Characterization of an ex vivo equine endometrial tissue culture model using next-generation RNA-sequencing technology

Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endo-metritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F2a as a biomarker. However, this model has yet to un-dergo a wide-ranging assessment through transcriptomics. In this study, we assessed the tran-scriptomes of cultured endometrial explants and the optimal temporal window for their use. En-dometrium harvested immediately post-mortem from native pony mares (n=8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNA-Later, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal Component Analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24-48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. STRING assessments of gene ontology suggested that DEGs between 24-48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction. Taken together our observations indicated that 24- 48 h is the optimal temporal window when the explant model can be used. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endo-metritis to persistent inflammation between 24-48 h. Overall design: In-depth transcriptomic assessment of endometrial explants over a culture period of 72 h