Layer‐Specific Quantitative Assessment of Collagen Distribution in Equine Uteri Using Masson's Trichrome and Digital Image Analysis: A Comparative Study

ABSTRACT

Equine endometrial fibrosis is a chronic and progressive equine uterine condition characterized by fibrosis that affects mares' fertility, with a wide range of fertility outcomes being reported for these mares. Therefore, assessing layer‐specific collagen (COL) distribution is essential for a comprehensive understanding of endometrial remodelling and its impact on equine reproductive potential. Full‐thickness uterine samples, including stratum functionalis (SF), stratum basalis (SB) and myometrium, were collected post‐mortem from healthy control mares (Category I; n  = 10) and those with moderate endometrial fibrosis (Category IIB; n  = 10). Samples were analysed using Masson's trichrome and digital imaging. Significant differences between categories were identified exclusively within the superficial SF, where category IIB displayed higher COL area fraction (%Area) and Normalized Integrated Density than healthy controls. When evaluating variations across layers, both groups exhibited a higher %Area and Normalized Integrated Density in the SB compared to the SF, followed by a significant decrease in Normalized Integrated Density towards the myometrium. Furthermore, the Mean Grey Value progressively decreased into the deeper compartments, indicating higher staining intensity, only in the group with moderate fibrosis. In conclusion, layer‐dependent COL deposition underscores the value of multiparametric, quantitative approaches for an objective characterization of equine endometrial fibrosis.