Proteomics of endometritis in Equine: identification of putative endometritis-associated marker proteins in serum

Project leaders

Annamaria Salzano, Amal Mahmoud Abo El-maaty

Agreement

EGITTO - ASRT - Academy of Scientific Research and Technology

Call

CNR-ASRT 2016-2017

Department

Biology, agriculture and food sciences

Thematic area

Biology, agriculture and food sciences

Status of the project

New

Research proposal

Equine infertility is a very common reproductive problem that is particularly relevant where horse breeding is a source of income. It is worth noting that the rate of equine infertility under human management is very high. Breeding mares, indeed, results in a transient physiological inflammation of the endometrium (Watson et al., 2001; Troedsson et al., 2001) that occurs primarily as a response to semen, seminal plasma, extender, and/or bacteria present within the uterine lumen. Commonly, mares that do not clear an infection after repeated treatments and do not become pregnant after repeated breeding lead to extended inter foaling interval. Persistent mating-induced endometritis was identified as the third most common medical disorder of adult horses. This condition causes huge economic losses due to the cost of insemination or breeding, veterinarian examinations and medication used for treatment.
Infectious infertility in the mare is clinically well described. Moreover, it has been reported that cytokines play a crucial role in the modulation of local and systemic inflammatory responses and are relevant for the resolution of the induced inflammation (Henderson and Wilson, 1996). Mares susceptible to developing persistent endometritis have higher endometrial mRNA expression of pro-inflammatory cytokines, lower level of the anti-inflammatory cytokine and sustained high number of inflammatory cells in their endometrium compared to resistance mares (Fumuso et al., 2007). However, little is known about the systemic acute phase reaction (APR) and local immunological responses accompanying equine endometritis (Christoffersen et al., 2010).
In order to diagnose and treat mares with endometrial inflammation, it is imperative to identify predisposing factors and challenges to the reproductive tract of the mare. Our joined proposal aims to address diagnostic and clinical aspects of equine endometritis by using different methodological approaches. In particular, clinical examinations on mares, suffering from subclinical and clinical infections, will be carried out by the Egyptian research team. Clinical examination will be performed by means of Ultrasound scanning methodologies (transrectal Doppler ultrasonographic exams) and uterine blood flow measurements. In the case of subclinical endometritis, about mares without clinical endometritis symptoms but with bacterial growth and positive cytology from an endometrial sample, the bacterial agents responsible for infection will be isolated and then identified by single and multiplex PCR Clinical data will then be correlated with microbiological diagnosis. The endometrium shows dramatic morphological and secretory changes as consequence of inflammations as well as throughout the estrous cycle or during pregnancy. These physiological changes are well represented by changes in serum protein composition because serum receives proteins released by all tissues in the organism, either through secretion or after membrane permeabilization and cell lysis. As serum proteins are potentially diagnostic of various physiological or pathological conditions, serum proteomics offers a very powerful approach to address early detection of animal diseases by identifying novel biomarkers. In this study blood serum samples from different groups of mares, will be analysed in order to detect quali-quantitative changes of proteome associated to different grade of endometritis conditions. For this purpose, an advanced analytical platform for proteomic analysis, based on MALDI-TOF MS profiling of serum samples and statistical analysis of acquired spectral data, will be set up by the Italian research team. For MALDI-TOF-MS analysis an UltraflexeXtreme mass spectrometer (Bruker Daltonics), equipped with proprietary software for machine control, mass spectral data analysis, and statistical analysis (ClinProt), will be used.
Final aim of our proteomic investigation will be to identify protein markers of clinical and subclinical endometritis and even markers of inflammation associated to a specific pathogen infection. The last condition appears extremely relevant for further investigation on response of animals to suggested veterinary treatment with specific antibiotics. The knowledge of protein markers will finally allow the development of reliable diagnostic tests for the early detection of the inflammation condition, thus preventing the onset of endometritis.
This project will establish a fruitful collaboration between research teams in Egypt and Italy thanks to the complimentary expertise owned by project coordinators. Prof. Amal M. Abo El-Maaty, coordinator of the Egyptian team, is specialized in physiology of reproduction and will cover all veterinary aspects of the project while Dr. Anna Maria Salzano, with a strong expertise in proteomics and mass spectrometry, will be in charge of the Italian research team and responsible for the serum proteome investigation.

Reference:
-Christoffersen M, Baagoe CD, Jacobsen S, et al. Evaluation of the systemic acute phase response and endometrial gene expression of serum amyloid A and pro- and anti-inflammatory cytokines in mares with experimentally induced endometritis. Vet Immunol Immunopathol. 2010 Nov 15;138(1-2):95-105.
-Fumuso, E.A., Aguilar, J., Giguire, S., et al., 2007. Immune parameters in mares resistant and susceptible to persistent post-breeding endometritis: effects of immunomodulation. Vet. Immunol. Immunopathol. 118, 30-39.
Henderson, B., Wilson, M., 1996. Cytokine induction by bacteria: beyond lipopolysaccharide. Cytokine 8, 269-282.
-Troedsson MH, Loset K, Alghamdi AM, Dahms B, Crabo BG. Interaction between equine semen and the endometrium: the inflammatory response to semen. Anim Reprod Sci 2001;68:273-8.
-Watson ED, Barbacini S, Berrocal B, et al. Effect of insemination time of frozen semen on incidence of uterine fluid in mares. Theriogenology 2001;56:123-31.

Research goals

Main goals of this projects are listed below:
1. Estimation of the prevalence of endometritis (clinical and subclinical) in Egyptian horses;
2. Definition of relations existing in healthy mares and mares with endometritis between uterine blood flow and stage of the estrous cycle;
3. Identification of the major bacterial causes of endometritis in Egyptian horses;
4. Highlight of significant differences in serum proteome between different groups of animals (healthy, subclinical and clinical endometritis), and proteome modifications related to the defense response of the endometrium to different pathogens;
5. Identification of putative protein biomarkers useful for monitoring endometritis and for the careful management of the condition.
6. Application of the genetic markers on the diagnosis of endometritis;
7. Correlation between the proteomic markers and genetic markers regarding the diagnosis stage and the type of the pathogen.

Last update: 25/04/2024