Endometrosis--significance for horse reproduction, pathogenesis, diagnosis, and proposed therapeutic methods

Equine endometrosis is a multifactorial disease considered to be a one of the most important causes of equine infertility, especially in older mares. This article reviews the current knowledge of equine endometrosis: pathogenesis, diagnosis, and optional treatment. Also describes the histomor- phological and immunohistochemical characterization of endometrosis as well as potential etiological factors which may influence disease progression. Unfortunately, the etiology and pathogenesis of endometrosis still remains unclear, and consequently no effective treatment has been proposed so far. Therefore future studies are needed for explanation of this disease. Key words: mare, endometrium, biopsy, endometrosis

According to the scheme proposed by Troedsson (1999), endometritis in mares is divided into: 1) sex- ually transmitted diseases, 2) chronic infectious en- dometritis, 3) persistent breeding-induced endometri- tis and 4) chronic degenerative endometritis (en- dometrosis). This paper will be devoted to the last of the above-mentioned diseases, to which more atten- tion has been drawn recently, shedding new light on its pathogenesis. Proposed therapeutic methods will be also discussed in this paper. Correspondence to: J. Buczkowska, e-mail: [email protected], tel.: +48 71 320 53 11 There are two terms: endometrosis and endomet- riosis used in the medical literature, which are defined differently and should not be confused. Endometriosis means extra-uterine implantation of endometrial tis- sue and refers to women (Snider et al. 2011), whereas the term endometrosis was introduced by Kenney in 1992 and means changes in the mare’s uterus previ- ously referred to as chronic degenerative endometritis (Allen 1993). At present, endometrosis is defined as active or inactive perigrandular and/or stromal en- dometrial fibrosis including glandular alterations within fibrotic foci (Hoffmann et al. 2009b). This pro- Polish Journal of Veterinary Sciences Vol. 17, No. 3 (2014), 547–554 Table 1. Types of endometrosis and histopathological view of endometrium (Lehmann et al. 2011). Types of endometrosis Histopathological changes in the endometrium active glands with several disordered fibrotic stromal cells showing a metabolically active dif- ferentiation with medium to large, ovoid hypochromatous nuclei and a pale cytoplasm inactive glandular nest with periglandular stromal cells showing a metabolically inactive differen- Nondestructive tiation, characterized by spindle shaped, hyperchromatous nuclei and an elongated cytoplasm mixed glands with periglandular stromal cells showing both 50% metabolically active and 50% inactive differentiation active glands surrounded by metabolically active stromal cells, in addition to the disordered appearance of different layers, the fibrotic stromal cells are invasive and they penetrate the lumen of the gland; different degenerated epithelial cells are visible Destructive inactive glands with metabolically inactive fibrotic stromal cells which lie parallely arranged to the axis of the adjacent gland, multifocal destruction of single epithelial cells and con- gestion of the uterine secretions in the lumina are visible mixed glands surrounded by active and inactive periglandular stromal cells which penetrate the glandular epithelia cess may affect single glands and/or glandular nests (Kenney 1978). The degree of endometrosis in mares increases with age; however, it is thought that this is not connected with the number of foalings (Ricketts and Alonso 1991, Kenney 1993, Hoffmann et al. 2009b, Aresu et al. 2012). Endometrosis is one of the causes of infertility in mares. Structural changes in the endometrium include the occurrence of progressive focal proliferation of the uterine glands, which are concentrated in the glandular nests encircled by more and more numerous layers of fibrous tissue as the pro- cess progresses. Inside the nests, glandular cysts oc- cur, whereas the number of normal endometrial glands decreases. Stromal fibrosis is a dominating dis- ease process, which results in disturbance of lymph circulation, as a result of which lymphatic extension occurs (Katkiewicz et al. 2007). Based on the morphology of the periglandular stromal cells, the fibrosis can be divided into different types, which can be classified as either destructive or non-destructive, with biopsies displaying more than 75% of the fibrotic foci of one of these groups being termed “active” or “inactive” fibrosis respectively. Bi- opsy with approximately equal ratios of active and in- active periglandular stromal cells are termed “mixed” endometrosis (Table 1) (Lehmann et al. 2011). In all of the above-mentioned types, single uterine glands and/or glandular nests may be affected by the process (Hoffmann et al. 2009b). Additionally, we can deter- mine the degree of endometrosis describing it from