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Metabolomics of bovine uterine fluid at the onset of conceptus elongation

ABSTRACT: The objective is to investigate changes in metabolomics of uterine lumen content of lactating dairy cows associated with the onset of conceptus (embryo and associated membranes) elongation. Lactating dairy cows had estrous cycles synchronized and were subjected to induced ovulation and timed artificial insemination (AI). The day of AI was considered study d 0. On d 15, uteri were flushed by transcervical catheterization and infusion of 20 mL of phosphate buffered solution with 0.1% of polyvinyl acetate. Recovered conceptuses were classified based on morphology/length as ovoid (OV; 1-4 mm), tubular (TUB; 5-19 mm) and filamentous (FIL; 20-85 mm). The first 20 mL infused in the uterus were recovered, placed in conical tubes and centrifuged at 2,000 × g at 4ᵒC. The supernatant was collect, aliquoted and stored at -80ᵒC for later analyses of fluid composition, including measurement of IFN-τ concentration. Cows with no conceptus recovered and no detection of IFN-τ in uterine flushing were considered as nonpregnant (NPREG). The experimental design was then considered a prospective cohort study with 4 independent groups (NPREG, OV, TUB, and FIL). The additional 5th group represents a specific physiological condition of cows within the study and it will be compared to TUB and FIL groups combined, working as a pilot study for future research.

ORGANISM(S): Cow Bos Taurus

TISSUE(S): Uterus

SUBMITTER: Eduardo Ribeiro

PROVIDER: ST000335 | MetabolomicsWorkbench | Sun Jan 17 00:00:00 GMT 2016

REPOSITORIES: MetabolomicsWorkbench

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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: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

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Metabolomics of bovine uterine fluid at the onset of conceptus elongation

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