotypes, mainly by HPV-16 [10]. The high detection rate of HPV-16, together with HPV-18 and the low-risk HPV-6, strongly supports the administration of HPV vaccines to prevent HPV-related cancers in both sexes [67].

Finally, our study showed a significant association between HPV and BCa (OR: 7.84), confirming the findings of recent meta-analyses on the risk of BCa [10,62,65]. However, these results are in contrast with a previous study published by Khatami et al. [61], who described a non-significant association between infection and cancer and highlighted the scientific need of larger case-control studies. Although the majority of case-control studies were classified as "medium-quality", controls showed a prevalence of infection <10%, regardless of HPV detection methods, suggesting the etiological role of HPV in BCa.

Previous systematic reviews investigated the role of HPV infection in BCa and described a moderate association. Our study selection and analysis showed an important role of HPV infection in SCC BCa. However, some limitations should be acknowledged. More stratified analyses related to demographic (i.e., geographical area, behavioral factors, occupational exposures) and clinical (i.e., stage, HPV detection methods, prognosis of patients) confounders should be performed. The cross-sectional assessment does not help prove the temporal relationship between an exposure and the development of cancer.