mild to severe (Kenney and Doig 1986, Hoffmann et al. 2009b). In severe endometrosis a significantly high- er incidence of glandular nest development was ob- served. Furthermore, a significant association of in- active endometrosis and cystic dilatation of the affected glands could be seen. However, within active non-destructive endometrosis, an asynchronous cycle, in particular irregular differentiation of the affected glandular epithelia, was most pronounced (Hoffmann et al. 2009b). Pathogenesis Atypical morphological and functional differenti- ation of periglandular endometrial stromal cells are the first sign of endometrosis. The first stage of fi- brosis is characterised by large, polygonal periglandu- lar stromal cells which synthesize collagen fibres, whereas in advanced fibrosis metabolic active or in- active stromal cells, without signs of collagen syn- thesis, as well as myofibroblasts, predominate (Walter et al. 2001, Hoffmann et al. 2009a,b). Periglandular fibrosis in endometrial tissue is not characterized by an increased incidence of collagen fibres, but by perig- landularly arranged fibroblasts in one or more layers, which produce the extracellular matrix proteins col- lagen IV, laminin, and fibronectin. In addition, these fibroblasts express h-smooth muscle actin, tropomyosin and occasionally desmin, which confirms their differentiation into myofibroblasts. It remains to be elucidated whether the above-mentioned differen- tiation has further consequences on the uterine gland epithelium or the stromal cells, as has been shown for various pathological processes (Walter et al. 2001). During the fibrosis process, the stromal cells of the basal lamina are capable of collecting collagen un- der the influence of different stimulating factors. The collection of collagen occurs most often around the glands or in connection with the basement membrane 548 J. Buczkowska et al. of the luminal epithelium (Kenney and Doig 1986). Fibrosis occurs most often without the presence of inflammatory cells (Kenney 1978). The first evidence of impending deposition of collagen is the loss of ran- domization of stromal cells (and their nuclei) in the stratum compactum and stratum spongiosum, particu- larly visible around the glands. The advancement of fibrosis may be evaluated by the degree (the number of periglandular layers) and frequency (the number of fibrotic foci per linear field) (Kenney and Doig 1986). From one to three layers of perigrandular fibrosis is considered to be a slight reaction, from four to ten – a moderate reaction, and more than ten layers indi- cate a severe degree of fibrosis (Kenney 1978). Potential etiological factors which may affect de- velopment of the disease are morphological and im- munohistochemical changes of the endometrium, which occur in endometrosis (Table 1) (Hoffmann et al. 2009b, Lehmann et al. 2011). In the unaffected endometrium, cyclical and seasonal endocrine changes result in cycle-synchronous morphological al- terations to epithelial and stromal cells. With regard to the morphology of the periglandular stromal cells within the fibrotic foci of the endometrosis an active and inactive differentiation can be distinguished (Hof- fmann et al. 2009b, Lehmann et al. 2011). However, in contrast to the unaltered stromal cells, neither sea- sonal nor cyclic endocrine changes seem to have a sig- nificant influence on the activity of the fibrotic stromal cells (Hoffmann et al. 2009b). Hoffmann et al. (2009b) showed that, in the biopsy material collected during the breeding/non-breeding season or on de- fined days during the oestrous cycle, both seasonal and cycle-associated endocrinological changes do not determine the activity of stromal cells. In explaining the connection between endometri- tis and endometrosis, both the impact of the endomet- ritis on further development of degenerative changes in the endometrium and the process of activation of inactive fibrotic foci were taken into account, and pre- dispositions for the occurrence of endometritis in a mare with endometrosis were examined (Lee et al. 2001, Atamas 2002, Keller et al. 2006, Hoffmann et al. 2009b). A higher frequency of endometritis in a mare with a destructive type of endometrosis was described (Hoffmann et al. 2009b), which indicates an important role of uterine glands in the physiological clearance of the endometrium, and disturbance of this function may cause endometritis (Hoffmann et al. 2009b). It was also shown that mares with a higher biopsy grade more frequently retain fluid in the uterus after insemi- nation (Woodward et al. 2012). It is suggested that temporary activation of fibrotic stromal cells, which could be observed as a result of experimentally in- duced bacterial endometritis, is presumably caused by profibrotic growth factors and cytokines released from inflammatory cells (Lee et al. 2001, Atamas 2002, Hoffmann et al. 2009b). However, it was shown that inflammation is poorly correlated