Integrated analysis of the local and systemic changes preceding the development of post-partum cytological endometritis

Abstract

BackgroundThe regulation of endometrial inflammation has important consequences for the resumption of bovine fertility postpartum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the postpartum dairy cow.MethodsEndometrial biopsy RNA was extracted from 15 cows at 7 and 21 days postpartum (DPP), using the Qiagen RNeasy® Plus Mini kit and quality determined using an Agilent 2100 bioanalyser. Disease status was determined by histpathology based on inflammatory cell infiltrate. RNA-seq of both mRNA and miRNA libraries were performed on an Illumina® HiSeq™ 2000. Paired reads were aligned to the bovine genome with Bowtie2 and differentially expressed genes were identified using EdgeR. Significantly over-represented Gene Ontology terms were identified using GO-seq, and pathway analysis was performed using KEGG. Quanititative real-time PCR was also performed for validation (ABI 7500 fast). Haematology was assessed using an automated ADVIA 2120 analyser. Serum proteins were evaluated by ELISA and metabolite analysis was performed using a Beckman Coulter AU 400 clinical analyser. Terminal-restriction fragment length polymorphism (T-RFLP) was used to obtain fingerprints of the microbial communities present.ResultsNext-generation sequencing from endometrial biopsies taken at 7 DPP identified significant induction of inflammatory gene expression in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor and NFκB pathways, 73 genes and 31 miRNAs were significantly differentially expressed between healthy cows (HC, n = 9) and cows which subsequently developed CE at 7 DPP (n = 6, FDR < 0.1). While significant differential expression of 4197 genes in the transcriptome of healthy cows between 7 and 21 DPP showed the transition from a proinflammatory to tissue profliferation and repair, only 31 genes were differentially expressed in cows with CE (FDR < 0.1), indicating the arrest of such a transition. A link betwene the dysregulated inflammatory response and the composition of the uterine microbial communities was suggested by the presence of significant differences in uterine bacterial tRFLP profiles between HC and CE groups. Furthermore, inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) expression levels were detected in plasma at 7 DPP in cows that developed CE.ConclusionOur data suggests that the IL1 and IL17 inflammatory cascade activated early postpartum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common early inflammatory profile, elevated and differe...

Analysis of transcriptomic changes in bovine endometrial stromal cells treated with lipopolysaccharide

“…Previous studies on RNA-seq analysis in mixed bovine epithelial and stromal endometrial cells treated with LPS showed 20 differentially-expressed miRNAs and 108 DEGS, as well as enriched 118 GO and 66 KEGG pathways connected to them [14]. In addition, a previous study on RNA-seq analysis of change in miRNA and mRNA expression in 15 cows at 7 and 21 days postpartum showed 4197 DEGs in healthy cows, while only 31 genes were differentially expressed in cows with cytological endometritis from proinflammatory to hyperplasia stages [15]. Other studies have been performed on RNA-seq analysis of effects of LPS on whole transcriptome of bovine endometrial epithelial cells with focus on genes involved in embryo-maternal interactions.…”

Uterine inflammation affects the reproductive performance of dairy cows: A review

“…A successful immune response in a healthy cow is able to resolve the inflammatory state postpartum to a regular or homeostatic state within a period of one to three weeks. Cows that develop endometritis or SCE are not able to do this and maintain the inflammatory response for a longer period (Foley et al, 2015).…”

Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle

“…IL-8 and acute phase proteins e.g. α1-Acid Glycoprotein (AGP), produced by endometrial epithelial cells and local immune populations are increased in inflamed uterine tissue and in vitro models [ 6 – 8 ]. These inflammatory biomarkers have been detected in uterine mucus and CVM [ 8 – 10 ].…”