Complimentary Diagnostic Tools for Endometrosis in Biopsies of Mares with Clinical Subfertility

Daniela Rojas, Joel Cabezas, Ghyslaine Ramírez, Fernando Saraiva, Rodriguez-Alvarez Lleretny, Fidel Ovidio Castro


Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiology and pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis with glandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary among individuals according to the disease progression. The aim of this research was to combine histology with new immune and histochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.

Materials, Methods & Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples were subjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specific techniques such  as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were present in those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods for the detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3  were used. Finally in order to detect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. As a result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb and III (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result we found specific staining in 15 samples coming from higher endometrial damage using Masson-Fontana, while acid staining indicative of acid mucopolysaccharides were detected in 97% of the samples. At immunostaining, we found cytokeratin and vimentin differentially expressed as a function of the degree of the lesions. Cytokeratin was detected in glands from healthy mares, but it was almost absent as the injury was increased. Vimentin in turn, was detected in 26 samples with different degree of intensity. In nine animals, vimentin expression was anomalous, and the marker was detected in fibrotic foci concentrically surrounding glands in biopsies of grade I and IIA only. Although  progesterone receptor was detected, there was no correlation with endometrosis. Finally, the lymphocyte T specific CD3 marker was positive in 100% of cases analyzed in which  moderate lymphocytic infiltration was found by hematoxylin-eosin; however no staining could be detected in mares with more advanced endometrosis.  By combining immunofluorescence with the detection of second harmonic it was possible to detect in the same sample two proteins and collagen deposition at the same time, this had not been reported earlier for endometrosis and mares.

Discussion: Our results suggest that the combination of the different methods mentioned is useful for validation and further characterization of the routine hematoxylin-eosin, and can be used as a complementary tool for the diagnosis. Of value, it was possible to combine immunofluorescence with SHG in a single sample in situ.

Full Text:



Aupperle H., Schoon D. & Schoon H.A. 2004. Physiological and pathological expression of intermediate filaments in the equine endometrium. Research in Veterinary Science. 76(3): 249-255.

Blanchard T.L., García M.C., Kintner L.D. & Kenney R.M. 1987. Investigation of the representativeness of a single endometrial sample and the use of trichrome staining to aid in the detection of endometrial fibrosis in the mare. Theriogenology. 28(4): 445-450.

Carlson M.A. & Longaker M.T. 2004. The fibroblast-populated collagen matrix as a model of wound healing: a review of the evidence. Wound Repair and Regeneration. 12(2): 134-147.

Chen X., Nadiarynkh O., Plotnikov S. & Campagnola P.J. 2012. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nature Protocols. 7(4): 654-669.

Christoffersen M., Woodward E.M., Bojesen A.M., Petersen M.R., Squires E.L., Lehn-Jensen H. & Troedsson M.H. 2012. Effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares. Theriogenology. 78(1): 991-1004.

Denk W., Strickler J.H. & Webb W.W. 1990. Two-photon laser scanning fluorescence microscopy. Science. 248(4951): 73-76.

Doig P. & Waelchli R. 1993. Endometrial biopsy. In: McKinnon A.O. & Voss J.L. (Eds). Equine Reproduction. Philadelphia: Lea & Febiger, pp.225-233.

Ferreira-Dias G., Nequin L.G. & King S.S. 1999. Morphologic comparisons among categories I, II, and III equine endometrium using light and transmission electron microscopy. American Journal of Veterinary Research. 60(1): 49-55.

Flores J.M., Rodríguez A., Sánchez J., Gómez-Cuétara C. & Ramiro F. 1995. Endometrosis in mares: incidence of histopathological alterations. Reproduction in Domestic Animals. 30(2): 61-65.

Franke W.W., Schmid E., Winter S., Osborn M. & Weber K. 1979. Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Experimental Cell Research. 123(1): 25-46.

Freund I., Deutsch M. & Sprecher A. 1986. Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. Biophysical Journal. 50(4): 693-712.

Gilles C., Polette M., Zahm J.M., Tournier J.M., Volders L., Foidart J.M. & Birembaut P. 1999. Vimentin contributes to human mammary epithelial cell migration. Journal of Cell Science. 112(24): 4615-4625.

Greiling D. & Clark R.A. 1997. Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix. Journal of Cell Science. 110(7): 861-870.

Hoffmann C., Bazer F.W., Klug J., Aupperle H., Ellenberger C. & Schoon H.A. 2009. Immunohistochemical and histochemical identification of proteins and carbohydrates in the equine endometrium: Expression patterns for mares suffering from endometrosis. Theriogenology. 71(2): 264-274.

Hoffmann C., Ellenberger C., Mattos R.C., Aupperle H., Dhein S., Stief B. & Schoon H.A 2009. The equine endometrosis: New insights into the pathogenesis. Animal Reproduction Science. 111(24): 261-278.

Jielsen J.M. 2005. Endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy. Theriogenology. 64(3): 510-518.

Kenney R.M. 1978. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. Journal of American Veterinary Medicine Association. 172(3): 241-262.

Kenney R.M. 1992. The etiology, diagnosis and classification of chronic degenerative endometritis. Equine Veterinary Journal. 25(3): 186-192.

