Proteomic profiling of tissue and plasma from mares with endometrial fibrosis

Endometrial fibrosis causes subfertility in mares, and is characterized by excessive deposition of extracellular matrix. It is diagnosed by a histopathologic evaluation of a biopsy, but the prognostic value of a single biopsy is limited, and a better understanding of the pathogenesis is needed. To gain a better understanding of protein dynamics underlying this disease, Data-Independent Acquisition (DIA) Mass Spectrometry was utilized to comprehensively character-ize and compare the equine endometrial and plasma proteome from these mares. Mares in die-strus were selected as controls (n=9), with moderate endometrial fibrosis (n=9), and severe endometrial fibrosis (n=9), based on tissue sections stained with hematoxylin and eosin and immunostained for alpha-smooth muscle actin. Approximately 10,000 proteins were identified in endometrial tissue and 500 in plasma, from which 310 and 75 were significantly regulated between moderate endometrial fibrosis and controls, and 585 and 71 between severe endome-trial fibrosis and controls. Pathways involved in extracellular matrix were over-represented among upregulated proteins in endometrial tissue and among downregulated proteins in plasma from moderate and severe endometrial fibrosis compared to controls. Several of the proteins involved appeared to be driving the development or progression of endometrial fibrosis. Path-ways involved in the immune response were over-represented among downregulated proteins in moderate endometrial fibrosis, and among upregulated proteins in severe endometrial fibro-sis, in both endometrial tissue and plasma. Several of the proteins highlighted by our analysis appear to have both immunologic and fibrotic properties. This extensive profiling of the endo-metrial and plasma proteome paves the way for further research in potential biomarkers and candidates for therapeutic use in endometrial fibrosis.