Histomorphometric characterization of the endometrium in mules (Equus mulus): An approach to...

Mules (Equus mulus), as sterile hybrids between mares and donkeys, present a unique uterine morphology and physiology that remains poorly characterized. This study provides the first histomorphometric evaluation and transcriptional profiling of the endometrium in adult mules. To achieve this, endometrial biopsies were analysed using quantitative stereology, histological classification (Kenney-Doig system), and qPCR of key immune-related and fibrotic genes. Histometric analyses revealed that the stratum spongiosum was the predominant component of the mule endometrium (mean volume density: 84.7 %), whereas the epithelium and compact layers accounted for smaller proportions. This distribution, more pronounced than what is typically reported in mares, suggests enhanced stromal expansion or edema. Endometrial glands were abundant, and the overall structural profile exhibited an intermediate phenotype with a clear asinine bias. Most samples were classified as grades I, IIA, or IIB, with no specimens reaching grade III. Mild to moderate stromal remodeling, preserved tissue architecture, and inflammatory signatures -characterized by increased relative abundance of IL1B, IL6, and TNFα transcripts- were observed in higher grades and were consistent with early stages of endometrial degeneration. Although these features indicate early degenerative changes, the hybrid reproductive physiology of mules, including limited hormonal cyclity, may contribute to reduced progression towards fibrosis. This integrated histological-molecular assessment reinforces the diagnostic value of the Kenney-Doig system in mules and supports the adaptation of a refined IIC grade to better capture hybrid-specific features. Overall, the mule endometrium displays distinct yet comparable traits relative to that of horses and donkeys, highlighting its potential as a comparative model for equine reproductive research. These findings provide a foundation for improved clinical decision-making and future studies on hybrid fertility and uterine health.