Project description:Co-ordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A non-receptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. To gain support for this hypothesis the correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and sub-fertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with transcription profiles which have been previously reported. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. The 1,000 most significant correlations were used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when comparing pregnant and cycling animals and 11% were differentially expressed comparing pregnant fertile and sub-fertile animals. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies. The estrous cycles of 24 lactating dairy cows were synchronized (at 58.8 (SEM 3.77) and 60.2 (SEM 1.51) days post calving in dairy cows of sub-fertile and fertile strains, respectively) and 14 received a single embryo transferred on day 7 of the estrous cycle. Animals were slaughtered at day 17 of the reproductive cycle and endometrial tissues (both caruncular and intercaruncular) were sampled. Selection criteria for the study included strain and calving date, and health postcalving was an exclusion criterion (cows with severe uterine infections or mastitis were excluded before being enrolled in the embryo transfer round). Cows in each strain were matched for calving number and age. A total of 10 cycling and 12 pregnant animals enrolled in the study were utilized, due to the associated costs of slaughtering the cows. These animals represented fertile (six pregnant and five cycling Holstein-Friesian cows with New Zealand ancestry/M-bM-^IM-$30% North American genetics, n=11, NZ) and sub-fertile (six pregnant and five cycling Holstein-Friesian cows with >87% North American ancestry, n=11, NA) phenotypes of Holstein-Friesian dairy cows

Project description:The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility post-partum. 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 post-partum dairy cow. Here, next-generation sequencing from endometrial biopsies taken at 7 days post-partum (DPP) identified significant expression of inflammatory genes in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor, NF?B and TNF signalling pathways, 73 genes and 31 miRNAs differentiated between healthy cows (HC, n=9) and cows which subsequently developed CE at 7 DPP (n=6, FDR<0.1). In healthy cows, 4197 differentially expressed genes between 7 and 21 DPP whereas only 31 genes were differentially expressed in samples from cows with CE. At 21 DPP, a further 1167 genes were differentially expressed between HC cows and cows diagnosed with CE (FDR<0.1). These changes in host gene expression reflected culture-independent microbiological analysis which showed significant differences in uterine bacterial profiles between groups. Inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) plasma expression levels were detected at 7 DPP in cows that developed CE. In conclusion, our data suggests that the major inflammatory cascade activated early post-partum 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 inflammatory profile, differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE post-partum. Sixteen Holstein Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking, over an eight week period.

Project description:The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility post-partum. 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 post-partum dairy cow. Here, next-generation sequencing from endometrial biopsies taken at 7 days post-partum (DPP) identified significant expression of inflammatory genes in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor, NFκB and TNF signalling pathways, 73 genes and 31 miRNAs differentiated between healthy cows (HC, n=9) and cows which subsequently developed CE at 7 DPP (n=6, FDR<0.1). In healthy cows, 4197 differentially expressed genes between 7 and 21 DPP whereas only 31 genes were differentially expressed in samples from cows with CE. At 21 DPP, a further 1167 genes were differentially expressed between HC cows and cows diagnosed with CE (FDR<0.1). These changes in host gene expression reflected culture-independent microbiological analysis which showed significant differences in uterine bacterial profiles between groups. Inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) plasma expression levels were detected at 7 DPP in cows that developed CE. In conclusion, our data suggests that the major inflammatory cascade activated early post-partum 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 inflammatory profile, differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE post-partum. Sixteen Holstein Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking, over an eight week period.

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