with grade of en- dometrosis, and bacterial infection does not increase intensification of this disease (Keller et al. 2006, Ar- esu et al. 2012). Additionally, in mares experimentally infected with Streptococcus equi subsp. Zooepidemicus, the degree of endometrosis did not change during the 2-year period of the observation (Hoffmann et al. 2009b). Recently it was shown that intrauterine enrofloxacin infusion in mares induced severe acute uterine mucosal necrosis, inflammation, and a significant increase in endometrial fibrosis (Rodriguez et al. 2012). Among other changes in the mare endometrium, a negative correlation between mild endometrosis and changes in vessels of severe angiosclerosis type was also observed, yet such a correlation between en- dometrosis and inflammation of the perivascular sheath and surrounding tissue (perivasculitis) was not noted (Hoffmann et al. 2009b). It was observed that a large percentage of both barren and foaling mares show angiosclerosis; however, this does not have an impact on the degree of endometrosis (Lehmann et al. 2011). An immunohistochemical study showed that stromal maldifferentiation which could be seen in all fibrotic foci is independent of the degree of endomet- rosis, and that the cells show a lower expression of steroid hormone receptors compared to unchanged stromal cells (Hoffmann et al. 2009b). Moreover, metabolically inactive stromal cells exhibited a de- creased proliferation activity as compared to the unal- tered endometrium, whereas no differences could be observed within active fibrotic foci (Hoffmann et al. 2009b). A considerable reduction of estrogen and progesterone receptor expression of fibrotic stromal cells as compared to the normal stroma is one of the hallmarks of endometrosis (Hoffmann et al. 2009b). This reduction indicates advanced changes in the stromal cells within a fibrotic focus, which lead to the loss of specialization in the cell structure and function. As a result, fibrotic stromal cells are unable to react to cyclic endocrine changes and become independent of hormonal control mechanisms in the uterus (Hof- fmann et al. 2009b). Maldifferentiation of the epithelial cells of the uterine glands within fibrotic foci was also character- ized by a distinct variability in glandular steroid hor- mone receptor expression. In active endometrosis, an increase in expression of estrogen and progesterone receptors dominates, as compared to the unchanged glands, whereas glandular epithelia in inactive en- dometrosis show a decrease in expression of the Endometrosis – significance for horse reproduction... 549 early stages of fibrosis active fibrosis destruction mechanical stressincrease of myofibroblasts and extracellular matrix endometritis ? ? periglandular fibrosis inactive fibrosis inactive destructive fibrosisactive destructive fibrosis stromal fibrosis endocrine changes Fig. 1. Schematic picture of the possible pathogenesis of endometrosis (adopted from Hoffmann et al., 2009b). above-mentioned receptors (Hoffmann et al. 2009b). Moreover, the concentrations of steroid receptors in the destructive type of endometrosis are also lower. A mild increase in expression of estrogen and proges- terone epithelial receptors was observed in active non-destructive fibrotic foci, whereas in the other types of endometrosis, a decrease in expression of the above-mentioned receptors was visible. This phenom- enon can be explained by paracrine mechanisms be- cause the effect of the impact of steroid hormones on the glandular epithelium is physiologically mediated by the adjacent stromal cells (Cooke et al. 1997, Kurita et al. 1998, Pierro et al. 2001). These cells are maldifferentiated in endometrosis and presumably re- lease other paracrine signals. Therefore, the asyn- chronous cycle differentiation of the affected glands is, initially, most likely the consequence of maldif- ferentiation of the stroma, and the decreased express- ion of steroid hormone receptors is likely to be caused by distinct degeneration of the epithelial cells. An- other possible explanation of the asynchronous cycle differentiation of glandular epithelia within fibrotic foci is the severe damage of the basal lamina connec- ted with altered expression of laminin. All of the stromal cells of fibrotic foci, in particular in active destructive fibrosis, show intracellular laminin ex- pression (Hoffmann et al. 2009b). Only an intact basal lamina is able to ensure complex paracrine interaction between the epithelia and the underlying stroma (Lin and Bissel 1993, Arnold et al. 2001), which inhibits direct stromal-epithelial contacts as well as an interac- tion between epithelial cell surface integrins and the fibrotic extracellular matrix. Furthermore, large dis- continuities of the glandular basal lamina were deter- mined predominantly in destructive endometrosis. Fi- brosis activity visibly affects the expression of estrogen receptors; however, it does not have an influence