Collagen is More Abundant and Structurally Altered in Lichen Sclerosus

Abstract

Objective: To test the hypothesis that genital skin and male urethra affected by lichen sclerosus (LS) has increased collagen content and altered collagen structure.

Methods: We used picrosirius red to stain and image collagen in human urethral, vulvar, and foreskin specimens with and without LS. Using Image J software, we quantified and compared (1) collagen content (using 2o metrics: collagen proportionate area [CPA] and collagen fiber count), (2) collagen fiber length and width, and (3) collagen structure using the texture analysis technique gray level co-localization matrix (GLCM) with respect to LS status and tissue type.

Results: We analyzed 23 LS specimens (vulva n=9, urethra n=7, foreskin n=7) and 29 non-LS specimens (vulva n=9, urethra n=7, foreskin n=13). Fiber count and CPA were significantly higher in all LS specimens compared to non-LS specimens (CPA: mean±SD 0.971±0.03 vs 0.948±0.02, P < .007; fiber count: mean±SD = 2906±127 vs 2509±78 fibers; P = .003). Collagen fiber width and length were similar with respect to LS status. GLCM analysis showed decreased inverse difference moment and increased entropy in LS tissues indicative of less homogeneous and more disorganized tissue structure (P<.001).

Conclusion: LS tissues have greater collagen content compared to non-LS tissues. Quantitative assessment of collagen organization, using GLCM, revealed less homogeneity and more disorganization of collagen in LS compared to non-LS tissues. Taken together, our findings suggest that alterations in physical tissue properties seen in LS may be due to both increased collagen abundance and altered structure.

Figure 1.

Comparison of collagen content in human vulva, urethra, and foreskin with and without LS. (A) Representative fluorescent microscopy images (20x) of PSR stained urethral collagen demonstrating increased collagen content in LS compared to non-LS tissue. (B) Comparison of mean collagen proportionate area in tissues with and without LS. Taken together, LS tissues had higher mean collagen proportionate area compared to non-LS tissues (0.971 vs 0.948, respectively; p=0.007). When segregated by tissue type urethra and foreskin LS tissues had significantly higher collagen proportionate areas (urethra – 0.979 vs 0.953, p=0.02; foreskin – 0.974 vs 0.951, p = 0.027) while vulvar tissues were no different with respect to LS status (0.961 vs 0.941, p = 0.385). (C) Comparison of collagen fiber count in tissues with and without LS. Taken together, LS tissues had higher mean collagen fiber counts compared to non LS tissues (2906 vs 2509 fibers, p = 0.003). When segregated by tissue type foreskin LS tissues had higher mean number of collagen fibers (2722 vs 2161 fibers, p = 0.004) while vulva and urethra were similar with respect to LS status (vulva – 3071 vs 2625 fibers, p = 0.07; urethra – 3036 vs 2707, p = 0.139). Brackets indicate pairwise comparisons with “*” indicating p < 0.05. LS = lichen sclerosus, PSR = picrosirius red. Scale bar = 100 μm.

Figure 2.

Comparison of collagen fiber width and length in human vulva, urethra, and foreskin tissue with and without LS. (A) Representative 40x image demonstrating an area of subepithelial collagen deposition with superimposed curvelets from the CT-FIRE software package. Each colored curvelet is fit to an individual collagen fiber and analysis of these curvelets yield the data presented in the remainder of the figure. (B) Mean collagen fiber width was similar across all tissue types with respect to LS status (p=0.111). However, when individual tissue types were examined foreskin tissues with LS were found to have significantly increased collagen fiber width compared to non-LS tissues (0.877 vs 0.93 microns, p = 0.006). (C) Comparison of collagen fiber lengths across vulvar, foreskin, and urethral tissues did not display significant differences with regard to LS status. Brackets indicate pairwise comparisons with “*” indicating p <0.05. LS = lichen sclerosus. Scale bar = 50 μm.

Figure 3.

Comparison of inverse difference moment (IDM) and entropy in human vulva, urethra, and foreskin tissues with and without LS. There is a consistent decrease in IDM and increase in entropy in LS compared to non-LS tissues. This reflects decreased homogeneity and disorganization of collagen structure and may imply alternative pathways for collagen production and remodeling in LS. * indicates p<0.001 in pairwise comparisons.