Fluorescence Spectroscopy for the Diagnosis of Endometritis in the Mare

SIMPLE SUMMARY: A new method is proposed for the diagnosis of equine endometritis, one of the main pathologies affecting the fertility of this species. Cytological diagnosis is commonly based on the microscopic identification of polymorphonuclear cells (PMNs) among the cells recovered from the uterus; when the PMN/endometrial cells ratio exceeds a threshold level, the diagnosis of endometritis may be achieved. PMNs are cells characterized by the content of enzymes capable of producing high quantities of reactive oxygen species (ROS) that are used to neutralize pathogens. Using ROS-sensitive fluorescent labeling, the incidence of ROS in uterine cells has been evaluated with a spectrofluorometer. Following preliminary tests carried out on endometrial cell samples collected from slaughtered animals and mixed with blood leukocytes, calibration lines were obtained for the relative fluorescence intensity. The study was, then, moved to field conditions on mares that were submitted to a uterine flushing before insemination for performing microbiological tests, endometrial cell smears, and spectrofluorometer reading. The here proposed method based on fluorescence spectroscopy demonstrated a high relationship with the microscopic cytological technique, offering easy practicality and speed of execution. ABSTRACT: By exploiting the PMN property to produce high quantities of oxygen peroxide to neutralize pathogens, the oxygen peroxide content of uterine cells was measured to diagnose endometritis. After preliminary in vitro studies in which endometrial cells from slaughtered mares were mixed with leukocytes from peripheral blood, endometrial samples were collected by uterine flushing from mares before insemination. Staining endometrial cells with H(2)DCF-DA was combined with hydroethidine to normalize the fluorescence intensity with the cellular content of the sample. Stained cell smears were assumed as the gold standard of endometritis, and based on this assay, the samples were considered positive (C+) and negative (C−) for endometritis. The amount and the turbidity of fluid recovered by uterine flushing were significantly (p < 0.01) higher in C+ than in C−. Moreover, the oxygen peroxide content of the endometrial cells was significantly higher in the C+ than in the C− group (6.31 ± 1.92 vs. 3.12 ± 1.26, p = 0.001). Using the value of 4.4 as the cutoff level of this fluorescence cytology assay, it was found that only one C− sample exceeded the cutoff level (false positives = 7.7%) while three C+ samples showed values below the cutoff level (false negative = 11.5%).