Kenney R.M., Bergman R.V., Cooper W.L. & Morse G.W. 1975. Minimal contamination techniques for breeding mares: techniques and preliminary findings. Proceedings of the American Association of Equine Practitioners. 21: 327-336.

Kenney R.M. & Doig P.A. 1986. Equine endometrial biopsy. In: Morrow D.A. (Ed). Current Therapy in Theriogenology. Philadelphia: Saunders W.B, pp.723-729.

Kenney R. & Ganjan V. 1975. Selected pathological changes of the mare uterus and ovary. Journal of Reproduction and Fertility. Suppl(23): 335-339.

Kidd M.E., Shumaker D.K. & Ridge K.M. 2014. The role of vimentin intermediate filaments in the progression of lung cancer. American Journal of Respiratory Cell and Molecular Biology. 50(1): 1-6.

Lehmann J., Ellenberger C., Hoffmann C., Bazer F.W., Klug J., Allen W.R., Sieme H. & Schoon H.A. 2011. Morpho-functional studies regarding the fertility prognosis of mares suffering from equine endometrosis. Theriogenology. 76(7): 1326-1336

Lorena D., Uchio K., Costa A.M. & Desmouliere A. 2002. Normal scarring: importance of myofibroblasts. Wound Repair and Regeneration. 10(2): 86-92.

Mansour G.D., Henry M. & Ferreira A.M. 2003. Immunohistochemical study of equine endometrial extracellular matrix during the oestrous cycle. Journal of Comparative Pathology. 129(4): 316-319.

Moll R., Franke W.W., Schiller D.L., Geiger B. & Krepler R. 1982. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 31(1): 11-24.

Mutsaers S.E., Bishop J.E., McGrouther G. & Laurent G.J. 1997. Mechanisms of tissue repair: from wound healing to fibrosis. International Journal of Biochemistry and Cell Biology. 29(1): 5-17.

Oddsdóttir C., Riley S.C., Leask R., Shaw D.J., Aurich C., Palm F., Fowden A.L., Ricketts SW. & Watson E.D. 2011. Dynamics of activities of matrix metalloproteinases-9 and -2, and the tissue inhibitors of MMPs in fetal fluid compartments during gestation and at parturition in the mare. Theriogenology. 75(6):1130-1138.

OverbeckW., JägerK., Schoon H.A. & Witte T.S. 2013. Comparison of cytological and histological examinations in different locations of the equine uterus-an in vitro study. Theriogenology. 79(9): 1262-1268.

Overbeck W., Witte T.S. & Heuwieser W. 2011. Comparison of three diagnostic methods to identify subclinical endometritis in mares. Theriogenology. 75(7): 1311-1318.

Rao R.A.R., Metha M.R., Leithem S. & Toussani K.C. 2009. Quantitative analysis of forward and backward second harmonic generation microscopy. Optical. Letters. 34(24): 3779-3381.

Ricketts S.W. & Alonso S. 1991. The effect of age and parity on the development of equine chronic endometrial disease. Equine Veterinary Journal. 23(3): 189-192.

Satelli A. & Li.S. 2011. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cellular and Molecular Life Science. 68(18): 3033-3046.

Schoon H.A., Schoon D. & Klug E. 1992. Uterusbiopsien als Hilfsmittel fur Diagnose und Prognose von Fertilitatsstorungen der Stute. Pferdeheilkunde. 8(6): 355-362.

Tilbury K., Hocker J., Wen B.L., Sandbo N., Singh V. & Campagnola P.J. 2014. Second harmonic generation microscopy analysis of extracellular matrix changes in human idiopathic pulmonary fibrosis. Journal of Biomed Optics. 19(8): 86014.

Turani H., Lurie B., Chaimoff C. & Kessler E. 1986. The diagnostic significance of sulfated acid mucin content in gastric intestinal metaplasia with early gastric cancer. American Journal of Gastroenterology. 81(5): 343-345.

Waelchli R. 1990. Endometrial biopsy in mares under non uniform breeding management conditions: Prognostic value and relationship with age. Canadian Veterinary Journal. 31(5): 379-384.

Walter I., Handler J., Reifinger M. & Aurich C. 2001. Association of endometrosis in horses with differentiation of periglandular myofibroblasts and changes of extracellular matrix proteins. Reproduction. 121(4): 581-586.

Watson E.D. & Thomson S.R.M. 1996. Lymphocyte subsets in the endometrium of genitally normal mares and mares susceptible to endometritis. Equine Veterinary Journal. 28(2): 106-111.

Williams R.M., Zipfel W.R. & Webb W.W. 2001. Multiphoton microscopy in biological research. Current Opinion in Chemical Biology. 5(1): 603-608.

Wynn T.A. 2007. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. Journal of Clinical Investigation. 117(3): 524-529.

Zent W.W. & Troedsson M.H. 1998. Post breeding uterine fluid accumulation in a normal population of thoroughbred mares: A field study. Proceedings of the American Association of Equine Practicioners. 44: 64-65.

Zipfel W.R., Williams R.M. & Webb W.W. 2003. Nonlinear magic: multiphoton microscopy in the biosciences. Nature Biotechnology. (21): 1369-1377.


Copyright (c) 2019 Daniela Rojas, Joel Cabezas, Ghyslaine Ramírez, Fernando Saraiva, Rodriguez-Alvarez Lleretny, Fidel Ovidio Castro

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.