Novel sampling procedure to characterize bovine subclinical endometritis by uterine secretions and tissue

Abstract

Subclinical endometritis (SE) in cattle is defined as clinically unapparent inflammation of the endometrium. It is reported to impair fertility in affected cows and causes economic loss within the dairy industry. A gold standard for diagnosis of SE has not been set. Uterine cytology and histopathology are both applied, but low agreement between these methods has been described. The objective of the present study was to assess the capability of uterine secretions (US) as a new medium for diagnosis of SE. A novel sampling tool was applied to retrieve US as well as cytological, histological and bacteriological samples of the endometrium after a singular passage through the cervix in 108 dairy cows (43-62 days post-partum [dpp]). To assess the quality of the US samples, a proteome analysis of samples from five healthy donors was performed, demonstrating that in vivo sampling of US was feasible and generated samples suitable for diagnostic purposes. Diagnosis of SE was realized by the combination of clinical, cytological, and histopathological findings. Quantitative analysis of pro- and anti-inflammatory cytokines (interleukin (IL)1B, IL6, IL8, IL17A, IL10) in US was conducted using AlphaLISA-technology. RNAlater-fixed endometrial biopsies were used for gene expression analysis of the cytokines IL1B, IL6, IL8, IL10 and tumor necrosis factor alpha (TNFα) as well as the prostaglandin-endoperoxide synthase 2 (PTGS2) and the antimicrobial peptide S100A9 by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Cows were assigned to groups according to their uterine health status. A large group of animals (n = 83) displayed no signs of endometritis (E.NEG). Cytological and histopathological examination revealed low agreement; hence, animals with SE were differentiated into SE(cyto) and SE(histo) groups (n = 7 and n = 13, respectively). One animal in group SE(cyto + histo) as well as four animals with signs of clinical endometritis (CE) were excluded from further analysis. SE(cyto) showed significantly higher median concentrations of IL1B, IL8 and IL17A in US as well as a significantly higher median expression of IL1B, IL8 and IL10 in endometrial biopsies compared to E.NEG. No significant differences were found for IL6 and IL10 in US and IL6, TNFα, PTGS2 and S100A9 in endometrial tissue between these groups. SE(histo) presented no differences concerning the analyzed parameters compared to E.NEG. In conclusion, a method to sample US was successfully established in dairy cows. The cytokines IL1B, IL8 and IL17A are promising candidates in diagnosing cytological endometritis by US. Further assessment of US might contribute to a better understanding of the pathological mechanisms leading to chronic endometrial inflammation and to impaired fertility in affected cows.

Keywords: Endometrial tissue; Inflammatory cytokine; PTGS2; S100A9; Uterine secretion.