on the concentration of intermediate filaments in the glandular epithelium, although in the destructive type of endometrosis the expression of filaments increases considerably compared to the non-destructive type (Hoffmann et al. 2009b). The immunohistochemical study of stromal cells indicates that distinct variability in all types of en- dometrosis regards coexpression of vimentin and de- smin intermediate filaments as well as h-actin in my- ofilaments. The stromal cells in destructive endomet- rosis, in particular in active destructive endometrosis, tended to express more h-actin. In addition, in the latter type of endometrosis, expression of laminins in the stromal cells is greater (Hoffmann et al. 2009b). The immunohistochemical study of extracellular matrix in fibrotic foci indicated the presence of more expression of proteoglycans and fibronectin in the de- structive type of endometrosis, in particular in severe active endometrosis, compared to other types (Hof- fmann et al. 2009b). Therefore, the observed accumu- lation of proteoglycans and fibronectin is probably due to an increased number of secretively active my- ofibroblasts (Hoffmann et al. 2009b). 550 J. Buczkowska et al. Summing up, partial thinning of a fragment of the basal lamina initiates changeability and activity of the glandular epithelium (Fig. 1). A few etiological factors may cause this process e.g. a periglandular localised endometritis, a local deficiency of oxygen caused by severe angiosclerosis, wound healing processes follow- ing mechanical damage, and disorders regarding physiological regeneration of the basal lamina. How- ever, only the intact basal lamina is able to suppress activation of epithelial cells and synthesis of profibrotic growth factors (Streuli et al. 1993). Endometritis which occurs simultaneously with endometrosis causes activa- tion of the metabolism of fibrotic stromal cells. In con- trast with the above, cyclical and seasonal endocrine changes seem to have no affect on the disease process. Further studies are necessary to determine the factors which are able to maintain active fibrosis. The pro- gressive nature of the disease is likely to be attributed to an increase in the number of myofibroblasts leading t ot h ed a m a g eo fu t e r i n eg l a n d si nt h ef i n a ls t a g eo f fibrosis. On the other hand, the ability of these cells to release enzymes which regulate matrix homeostasis may contribute to progressive damage of the basal lamina and may lead to the collection of extracellular matrix, which in turn causes maintenance of the fi- brosis process (Ramos et al. 2001). For the develop- ment of metabolically inactive fibrosis, etiological fac- tors are particularly interesting, although they have not yet been determined. The contractibility of myofibrob- lasts and their arrangement parallel to the adjacent glands improves the ability of the fibrotic tissue to con- tract. Mechanical stress may explain cystic expansion of the glands and focal damage of the epithelium, which o c c u r sm o r eo f t e ni nt h i st y p eo fe n d o m e t r o s i s .I ti s assumed that activation of inactive fibrosis may occur at any time during endometritis and, probably, via other, still unknown, factors (Hoffmann et al. 2009b). Diagnosis Most degenerative changes typical for endometro- sis can be diagnosed only through the histological evaluation of an endometrial biopsy (Kenney 1978, Kenney and Doig 1986, Ricketts and Alonso 1991, Ricketts and Barrelet 1997, Katkiewicz et al. 2007, Zajac et al. 2008, Schlafer 2009, Snider et al. 2011, Aresu et al. 2012). Endometrial biopsy has been a stan- dard procedure for evaluation of the health of the mare’s uterus for more than 40 years. It is a safe, prac- tical and very useful method (Kenney 1978, Ricketts and Barrelet 1997). Nevertheless, it should be noted that some researchers draw the conclusion that collec- tion of an endometrial specimen from one uterine por- tion does not necessarily reflect the state of its remain- ing areas, as significant differences were observed re- garding both the size of a fibrosis area and the grade of endometrosis in a biopsy collected from the same mare yet from different portions of its uterus. In more than half of the mares variation in the endometrial grade in biopsies collected from different uterine portions was revealed (Fiala et al. 2010). Keller et al. (2006) also observed that a single biopsy does not represent the whole endometrium in the diagnostics of degenerative changes. However, according to the other researchers, a single biopsy reflects very well the condition of the whole uterus if the biopsy material has proper size and s h a p e ,a n di ti sp r e p a r e da n de v a l u a t e di nt h ea p p r o - priate way (Kenney 1978, Kenney and Doig 1986). The purpose of performing the biopsy is to detect changes in the endometrium and to determine the fer- tility potential of the mare. The biopsy should be taken from barren