Confocal laser endomicroscopic imaging of equine endometrium: evaluating image quality

Abstract

A heathy uterus is a key piece of the fertility jigsaw and assessment of the endometrium is an essential component of the breeding soundness examination in mares. The current gold standard for evaluating uterine health is histopathological assessment of an endometrial biopsy. The Kenney-Doig (KD) grading scale is most used and scores both fibrotic and inflammatory changes present within the endometrium. The KD grade provides a prognostic indicator of the probability a mare can carry a pregnancy to full-term. Inconsistencies may arise when a single blinded biopsy is used, especially when uterine pathologic changes don't have a widespread distribution pattern. Confocal laser endomicroscopy (CLE) is a novel imaging modality that enables real-time in-vivo imaging of the endometrium. Multiple subcellular endometrial images (“virtual biopsies”) can be captured in quick succession in multiple locations within the uterine lumen noninvasively. Image depth in the z-axis can be controlled (0-100µm). Image quality was noted to be variable between mares in a previous pilot study (Gallacher.et.al. Journal of Equine Veterinary Science. 2018;66:106). The objective was to determine if physiological endometrial oedema present during oestrus has a negative effect on image quality. Twenty-four mares were examined with CLE (ViewnVivo, Optiscan, Australia) on two separate occasions, once during oestrus and once in dioestrus. A total of 4294 CLE images were captured, witha mean of 89.5 images saved per exam. A 4-point grading scale (1=poor and 4=excellent) was developed for quality assessment. A subset of ∼10 representative images from each of the 48 examinations were selected. Four examiners were blinded to the phase of the oestrous cycle and graded the images using the quality criteria assigned to the 4-point grading scale. An intraclass correlation (ICC) was estimated to determine reliability between the 4 scorers. The ICC coefficient was estimated using the irr package in R. Moderate agreement (ICCC(C,4) = 0.69) between scorers was observed while using the two-way random effect models and average rater where p<0.001. Quality data were analysed using a mixed model in asreml-R. The fixed factors included in the model were depth (1-luminal epithelium, 2-subluminal), cycle (oestrus and dioestrus) with brightness included as a covariate. Scorer and horse were included as random terms. There was a difference (p<0.001) between depth 1 and 2 (2.51 vs 2.71 respectively), and cycle (p<0.001) dioestrus = 2.71 oestrus = 2.51. The brightness of the image was also related to quality score (p<0.001) where a 100unit increase in brightness was associated with a reduction in quality score of ∼0.3. Image quality appears to be influenced by the phase of the oestrous cycle and this may guide the clinician to perform CLE examination during dioestrus.