Project description:Cattle with subclinical endometritis (SCE) are sub-fertile, but there is no predictive or diagnosis tool for subclinical uterine disease. The hypothesis for this study was that endometrial inflammation is reflected in peripheral blood leucocytes gene expression. Transcriptome patterns in healthy cows and in cows with SCE at 45-55 days postpartum were evaluated using circulating white blood cells and endometrial biopsies samples collected from the same animals. Bioinformatics analyses of microarray-based transcriptional data identified gene profiles associated with distinct biological functions in circulating white blood cells and endometrium. In circulating white blood cells SCE promote a pro-inflammatory environment related to the activation of inflammation, whereas in the endometrium functions also related to tissue remodeling. Nineteen differentially expressed genes were altered both in circulating white blood cells and in the endometrium of SCE cows compared with healthy cows. Among these genes, transcript abundance of immune factors C3, C2, LTF, PF4 and TRAPPC13 were up-regulated in SCE cows at 45-55 days postpartum. Moreover, the mRNA expression of C3, CXCL8, LTF, TLR2 and TRAPPC13 was temporally regulated during the postpartum period in circulating white blood cells from healthy cows compared with SCE cows. This observation might indicate an advantageous activation of the immune system in healthy animals. The transcript abundance of these genes might also be used as an indicator for subsequent postpartum uterine health.
Project description:Background: Physiological inflammation of the uterus postpartum is essential for the reparative processes of involution after calving. In the majority of cows, this inflammation is resolved and homeostasis is restored. However, in a significant subset, inflammation persists and contributes to tissue damage, pathology and subfertility. Transcriptomic differences of immune genes between cattle that resolve inflammation and those that develop uterine disease have been detected as early as 7 days postpartum (DPP) suggesting that the host immune response plays an important role in disease outcome. Results: Here, we extensively characterise the immune response at the transcriptomic level in endometrial epithelial cells from post-partum dairy cows phenotyped for both clinical and sub-clinical forms of uterine disease. We address the hypothesis that excessive expression of endometrial inflammatory molecules contributes to development of endometritis. Classification of cattle (n=112) as healthy or with uterine disease (purulent vaginal discharge; PVD and cytological endometritis; CYTO) was based on vaginal mucus score and >18% polymorphonuclear cell infiltrate into the endometrium at 21 DPP. RNA-seq analysis of endometrial epithelial cells collected using cytobrushes identified differential expression of 294 genes (FDR <0.05) between cows that subsequently resolved inflammation (n=10) and those that developed disease (n=20). Pathway over-representation analysis of differentially expressed genes (DEG) identified significant changes in immune-related pathways, including the NOD-like receptor signalling pathway, cytokine-cytokine receptor interaction pathway and the Toll-like receptor signalling pathway which were up-regulated in cattle that subsequently developed disease. The majority of the DEG were upregulated in cows that developed PVD, and included all genes upregulated in CYTO cows, suggesting a core inflammatory gene signature early post-partum contributes to the onset of uterine disease. This inflammatory signature was validated by qPCR in an independent group of cows (n=56) and included upregulation of pro-inflammatory genes (including TLR2, TLR4, NLRP3, IL1A, IL1B, IL8, and S100A8) at day 7 postpartum in cows that failed to resolve inflammation. Conclusions: Despite a large amount of inter-animal heterogeneity, these results suggest that excessive activation and inappropriate regulation of the inflammatory response early postpartum is a key feature of the subsequent development of uterine disease. Keywords: Endometritis, Inflammation, Transcriptome, Next generation sequencing, Dairy cattle, Uterine involution, Immune response
Project description:The impact of CRTAM modulation on the balance between gut microbiota and the mucosal immune system under both homeostatic conditions and during enteric infections
Project description:In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, which exerts profound detrimental effects on subsequent reproductive performance. So far, transcriptomic studies related to this condition were either based on biopsy-derived whole endometrium tissue or endometrial swab/cytobrush samples, thus neglecting cell type-specific variations in gene expression. This study tested the hypothesis that different endometrial health statuses are associated with distinct transcription profiles of endometrial stromal, glandular and luminal epithelial cells. In conclusion, this study evidences that endometrial inflammation recovery or persistence is associated with gene expression patterns involved in immune function, tissue remodelling, and uterine receptivity in a cell type-specific manner. Identifying these signatures may prove instrumental to developing novel diagnostic and therapeutic targets, either to prevent persistence or speed recovery from endometrial inflammation, thus restoring the fertility of postpartum dairy cows.