mares, “repeat breeder” mares, from mares showing anoestrus during the breeding season, and from mares with a pathologic content of the uterus (Kenney and Doig 1986, Schlafer 2007, Snider et al. 2011). Another group consists of mares with a history of early embryonic or foetal death, mares with poor perineal conformation before surgical correction, and recipient mares in the embryotransfer programme. Pregnancy is the only known contraindication for the performance of the biopsy (Kenney and Doig 1986, Schlafer 2007, Snider et al. 2011). In the image of the uterine wall, seven components can be distinguished: the uterine lumen, endometrial epithelial interface, superficial stroma (stratum com- pactum), the glands and the glandular epithelium, the stroma – the mid and deep endometrium (stratum spongiosum), vessels (arteries, veins and lymphatics), myometrium and larger vessels. An assessment of these components helps the pathophysiology of the uterine wall to be determined. Apart from microscopic examin- ation of the biopsy material, immunohistochemical examination can be also performed (Schlafer 2007). Evaluation of the degree of endometrial fibrosis is essential as, in contrast to the inflammatory changes, fibrosis is of a permanent nature and, if it is intense, it becomes the main factor that reduces the reproductive performance of the mare (Kenney and Doig 1986). De- pending on the degree of intensification of structural changes in the endometrium, Kenney and Doig (1986) divided endometrosis into four categories (Table 2). Inflammation, fibrosis, lymphatic system and atrophic changes may coexist, and the more changes occur and the more advanced they are, the higher the category is. For example, an occurrence of inflammatory changes or fibrosis signifies category IIA; however, if they occur simultaneously, it will signify category IIB. If the in- flammatory condition is eliminated, the endometrium may return to category IIA. Moreover, if inflammatory Endometrosis – significance for horse reproduction... 551 Table 2. Standard classification system for histologic changes in the endometrium (Kenney and Doig 1986). Percentage of altered glandsCategory Structural changes in the endometrium I Healthy mares; no pathologic changes or any existing changes (such as inflammation or fibrosis) are slight and sparsely scattered – IIA A small degree of stromal fibrosis around the individual gland branches; a lack of glandular nests in 4 adjacent fields; slight to moderate inflammatory changes, lymphatic lacunae occurs; partial endometrial atrophy 10-35% IIB Fibrotic changes are more severe and extensive than in IIA; an average of 2-4 fibrotic nests of glands, usually with 2-4 layers in 4 adjacent fields; inflammatory and lymphatic changes are widespread, diffuse and moderately severe 35-60% III Widespread, diffuse, severe inflammatory changes; widespread fibrosis of gland branches of 5 or more fibrotic nests; severe lymphatic lacunae > 60% Table 3. Expected foaling rates of mares according to categorization of endometrium (Kenney and Doig 1986). Category Degree of endometrial change Expected foaling rate I Absent 80-90% IIA Mild 50-80% IIB Moderate 10-50% III Severe 10% changes, fibrosis, and lymphatic changes coexist, the endometrium belongs to category III. The more changes occur in the endometrium, the worse the prog- nosis of pregnancy and maintenance of pregnancy is (Table 3) (Kenney and Doig 1986). The lymphatic lacunae observed in the biopsy are derived from dilated lymphatic vessels and can occur in the lamina propria or core of the endometrium folds singly or in clusters and they can be focal or diffuse. When single lacunae are joined and expand, they be- come endometrial cysts, which achieve a size of even up to a few centimetres in diameter (Kenney 1978, Kenney and Doig 1986). Large uterine cysts may dis- turb embryo mobility, and consequently the process of maternal recognition of pregnancy (Kenney 1978). Cyst-like expanded glands are often noticed within the fibrotic nests. This phenomenon can be also ob- served during the seasonal atrophy; however, in that case, there is a thick secretion inside. Cystic distention without fibrosis and without inspissation also occurs during the physiological breeding season. When the above-mentioned changes are intensified to a great ex- tent, they seem to have a negative impact on reproduc- tion (Kenney 1978, Kenney and Doig 1986). In about 30% of cases of fibrosis, acute and/or chronic inflammation is observed (Ricketts and Bar- relet 1997). Then, in the biopsy, inflammatory cells (mainly lymphocytes and neutrophils, and a lower number of eosinophils and mastocytes which are concentrated around the glandular nests and vessels, and dispersed within the stroma) are observed (Kenney 1978, Zajac et al. 2008, Aresu et al. 2012). Impact of endometrosis on fertility Fertility impairment is the main problem connec- ted with endometrosis, and therefore Lehmann et