Endometrial gene expression of DEFB4B, LYZ, and SLPI in biopsy samples of mares resistant or susceptible to endometritis

Abstract

Endometritis is one of the main causes of subfertility in mares and therefore one of the most common reasons for economic losses in the horse breeding industry. Multiparous older mares are more susceptible to the development of persistent endometritis after mating, artificial insemination, or foaling since aging is associated with an altered systemic immune response and anatomical defects that interfere with physiological uterine clearance (Katila et.al. Animals. 2022; 12:779). In this context, their inability to recover from endometritis can lead to degenerative changes in the endometrium (e.g., glandular degenerative changes and associated peri-glandular and/or diffuse stromal fibrosis) (Canisso et.al. International Journal of Molecular Sciences 2020; 21:1432). This study aimed to evaluate possible variations in endometrial gene expression of the genes Defensin Beta 4B (DEFB4B), Lysozyme (LYZ), and Secretory Leukocyte Peptidase Inhibitor (SLPI) between resistant and susceptible mares. These three genes have already been identified as involved in the equine innate immune response and in the chronic uterine inflammatory response (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). Endometrial biopsy samples were collected from 19 mares. Mares were divided into two groups concerning their age and reproductive history: the "resistant group" (RES) (N=4) consisted of maiden mares 3.00±0.00 years old with endometrial biopsy grade I according to Kenney and Doig classification (1986); the "susceptible group" (SUS) (N=15)was composed of multiparous mares 17.33±3.28 years old, with endometrial biopsy of grade IIA, IIB or III. Total RNA was extracted from each sample and the corresponding cDNAs were prepared by retro-transcription using dedicated commercial kits. The samples were then amplified by qRT-PCR procedure as described by Marth and colleagues (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). The housekeeping gene GADPH was amplified in each sample as internal control and for data normalization. To minimize possible experimental errors, for each sample three technical replicates were performed and the quality of the extracted RNA was evaluated by spectrophotometry and gel electrophoresis. The results showed a statistically significant difference in the expression of the genes LYZ (p=0.0003) and DEFB4B (p=0.0273) which were upregulated in SUS versus RES mares. This suggests that lower gene expression found in young mares are attributable to a more balanced local immunomodulation, with consequent reduction of the inflammatory pathways and slower progression towards the deposition of fibrotic tissue compared to older mares. No differences in the expression of the SLPI gene were found between the two groups (p=0.8854). In conclusion, despite these promising results, larger studies are needed before LYZ and DEFB4B genes could be used as biomarkers to identify the mares susceptible to endometritis.