Background: Clinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42-60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS).

In the present study, we used an in vitro model to investigate the response of the oviduct with respect to inflammatory mediators and their regulatory microRNAs in case of bacterial infection and subsequent association with embryo survival. For this, we conducted two experiments. In the first experiment, cultured primary bovine oviductal cells (BOEC) were challenged with lipopolysaccharide (LPS) for 24h and the temporal expression pattern of inflammatory mediators and their regulatory microRNAs were measured at 0, 3, 6, 12, 24 and 48h after LPS treatment. Intriguingly, the temporal patterns of all miRNAs except miR-21 were significantly up-regulated at 6h after LPS treatment. Whereas, we observed significant overexpression of pro-inflammatory mediators as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) after LPS challenge for 24h. On the other hand, the expression level of essential elements like oviductal glycoprotein 1 (OVGP1) and insulin-like growth factor 2 (IGF2) was significantly decreased in challenged groups compared with control. Moreover, miR-155, miR-146a, miR-223, miR-21, miR-16 and miR-215 have shown a clear suppression in challenged group after LPS treatment. In the 2nd experiment there were four groups of blastocysts produced, namely embryo+LPS free media, embryo+LPS, BOEC+embryo and BOEC+embryo+LPS. The suboptimal oviduct environment due to LPS challenge is found to have a significant influence on the expression of inflammatory response genes (TNFα and CSF1), stress response genes (SOD and CAT), mitochondrial activity, reactive oxygen species (ROS) accumulation and apoptotic level either in cultured or co-cultured blastocysts. Collectively, LPS challenge led to aberrant changes in oviductal transcriptome profile, which could lead to a suboptimal environment for embryo development.

An impaired uterine environment triggered by the incidence of subclinical endometritis often compromises fertility in the bovine. The uterus is a dynamic organ with tight regulation of specific genes at the transcriptional and translational levels. Herein, we hypothesised that subclinical endometritis alters the expression of uterine microRNAs (miRNAs), which may result in the dysregulation of corresponding target genes and biological pathways. To test this hypothesis, we used a genome-wide RT(2) (Exiqon, Vedbaek, Denmark) miRNA PCR array consisting of 354 miRNA primers and analysed miRNA expression in uterine cytobrush samples taken from cows with and without subclinical endometritis. The results revealed aberrant expression of 23 miRNAs in cows with subclinical endometritis compared with healthy cows. Furthermore, we designed an in vitro endometrial cell culture model challenged by lipopolysaccharide (LPS) to validate the differential regulation of miRNAs in cytobrush samples. Interestingly, we observed similar expression miRNA patterns in cytobrush samples taken from cows with or without subclinical endometritis and in vitro cultured endometrial cells challenged by LPS. To trace signalling pathways and biological functions potentially controlled by the aberrantly expressed miRNAs, we filtered high-ranking target genes from miRBase and analysed them using ingenuity pathway analysis. The gene networks, canonical pathways and biological functions strikingly converged to signalling pathways that mediate inflammatory responses, cellular proliferation, cell movement, the cell cycle and apoptosis in the bovine endometrium. In addition, expression analysis of key genes from the gene networks confirmed their presence and the potential regulation of these genes by uterine miRNAs. Furthermore, luciferase assay data substantiated the primary information from bioinformatic prediction that generated potential target genes for the dysregulated miRNAs in subclinical endometritis. Together, these data suggest the potential regulatory role of uterine miRNAs in the development and progression of bovine subclinical endometritis.

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