Use of endometrial swab and cytobrush for detection of inflammatory diagnostic markers of endometritis

Abstract

Screening methods for endometritis are often performed through endometrial culture and cytology with a variety of techniques including endometrial swab, cytobrush, low-volume lavage, or biopsy. With growing research interest in equine reproductive immunology and low sensitivity of current screening tools for endometritis, one area that would be of immediate benefit to clinicians and clients is inflammatory diagnostic development. Previous research detected inflammatory markers in low-volume lavage fluid in mares with chronic and acute endometritis (Lection et al. AAEP Proceedings. 2020; 66: 150-151). Therefore, our objective was to investigate the utility of the endometrial swab and cytobrush to screen mares for endometritis. Mares (n=84) of reproductive age ranging 3-20 years of age had an endometrial swab (n=103) and/or followed by cytobrush (n=94) taken once in the same estrous cycle and placed in Amies media (n=172) or PBS (n=25). Aerobic culture was performed in 139 of those samples. An endometrial biopsy was taken on a subset of mares (n=46) and graded by a board-certified veterinary pathologist. The media in which the cytobrushes and swabs were stored was used in a bead-based multiplex assay (Luminex Corp. Austin, TX) to quantify concentration of the following equine specific biomarkers: IFN-α, IFN-γ, IL-1β, IL-4, IL-10, IL-17, sCD14, TNF-α, CCL2, CCL3, CCL5, and CCL11. A Shapiro-Wilk test was used to check normality, followed by a Wilcoxon testperformed in JMP Pro 16 (Cary, NC) to assess for significant differences in inflammatory marker concentrations between healthy mares and those with either cytology-diagnosed endometritis (≥1 neutrophil/hpf), positive bacterial culture, or poor biopsy scores (IIB or III on the Kenney-Doig scale). IFN-γ (P≤0.02) and IL-17 (P≤0.04) were significantly increased in mares with poor biopsy scores in both swab and cytobrush compared to mares with biopsy I or IIA. Mares with inflammation on cytology showed increased concentration of IL-1β (P≤0.05) and decreased levels of IFN-γ (P≤0.02) and CCL5 (P≤0.03) in swab. Mares with intrauterine fluid on ultrasound had increased IL-17 (P≤0.05) on cytobrush. Bacterial growth was detected in 62/139 samples, with 31 of those being gram-positive. sCD14 and TNF-α tended to be increased in cytobrush samples from mares with a positive culture, and with gram-positive bacteria compared to gram-negative, respectively (P=0.06). Cytokine levels significantly differed between cytobrush and swab for IFN-γ (P≤0.004) with increased levels for cytobrush, likely due to the higher amount of cellular exfoliation. Several inflammatory markers show promise as an ancillary screening test for equine endometritis. In conclusion, as equine reproductive immunology expands as a field, our understanding of patterns of inflammatory cytokines and chemokines can allow for new diagnostic techniques for endometritis in mares.