Equine endometritis is one of the most common and important reproductive conditions causing infertility in mares. After mating there is a normal, transient inflammation in response to sperm, seminal plasma and bacteria in the uterus, termed mating-induced endometritis (MIE). Mares are classified as susceptible to persistent mating-induced endometritis (PMIE) when they fail to clear the uterus from MIE within 36-48 hours post mating. PMIE creates an adverse uterine environment, resulting in subfertility due to inflammation and disruption of the endocrine maintenance of pregnancy. An equine endometrial explant system was previously established to measure uterine inflammation via Prostaglandin F2α as a biomarker. However, it was not determined if the transcriptome from explants was altered once in culture. The aim of this study was to characterise the genetic profiles of equine endometritis in vitro to better understand the transition from MIE to PMIE. The objectives were 1) to determine if the transcriptome of cultured endometrial explants collected from native pony mares represent the transcriptome of the whole mare in the prebreeding, non-inflammatory state and thus, determine how the transcriptome of explants is modulated once in culture; 2) compare the transcriptome profiles from unchallenged cultured explants from the follicular, luteal and anoestrous phases to characterise the global uterine transcriptomic changes throughout the oestrous cycle and anoestrous; 3) determine the RNA-Sequencing (RNA-Seq) changes in cultured explants after challenge with E. coli lipopolysaccharide (LPS) to better understand transcriptome modulation during inflammation; 4) compare the transcriptome profiles of mares resistant and susceptible to PMIE before breeding. Endometrium was collected from mares sent to a commercial abattoir, during different stages of the oestrous cycle or anoestrous, and explants established as appropriate to address each objective. RNA-Seq was performed for all experiments and differentially expressed genes (DEGs) were investigated in terms of significantly enriched biological pathways. In vitro explants cultured for 24 hours demonstrated significant transcriptomic changes compared to the ex vivo 0 hours biopsies, but thereafter remained similar up to 48 hours in culture. DEGs related to inflammation differing between 0 and 24 hours were set as the baseline changes between the ex vivo biopsies and the in vitro explants. The gene expression of innate immunity genes was greatest during the follicular phase of the oestrous cycle, followed by the anoestrous period and lower during the luteal phase in accordance with the endocrine milieu. Cultured explants did not exhibit large scale gene expression changes in response to LPS challenge and it was not in agreement with other studies. In addition, the transcriptome of resistant and susceptible mares showed different expression profiles of immune genes even before breeding. The thesis investigated the use of an explant tissue culture system as a future model for studies of equine endometritis at the level of the transcriptome. Innate immuneassociated genes exhibited increased expression during the follicular phase compared to the luteal phase of the oestrous cycle and anoestrous period. Furthermore, four genes were suggested to have the potential of distinguishing mares likely to be resistant or susceptible to PMIE, which may be identified in practice at pre-breeding examination.
The Ex Vivo and In Vitro Transcriptomic Profiles of Equine Endometrium Using an Explant Model to Further Study Endometritis
De Barros, M. (Author). 2019
Student thesis: Doctoral Thesis › Doctor of Philosophy