An Integrated Investigation of Uterine Disease

Teagasc researchers and University partners are developing biomarkers for the early detection of uterine disease.
calendar icon 28 May 2013
clock icon 6 minute read

Dr Kieran Meade, Senior Research Offi cer and
Cathriona Foley, Teagasc Walsh Fellow, Teagasc Animal and Bioscience Research Department, Grange.
Professor Cliona O’Farrelly, School of Biochemistry and Immunology, Trinity College Dublin. Correspondence: [email protected]

Economic Cost of Uterine Disease

All cows experience uterine bacterial contamination of the uterus after they give birth and approximately 40 per cent of dairy cattle develop uterine disease such as metritis and/or endometritis. Unresolved bacterial infection of the uterine lining (known as the endometrium), induces chronic inflammation, reduces subsequent fertility and is associated with lower milk yields and increased veterinary intervention costs.

Uterine disease costs the European Union €1.4 billion per annum. In Ireland, assuming a cost of €292 per cow and a conservative incidence rate of 20 per cent in the dairy cow population, we estimate the economic impact of metritis alone to be in the region of €64 million per annum (Figure 1, adapted from Sheldon et al., 2008).

Diagnosis and treatment of Uterine Disease

Regardless of the risks that predispose cows to uterine infection and different classifications of uterine disease, an inflamed endometrium and high uterine bacterial load is a typical phenotype of metritis. Twenty-one days postpartum (DPP), clinical endometritis is diagnosed by the presence of purulent material in the vagina and subclinical endometritis can be diagnosed by uterine cytology or histopathological analysis. However, the diagnosis of subclinically infected animals remains a challenge on farm and sub/infertile animals represent a major problem for the industry.

Recent studies have found increasing incidence of multidrug resistance strains of Arcanobacterium pyogenes and Escherichia coli isolated from bovine uteri, which are two of the most common causes of endometritis in cattle. Therefore, the long term use of wide-spectrum antibiotics is not a sustainable therapeutic approach.

Uterine Inflammation And Immunity–Joining the Plots!

The current understanding of mechanisms linking bacterial infection, inflammation and subfertility involves the recognition of bacteria by pathogen receptors on uterine cells stimulating accumulation of prostaglandin E in the postpartum uterus resulting in a prolonged luteal phase, which is associated with postpartum uterine disease and subfertility.

However, as all cows experience bacterial contamination of the postpartum uterus, why do some cows clear these pathogens effectively and others go on to develop uterine disease? Is the host-specific reaction to the same pathogens different between cows and, if so, is it either an over-reaction, resulting in too much inflammation and damage to host tissues; or a suboptimal immune response allowing bacteria to survive and grow? For useful answers to these questions one has to perform integrated analyses at several levels, from uterine tissue to cells to molecules to genes with a particular focus on the interface between the uterine microbes and the endometrium lining the uterus.

Uterine Biopsy and Histological Analysis

The uterus is a complex organ, composed of multiple layers of specialised cell types. In order to capture the contribution from each of these layers, uterine biopsies are essential. Taken correctly and carefully, these valuable research tools do not have a detrimental impact on subsequent fertility. Histological analysis can assess the degree of inflammation in a uterine sample, classified based on numbers of polymorphonuclear cells, including neutrophils, under microscopic magnification. This technique allows the categorisation of uterine biopsies as either inflamed and endometritic, or non-infl amed and healthy.

Culture-Independent Identification of Uterine Bacteria

Although numerous studies have identified specific bacterial species associated with endometritic uterine environments, the majority have used culture-dependent techniques. More than 90 per cent of microbial species have therefore been excluded using these analyses. This underestimation can be dramatically improved by using culture-independent techniques to characterise bacterial diversity of the uterus.

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a culture independent technique that assesses the DNA sequence encoding the 16S rRNA gene to discriminate and identify the different populations of bacteria in a uterine swab. Our group are currently analysing uterine swabs taken at 7 and 21 DPP with T-RFLP in both endometritic and healthy cows to determine the bacteria present and to measure relative changes in these populations over time and in response to disease.

Figure 2

Advances In Transcriptomic and Related Technologies

Transcriptomics is the study of the transriptome, the entire complement of expressed genes in a cell or tissue, inclusive of mRNAs, non-coding RNAs and small RNAs at a given time point. Advances in technology now enable the identification of all genes present in the tissue of choice in an unbiased manner.

A particular strength of RNA-Seq technology is that, unlike Real-Time RT-qPCR and microarrays, it does not rely upon existing knowledge of the genome sequence, and because it sequences what is present rather than what is captured, it can identify entirely new genes and expression products. Similar technological developments also facilitate the identification of other regulatory layers and molecules controlling gene expression including microRNAs and the chemical modification of DNA (epigenetics).

Recent Studies Of Uterine Immunology in Teagasc

Recent work performed in Teagasc, and in collaboration with University partners, has led to the concept that inflammatory responses that occur in the post-partum cow, are critical to the return of the uterus to a normal physiological state capable of supporting a subsequent pregnancy. Large numbers of genes have been shown to be differentially expressed in the post-partum uterus; and our work has shown a shift from the activation of immune pathways early post-partum to the upregulation of tissue repair and proliferation pathways in healthy cattle.

Uterine inflammation is therefore part of the normal tissue remodelling process that occurs post-partum in healthy animals and is driven by bacterial colonisation. Dysregulation of this immune response may contribute to sustained infl ammation and development of chronic infection.

We propose that by using all the tools currently available in the Teagasc/Irish University research arsenal, that a comprehensive picture of the role of local immunity, as well as interactions between the immune and other systems in the post-partum bovine uterus, will identify potential targets for early therapeutic intervention and improving downstream fertility.

The Future Of Diagnostics and Therapeutics for Uterine Disease

At present, there are no reliable diagnostic techniques for subclinical uterine infection or disease in cows. What is emerging from our collaborative research is that the environment in the postpartum uterus is immensely complex, with contributions from the host, the pathogen(s) and the farm environment.

We aim to integrate multiple layers of clinical, cellular, microbiological and molecular information (Figure 2) to define the mechanisms responsible for effective clearance of bacteria from the post-partum uterus. In particular, we aim to define the signature of response that predicts failure to clear bacteria, resulting in chronic infection, inflammation and subsequent infertility.

No single system acts in isolation; integration of metabolic, endocrine and immune data will be critical to defining pathological and healthy signatures. It is clear that unresolved inflammation or failure of normal ‘cleansing’ of uterine bacteria post-partum can have knock-on downstream consequences for fertility.

What is also clear is that the emergence of new reliable diagnostics will depend on a detailed understanding of the role the immune response plays in this process, and how dysregulation of the immune response contributes to the development of infection.

The ultimate goal of our research is to develop biomarkers of early detection of uterine disease in particular subclinical endometritis. Systemic detection of these biomarkers (e.g., in serum) would lead the way towards the development of diagnostics for uterine disease in cows. Identifying infection earlier prevents the reliance on the development of clinical symptoms, and is more likely to result in a favourable outcome in terms of fertility as well as cost.

Further Reading

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May 2013

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