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Diagnostic Aspects and Proteomic Anlysis of Uterine Flush Fluid from Mares with Endometritis

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Title: Diagnostic Aspects and Proteomic Anlysis of Uterine Flush Fluid from Mares with Endometritis
Author: Amorim, Mariana
Department: Department of Population Medicine
Program: Population Medicine
Advisor: Chenier, Tracey
Abstract: Endometritis is a common cause of infertility and early embryonic death in mares. Many mares fail to be diagnosed with endometritis despite different diagnostic tests available, especially in subclinical cases. The purpose of this study was to compare diagnostic methods for endometritis and identify the major proteins in endometrial flush fluids of mares with endometrial disease. Endometrial swab, low volume lavage, culture and biopsy were obtained in estrus and diestrus mares, and Kappa coefficient was used to determine the level of agreement among tests. Final diagnosis of endometritis was performed using a checklist where mares with two or more of five criteria (1. uterine fluid on ultrasound or excessive edema or history of subfertility; 2. one or more neutrophils per high power field on cytology; 3. cloudy LVL; 4. positive bacterial culture; and 5. active endometrial inflammation on biopsy) were considered positive. Proteins in supernatant were identified by LC-MS/MS after in-solution trypsin digestion and ranked in amounts based on spectral counting using Scaffold software. Endometritis was diagnosed in 35/44 mares by biopsy, and 33/51 based on the endometritis checklist. Endometrial culture was the least sensitive test (29.71%), and endometrial biopsy was the most sensitive method (85.71%) when the endometritis checklist was used as the Gold Standard. The optimal cut off-value was ≥1 neutrophils to epithelial cell ratio for classifying mares with endomodetritis on cytology. Over 2000 proteins were identified, and a subset was compared among the groups by 2-way ANOVA with SAS 9.3 software. Proteins that were decreased (p ≤ 0.05) in mares with endometritis included vanins 1-3, and connective tissue growth factor (CTGF). Major proteins that increased significantly in diestrus but were not associated with endometritis included P19 lipocalin (uterocalin), uteroferrin (TRAP5), secretory phospholipase A2 (sPLA2), and secretoglobin 1A1 (uteroglobin). These studies have shown that no single diagnostic test is sensitive enough to reliably diagnose mares with endometritis, and that we have identified several changes in the protein composition of uterine flush fluids in mares with endometritis. Some of these might become useful biomarkers in the clinical and biopsy assessment of uterine health in mares.
Date: 2014-01
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