Male circumcision has been shown to prevent HIV and other sexually transmitted infections (STIs) [12
]. The dominant theories regarding the underlying mechanism of this protective effect focus on the keratin layer of the inner foreskin. During sexual intercourse, this thinner keratin layer potentially increases the exposure of target cells to HIV. However, studies concerning foreskin keratinization have been inconclusive and contradictory. Our study addresses this critical impasse by quantifying the keratin thickness of 16 foreskin specimens using a novel method for keratin measurements. We found no difference between the inner and outer foreskin keratin layers, invalidating the hypothesis that the thinner keratin barrier of the inner foreskin leads to increased HIV infection in uncircumcised men. Our findings are in agreement with Qin et al., who measured the keratin layer of foreskins from Chinese men and boys [6
Previous studies have lacked description of the heterogeneous nature of the foreskin’s keratin layer. We observed significant heterogeneity between the donors in our study, as seen in the measurements presented in and which were determined with multiple samplings (>50/specimen). This phenomenon has been seen in dermatologic studies when skin samples are compared across a group of individuals, and may be related to genetic differences in skin composition [11
]. In addition, exposure to environmental stimuli over an individual’s lifespan might also induce changes in the expression of epidermal proteins, such as filaggrin or keratin.
In order to address potential differences in keratin thickness that may have been introduced by our methods, we conducted a detailed analysis of the methods used for fixing and staining the foreskin tissue. Previous analyses of foreskin keratinization have relied on formalin-fixed, paraffin-embedded tissue stained with H&E [2
]. These stains are non-specific; they depend on differences in chemical composition, which may render it difficult to distinguish each epithelial stratum. Standard immunohistochemical stains against epidermal cytokeratins (including AE1/AE3 and CK5/6) are also non-specific for our area of interest—they stain for keratin production in basal layers of the epidermis [21
]. The method developed by our group allows for high-resolution images based on the expression of proteins that highlight the keratin layer. In comparing sections from the same donor using different fixes and stains, we found no difference between the different methods, confirming the accuracy of our technique in measuring keratin thicknesses.
Although our study is the second largest to compare inner and outer foreskin keratinization, it remains limited by its small sample size. Future studies with a larger number of specimens (including penile specimens) will more clearly highlight similarities and differences between tissue types. Another drawback was the statistical analysis undertaken for this study, which may not completely take into account the intra-individual variability in measurements. Additionally, these measurements were taken from explanted tissue that is devitalized, though any effects to the keratin layer from this are likely minimal. Lastly, we did not know the donors’ exact medical indication for male circumcision. Most donors undergo male circumcision for phimosis (difficulty retracting the foreskin), which is commonly due to recurrent episodes of balanitis (or inflammation of the foreskin due to infections or diabetes mellitus) [22
]. It is unknown how these underlying conditions affect foreskin keratinization, and hence our findings. However, Qin et al. also evaluated the foreskins of healthy pre-school boys and found similar results [9
]. We have also directly compared the foreskin to the corresponding penile tissue from two uncircumcised cadaveric donors and found only marginal differences in the keratin thickness between foreskin and penile epithelia (data not shown). Additionally, circumcised penile epithelia tended to be more thinly keratinized than uncircumcised penile epithelia, further refuting the hypothesis that thinner keratin layers correspond to easier HIV transmission.
We propose that the mechanism responsible for the protective effect of male circumcision on HIV transmission involves more than keratin alone. A recent study by Kigozi et al. evaluating Ugandan men who underwent male circumcision described differences in HIV susceptibility that correlated with the size of their excised foreskin [23
]. These findings suggest that the larger area of squamous epithelium provided by the presence of the foreskin may play a more important role in HIV transmission. Features such as epithelial permeability and inflammatory responses also vary widely and may have epidemiological associations with HIV infection [19
]. Other studies have demonstrated a correlation between stricter preputial (the area between the foreskin and the glans penis) hygiene and lower HIV prevalence rates, suggesting that the dynamic environment created by the foreskin also plays an important role [25
]. In addition, the results of the Merck HIV-1 gag/nef/pol vaccine trial imply that being uncircumcised alone did not confer the highest risk of HIV infection; vaccination further enhanced that risk in a way that was not evident in the serologic response to the vaccine [27
]. These observations together argue against an overly simplistic model relating to keratin layers in the foreskin alone.
Ultimately, we need to better understand how HIV dynamically interacts with human genital mucosal surfaces, particularly in response to conditions that alter the mucosal environment created by the foreskin. Further studies aimed at clarifying these interactions will also help determine whether the protective effect of male circumcision, as seen in the African trials, can be applied to different at-risk groups, such as men who have sex with men. Only with a thorough understanding of the mechanism underlying the protective effects of male circumcision will it be possible to create effective topical agents or systemic vaccines to help finally stem the global epidemic.