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

Abstract

Background: The 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.

Methods: Endometrial 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.

Results: Next-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.

Conclusion: Our 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 differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE postpartum.

Fig. 1

Histological Analysis of Biospies Acccording to Leukocyte Infiltration into the Epithelial and Stromal Layers of the Uterus. a and c Representative histological classification of endometrial biopsies at 7 DPP shows significant immune cell infiltrate. b This influx of leukocytes is not apparent in HC cows, which have resolved inflammation by 21 DPP. d Cows that develop CE however, have sustained immune cell infiltrate in the endometrium at 21 DPP. Magnification shown in 400×

Fig. 2

Next-Generation Sequencing (NGS) of messenger RNA from Uterine Biopsies at both 7 and 21 DPP. a top panel High numbers of genes are differentially expressed in the endometrium from the HC (healthy) group between 7 and 21 DPP, whereas (bottom panel) the same transition in gene expression profile between time points is not present in cows that develop CE. The numbers of significantly differentially expressed genes between groups at both postpartum time points are shown (FDR < 0.1). Within group comparisons are shown in red boxes, and between group comparisons are in blue boxes. The numbers in green ovals show the direction in relative gene expression between comparisons. b MDS plots generated from endometrial RNA-seq data shows clearer clustering of 7 and 21 DPP time points for the HC group (top panel) than for the CE group (bottom panel). Nine HC (healthy control) endometrial samples at both 7 DPP (red) and corresponding same animal sample at 21 DPP (green) and b six CE (cytologically endometritic) cows at the same two time points are shown. Corresponding gene lists are shown in Additional file 2: Table S2

Fig. 3

Top 10 Significantly Enriched Biological Processes in the Endometrium Identified by Gene Ontological Analysis. Using significantly differentially expressed gene datasets, gene ontology analysis identifed the enriched biological processes (a) in HC cows between 7 and 21 DPP and b between HC and CE samples at 21 DPP. The resolution of the inflammatory response in HC cows (a) is evident as these defence and innate immune response processes are switched off. The sustained inflammatory response (lack of transition) is evident in (b) as these processes are enriched in CE compared to HC at 21 DPP. For full list of enriched gene ontologies, see Additional file 4: Table S4

Fig. 4

The Kyoto Encyclopaedia of Genes and Genomes (KEGG) annotated the Toll-Like Receptor Signalling Pathway as significantly enriched in the RNA-seq dataset for HC. Green boxes highlight the genes within the pathway that are significantly elevated in expression 7 DPP. For other significantly enriched pathways see Figure S1 (NFkB Signalling Pathway) and Figure S2 (Cytokine-cytokine receptor interaction). For full list of differentially expressed pathways see also Additional file 3: Table S3

Fig. 5

Heat map representation depicting the temporal changes in significantly differentially expressed genes from uterine biopsies between 7 and 21 DPP in the a HC and b CE cows. All 31 significantly DEGs were used to generate the heatmap in the CE group, and a similar number of the top DEGs (ranked on basis of P value) were used for comparative purposes for the HC group. Scale: Yellow indicates high expression and red is low expression. Unsupervised hierarchical clustering dendograms are included for these genes. a – DE genes between day 7 and 21 DPP in HC animals and b – DE genes between day 7 and 21 DPP in CE cows

Fig. 6

RT-qPCR validation of gene expression changes detected using mRNA-seq. Confirmation of differentially expressed genes from NGS results using quantitative real-time PCR. Significant changes in gene expression of both a pro- and anti-inflammatory cytokines as well as other b effector molecules of the immune response confirmed the findings from NGS. Results are colour-coded according to comparison and levels of expression of each gene of interest was normalised to expression levels of PPIA; D7PP between SCE and HC cows (black), D21PP between SCE and HC (blue), between D7 and D21PP in SCE (red) and between D7 and D21PP in HC (green). Between and within group comparisons are separated by a dotted line. *P < 0.05; **P < 0.01. n = 5–8 samples per time point, bars represent mean ± SEM

Fig. 7

Multi-Dimensional Scaling (MDS) Plots Generated from Endometrial microRNA-seq Data. a MDS-plot shown for HC cows at both 7 (red) and 21 DPP (green). Similarly, b MDS-plot shown for CE cows at both 7 (red) and 21 DPP (green). Clustering of the D7 and D21 profiles is apparent at both time points for HC and CE cows although tighter clustering of 7 DPP samples for the HC group than for the CE group (b) shows a higher degree of variation between samples at 7 DPP (b, red). Five HC (healthy control) samples at both 7 DPP (D7, red) and corresponding same animal sample at 21 DPP (D21, green) and B) five CE (cytologically endometritic) cows at the same two time points are shown

Fig. 8

Principal Coordinates Ordination of Endometrial Bacterial Communities. Bacterial community analysis, performed using culture-independent Terminal Restriction Fragment Length Polymorphism (T-RFLP), shows a significant clustering of samples according to their respective microbial communities. HC and CE cows are shown as blue and red circles, respectively

Fig. 9

Analysis of Systemic Immune and Metabolite Parameters in HC and CE cows. Significant early reductions in circulating granulocyte numbers in cows that subsequently developed CE. Absolute cell counts in Peripheral blood leukocytes (PBL) for a neutrophils and b eosinophils. Elevated plasma expression levels for c Haptoglobin (HP), d Serum Amyloid A (SAA) but not e Interleukin 6 (IL-6) were also detected in cows with CE. No significant differences in serum metabolite profile for f β-hydroxybutyrate (BHB), g non-esterified fatty acids (NEFA) and h Urea between cows that developed CE and HC, although temporal changes were significantly changed. Samples labelled as PC (pre-calving), and at 7 and 21 DPP in HC and CE cows.*P < 0.05; **P < 0.01. n = 5–9 samples per timepoint and bars represent mean ± SEM