al. (2011) decided to use histopathological and im- munocytochemical examination of the endometrium in order to determine reproductive abilities in fertile and barren mares. They showed abnormal secretion of the endometrial glands during endometrosis. In the barren mares, there was lower expression of proteins: uterog- lobin and uterocalin were detected in the fibrosis areas, especially in those mares which suffered from moder- ate destructive endometrosis. Uterocalins take part in the supply of proteins to the embryo (Crossett et al. 1998) and deficiency of this protein can contribute to early embryonic death (Lehmann et al. 2011), whereas deficiency of uteroglobins may lead to inhibition of the embryo protection against a maternal immune re- sponse which results in pregnancy loss (Zhang et al. 2000). In the destructive type of endometrosis, both quantity expression and quality expression of endomet- rial proteins are decreased (Hoffmann 2009a). In re- search into the fertility of mares suffering from en- dometrosis, Lehmann et al. (2011) showed that mild 552 J. Buczkowska et al. non-destructive endometrosis dominates in fertile mares, whereas most barren mares show a moderate degree of endometrosis. In addition, destructive changes in the fibrotic foci are observed in most barren mares. Thus, barren mares much more often suffer from moderate destructive endometrosis compared to fertile mares. It seems that whether fibrosis is active or inactive does not affect fertility. Recently, Szostek et al. (2012) showed that changes in mRNA transcription of prostaglandin synthases and prostaglandin production which occur in the equine endometrium during the course of fibrosis may lead to early embryonic death. Treatment It is difficult to provide satisfactory treatment for endometrosis. It is commonly thought that changes caused by this disease are irreversible (Kenney and Doig 1986). For its treatment, mechanical curettage or chemical agents (kerosene, DMSO, isotonic salt) can be applied (Keller et al. 2006). On the proceedings of J.P. Hughes international workshop on equine endometritis different therapeutic

were presented and summarised by Allen (1993). For example, one of the authors analyzed the effect of physical curettage applied to mares with vari- ous degrees of endometrosis, leading to hyperaemia a n dm i l da c u t ee n d o m e t r i t i s ,c o m b i n e dw i t hi n - trauterine administration of antibiotics to prevent in- fection. After treatment they noticed improvement in endometrial biopsy grade in 44% of mares, in 51% of mares there was no effect, and in 5% of mares it was worse. Additionally, there was no significant effect on the fertility rates. It was also showed that irrigating the uterus with 250-500 ml of kerosene, which causes uter- ine oedema lasting for 1-2 days and results in expulsion of retained excretions from the uterine glands, is able to improve fertility rates. The best results were ob- tained when mares were mated during the first oestrus after treatment. Other research, in which kerosene was also used, showed that the application of this therapy brings rather a short-term effect, as half of the mares which were pregnant after treatment with kerosene miscarried later (Allen 1993). On the other hand, Ley et al. (1989) studied the effect of intrauterine adminis- t r a t i o no f1 0 - 3 0 %D M S Oi ns t e r i l es a l i n es o l u t i o n , compared to the control group i.e. mares treated with sterile saline solution alone. This therapy was carried out during oestrus for five consecutive days. No nega- tive changes were found in the endometrium after any treatment protocols. It was shown that 30% DMSO solution caused a reduction of inflammation and/or a reduction of periglandular fibrosis. However, no sig- nificant difference was found in pregnancy rates be- tween mares treated with 30% DMSO solution and mares treated with saline solution alone, although a growing tendency was observed following DMSO therapy. Furthermore, Griffin and Bennet (2002) ob- served an improvement of fertility following photoabla- tion of endometrial cysts in barren mares.

Continuous progress and modern laboratory

enable a more in-depth analysis of changes in the process of endometrosis, and they also make it possible to understand accurate genesis and show the impact of different factors on the development and progression of degenerative changes in the mare’s uterus. However, further studies are still needed to ex- plain the etiology of endometrosis process initiation, and the improvement of knowledge concerning eti- ology and pathogenesis of endometrosis will enable the development of both prevention and more effective treatment of this disease. Conflict of interests The authors declare that there is no conflict of in- terests regarding the publication of this article. Acknowledgments This paper was supported by NCN grant, DEC-2011/01/B/NZ5/04173.

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