Endometrial fibrosis in mares: Picrosirius Red and Collagen-2 and 3

Abstract

In mares, endometrial fibrosis results from abnormal collagen deposition in the stroma and around the endometrial glands. The pathogenesis of endometrial fibrosis remains unknown, but the different types of collagens are a fundamental part of the process. The Picrosirius Red (PSR) staining is widely studied and necessary for collagen fiber and tissue remodeling. The present study aimed to determine the collagen deposition and the gene expression of collagen-2(COL-2) and collagen-3(COL-3) in the endometrium of mares with different degrees of fibrosis. Cyclic mares (n = 34) with a mean age of 14 ± 6.4 years old were used. A uterine biopsy from each mare was obtained at diestrus (D5–D10). Mares were grouped according to the degree of uterine fibrosis described by Kenney (1978): Grade I (n = 12), Grade II (n = 12), and Grade III (n = 10). Collagen in the equine endometrium was morphometrically quantified under PSR staining. The morphometric quantification of the images was performed from each sample after digital microphotographs were taken at 200x magnification, processed, and evaluated using the ImageJ 1.52a software. COL-2 and COL-3 expression was performed using qPCR. Grades were considered independent factors. Collagen-stained area and transcript expression were considered dependent factors. Data were analyzed with ANOVA andt-test. A Pearson correlation was performed between age and collagen deposition. The percentage of tissue collagen area stained with PSR revealed a significant increase between all groups in collagen deposition according to the degree of fibrosis: Grade I (11.7 ± 1.4%), Grade II (17.8 ± 1.3%), and Grade III (24.1 ± 1.9%). Age and the percentage of collagen deposited in the endometrial tissue correlated (r = 0.5) (P = 0.015). Grade II samples presented higher expression of COL-2 transcripts compared to Grade I (P = 0.026) and Grade III (P = 0.003). However, COL-3 did not show differences between groups. This study agrees with other studies that indicate that the worsening of degenerative changes coincides with advanced mare age. Sample evaluations emphasizing degenerative changes rely on an experienced evaluator, while the PSR, followed by computerized analysis, presents quantitative data. In conclusion, the increase in PSR analysis in Grade II and Grade III mares compared to Grade I demonstrates that this technique could play an important role in routine analyses for the detection, quantification, and dynamics of collagen in specific tissues. The study revealed that COL-2 might be involved in the progression and differentiation of healthy endometrium into an endometrium with severe fibrosis.