Project description:Post-partum uterine inflammation (endometritis) is associated with lower fertility at both the time of infection and after the inflammation has resolved. It was hypothesized that aberrant DNA methylation may be involved in the sub-fertility associated with post-partum uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with sub-clinical endometritis. Endometrial tissues were obtained at 29 days post-partum (n=12) and Agilent two-colour microarrays were used to characterize transcription and DNA methylation profiles. Analyses revealed 1,856 probes to be differentially expressed in animals with subclinical endometritis (SUI, n=6) compared with control cows (NUI, n=6, P<0.05, Storey Multiple testing correction). No significant associations among DNA methylation and gene expression were detected. Further analysis of gene expression data using GeneGo Metacore and Gene Set Enrichment Analysis identified several pathways and processes enriched in the comparison. Several pathways that are involved in the innate immune response were enriched in SUI cows. Consistent with the presence of microorganisms in the uterus, there was enrichment for the Toll-like receptor (TLR) signaling pathway, including increased expression of the transcription factor NFKB1, the pro-inflammatory cytokines IL1A and IL1B, downstream chemokines, cytokines, and acute phase and antimicrobial proteins in the endometrium of SUI cows. Furthermore, the chemokine signaling pathway was enriched in SUI cows, with increased expression of genes that attract cells of the innate immune system. Increased expression of IL-8 and CXCL6, chemotactic factors for recruitment of neutrophils along with the immune cell surface marker PTPRC in SUI cows is consistent with the greater number of polymorphonuclear cells present in the uterus of these cows. Several antimicrobial peptides (LAP, TAP, DEFB1, DEFB10, DEFB103B, DEFB7) and acute phase proteins, including SAA3, LBP, and the complement gene CFB, had greater expression in SUI cows. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local pro-inflammatory environment in the uterus. If this period of inflammation is prolonged, it could result in tissue damage or failure to complete involution of the uterus, which may create a sub-optimal environment for future pregnancy. Agilent two-colour microarrays were used to characterize DNA methylation profiles in cows with subclinical endometritis (SUI, n=6) compared to control cows (NUI, n=6). Endometrial tissues (caruncular, intercaruncular) were obtained at 29 days post-partum.

Project description:Post-partum uterine inflammation (endometritis) is associated with lower fertility at both the time of infection and after the inflammation has resolved. It was hypothesized that aberrant DNA methylation may be involved in the sub-fertility associated with post-partum uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with sub-clinical endometritis. Endometrial tissues were obtained at 29 days post-partum (n=12) and Agilent two-colour microarrays were used to characterize transcription and DNA methylation profiles. Analyses revealed 1,856 probes to be differentially expressed in animals with subclinical endometritis (SUI, n=6) compared with control cows (NUI, n=6, P<0.05, Storey Multiple testing correction). No significant associations among DNA methylation and gene expression were detected. Further analysis of gene expression data using GeneGo Metacore and Gene Set Enrichment Analysis identified several pathways and processes enriched in the comparison. Several pathways that are involved in the innate immune response were enriched in SUI cows. Consistent with the presence of microorganisms in the uterus, there was enrichment for the Toll-like receptor (TLR) signaling pathway, including increased expression of the transcription factor NFKB1, the pro-inflammatory cytokines IL1A and IL1B, downstream chemokines, cytokines, and acute phase and antimicrobial proteins in the endometrium of SUI cows. Furthermore, the chemokine signaling pathway was enriched in SUI cows, with increased expression of genes that attract cells of the innate immune system. Increased expression of IL-8 and CXCL6, chemotactic factors for recruitment of neutrophils along with the immune cell surface marker PTPRC in SUI cows is consistent with the greater number of polymorphonuclear cells present in the uterus of these cows. Several antimicrobial peptides (LAP, TAP, DEFB1, DEFB10, DEFB103B, DEFB7) and acute phase proteins, including SAA3, LBP, and the complement gene CFB, had greater expression in SUI cows. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local pro-inflammatory environment in the uterus. If this period of inflammation is prolonged, it could result in tissue damage or failure to complete involution of the uterus, which may create a sub-optimal environment for future pregnancy. 12 animals were enrolled in this study, 2 samples from each animal were used for microarray analysis consisting of caruncular and intercaruncular endometrium. A reference design was used for hybridisation, whereby each of the 24 samples were hybridised to individual microarrays along with a pooled reference sample. The reference sample was generated by taking equal concentrations of each of the experimental samples.

Project description:Post-partum uterine inflammation (endometritis) is associated with lower fertility at both the time of infection and after the inflammation has resolved. It was hypothesized that aberrant DNA methylation may be involved in the sub-fertility associated with post-partum uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with sub-clinical endometritis. Endometrial tissues were obtained at 29 days post-partum (n=12) and Agilent two-colour microarrays were used to characterize transcription and DNA methylation profiles. Analyses revealed 1,856 probes to be differentially expressed in animals with subclinical endometritis (SUI, n=6) compared with control cows (NUI, n=6, P<0.05, Storey Multiple testing correction). No significant associations among DNA methylation and gene expression were detected. Further analysis of gene expression data using GeneGo Metacore and Gene Set Enrichment Analysis identified several pathways and processes enriched in the comparison. Several pathways that are involved in the innate immune response were enriched in SUI cows. Consistent with the presence of microorganisms in the uterus, there was enrichment for the Toll-like receptor (TLR) signaling pathway, including increased expression of the transcription factor NFKB1, the pro-inflammatory cytokines IL1A and IL1B, downstream chemokines, cytokines, and acute phase and antimicrobial proteins in the endometrium of SUI cows. Furthermore, the chemokine signaling pathway was enriched in SUI cows, with increased expression of genes that attract cells of the innate immune system. Increased expression of IL-8 and CXCL6, chemotactic factors for recruitment of neutrophils along with the immune cell surface marker PTPRC in SUI cows is consistent with the greater number of polymorphonuclear cells present in the uterus of these cows. Several antimicrobial peptides (LAP, TAP, DEFB1, DEFB10, DEFB103B, DEFB7) and acute phase proteins, including SAA3, LBP, and the complement gene CFB, had greater expression in SUI cows. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local pro-inflammatory environment in the uterus. If this period of inflammation is prolonged, it could result in tissue damage or failure to complete involution of the uterus, which may create a sub-optimal environment for future pregnancy.

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

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Transcriptional response of endometrium and the bovine preimplantative embryo to subclinical inflamation of the uterine environment

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