Project description:All cows experience bacterial contamination and tissue injury in the uterus postpartum, instigating a local inflammatory immune response. However mechanisms that control inflammation and achieve a physiologically functioning endometrium, while avoiding disease in the postpartum cow are not succinctly defined. This study aimed to identify novel candidate genes indicative of inflammation resolution during involution in healthy beef cows. Previous histological analysis of endometrial inflammation showed a great degree of inflammation 15 days postpartum (DPP) which significantly decreased by 30 DPP. The current study generated a genome-wide transcriptomic profile of endometrial biopsies at both time points using mRNA-Seq. The pathway analysis tool GoSeq identified KEGG pathways enriched by significantly differentially expressed genes elevated at both time points. Novel candidate genes of inflammatory resolution were subsequently validated in additional postpartum animals using quantitative real-time PCR (qRT-PCR).
Project description:Dairy cow subfertility is a worldwide issue arising from multiple factors. It manifests in >30% early pregnancy losses in seasonal pasture-grazed herds, especially when cows are inseminated in the early post-partum period. Most losses occur before implantation, when embryo growth depends on factors present in maternal tract fluids. Here we used label-free LC-MS/MS to examine the proteomic composition of uterine luminal fluid in 87 day-7 pregnant cows to identify potential biomarkers of fertility. We also determined changes in uterine luminal fluid from first to third oestrus cycles postpartum in individual cows and linked those changes with divergent embryo development. Out of 1563 proteins detected, 472 had not been previously reported in this fluid, and 408 were predicted to be actively secreted by bioinformatic analysis. The abundance of 19 proteins with roles in immune regulation and metabolic function was associated with contrasting embryo quality (e.g. cystatin B, pyruvate kinase M2). Matched-paired pathway analysis indicated that, from first to third oestrus postpartum, upregulation of metabolic (e.g. creatine and carbohydrate) and immune (e.g. complement system regulation and antiviral defence) processes were related to poorer quality embryos in the third oestrus cycle postpartum. Conversely, upregulated signal transduction and protein trafficking appeared related to improved embryo quality in third oestrus. These results advance the characterisation of the molecular environment of bovine uterine luminal fluid and may aid in understanding fertility issues in other mammals, including humans.
Project description:All cows experience bacterial contamination and tissue injury in the uterus postpartum, instigating a local inflammatory immune response. However mechanisms that control inflammation and achieve a physiologically functioning endometrium, while avoiding disease in the postpartum cow are not succinctly defined. This study aimed to identify novel candidate genes indicative of inflammation resolution during involution in healthy beef cows. Previous histological analysis of endometrial inflammation showed a great degree of inflammation 15 days postpartum (DPP) which significantly decreased by 30 DPP. The current study generated a genome-wide transcriptomic profile of endometrial biopsies at both time points using mRNA-Seq. The pathway analysis tool GoSeq identified KEGG pathways enriched by significantly differentially expressed genes elevated at both time points. Novel candidate genes of inflammatory resolution were subsequently validated in additional postpartum animals using quantitative real-time PCR (qRT-PCR). Endometrial biopsies were collected as part of a previous study 15 and 30 days postpartum (DPP) from 13 mixed breed beef multiparous cows. The endometrial transcriptomic profiles from endometrial biopsies were assessed by mRNA-Seq (n=3) and candidate gene expression was measured by qRT-PCR (n=5) comparing 15 DPP to 30 DPP samples. Reads were mapped to the bovine genome with TopHat, Htseq-count summarized the number of aligned reads per exon and EdgeR normalized the data and returned significantly differentially expressed genes. GoSeq identified KEGG pathways enriched by significantly differentially expressed genes elevated at both time points.
Project description:Infection with non-cytopathic bovine viral diarrhea virus (ncpBVDV) is associated with uterine disease and infertility. This study investigated the influence of ncpBVDV on immune functions of the bovine endometrium by testing the response to bacterial lipopolysaccharide (LPS) at the level of whole-transcriptomic gene expression. Analysis showed that approximately 30% of the 1,006 genes altered by LPS are involved in immune response. Many innate immune genes that typically respond to LPS were inhibited by ncpBVDV including those involved in pathogen recognition, inflammation, interferon response, chemokines, tissue remodeling, cell migration and cell death/survival. Infection with ncpBVDV can thus compromise immune function and pregnancy recognition thereby potentially predisposing infected cows to postpartum bacterial endometritis and reduced fertility.