**2. Etiology and pathophysiology of equine endometriosis**

Endometriosis is a debilitating condition leading to significant subfertility and often infertility in the mare, causing significant economic loss in our horse breeding population [1, 2]. The term endometriosis is somewhat misleading in our equine clinics. Endometriosis in the mare is a degenerative, chronic condition demonstrated by fibrosis [3] within the endometrium often as a sequelae to chronic deep

rooted infection, typically associated with a number of bacterial species. Although this etiology is not universally recognized [4]. This fibrotic condition in the mare is considered irreversible [5–7], and sadly clinicians are limited in their treatment options for this condition. In our human patients, endometriosis is the presence of endometrial glands and stroma-like lesions occurring outside the uterus [8]. These lesions can be peritoneal lesions, superficial implants, cysts on the ovary or deep infiltrating disease in nature [9]. Thus for the academics amongst us, we must understand the difference between our human and non-human patients with regards to the terminology of endometriosis.

Kenney [10] termed endometriosis in our equine patients based on different alterations to the endometrium based on histopathology [11]. A grading system came into effect in the late 1980s [12]; this was later modified by Schoon [12, 13]. Currently endometriosis is defined as active or inactive periglandular and/or stromal endometrial fibrosis including glandular alterations within fibrotic foci [14]. Single glands and/or glandular nests may be affected in equine edometriosis [15, 16]. In our equine patients, endometriosis is further classified as either destructive or non-destructive forms of fibrosis [17]. Endometriosis is a condition that involves the stroma, progressing to involve around the endometrial glands and is characterized by periglandular fibrosis associated with dysfunction of affected glandular epithelial cells [18–20]. Distinguishing between active and inactive, destructive or non-destructive, is based on the morphology of stromal cells involved in fibrotic foci via pathological examination. For the attending veterinarian, regardless of type of endometriosis, the clinical picture would be one of an aged mare, with a history of multiple pregnancies, and pregnancy failure, often with a number of years (recently) of bareness. In tandem these mares are often observed clinically to have endometritis, the hallmark often being hyperechogenic particular fluid within the uterine lumen on transrectal ultrasonography. Destructive endometriosis is characterized by a strong epithelial vimentin expression, excessive extracellular matrix accumulation, cystic gland dilations and mechanical destruction of those glands [21]. Non-destructive endometriosis is characterized by glandular epithelial cells that are intact whereas their degeneration and necrosis are features of destructive endometriosis [22]. Active stromal cells are characterized by an oval shape, pale cytoplasm, and ovoid hypochromatic nuclei whereas inactive stromal cells are spindle shaped with elongated hyperchromatic nuclei [23]. The cyclicity of our mare patients (mares being long day breeders) and associated seasonal endocrine changes appear to have no effect on the disease process.

Mare's age, repeated insult on the endometrium, multiple pregnancies and parturition have been implicated as etiological factors for the development of the degenerative changes within the endometrium seen in endometriosis [24–27]. Nonetheless, not all of these are universally accepted as etiologic factors, as Hoffman [28] found no correlation between endometriosis and number of foaling (i.e. number of pregnancies carried to term), and indeed that of season and estrous cycle associated changes. Older mares with endometriosis have been shown to have a deficient uterine blood flow during pregnancy with poor placenta microvillus development contributing to increased pregnancy loss, or the birth of weak foals [29]; which also reduced fertility in our managed broodmare populations. Further etiologic factors have been described include periglandular localized endometriosis, focal oxygen deficiency (caused by angiosclerosis) and wound healing after mechanical damage leading to physiological turnover of the basal lamina [30]. In a retrospective study by Ebert et al. [31], found that 90 to 92.5% of uterine biopsies showed signs for endometriosis in age categories of 16–20 years and > 20 years of age, respectively.

Following deposition of semen in the uterus, whether by natural service or artificial insemination, a transient inflammatory process is initiated within the uterus. This

#### *Endometriosis in Mare; What the Mare Can Teach Us When Dealing with Endometriosis... DOI: http://dx.doi.org/10.5772/intechopen.100515*

is a normal physiologic event in the mare. This process removes excessive spermatozoa and bacteria that may be induced in the uterus are removed. This process peaks some 12 hours after deposition of semen within the uterus, and is usually completed within 48 hours after insult [32]. It has been suggested that this mechanism of uterine clearance is a critical factor in the uterine defense against infection [33].

Mares susceptible to endometritis have difficulty in clearing inflammatory debris either due to anatomical and/or degenerative defects that interfere with uterine drainage. These defects include a pendulous uterus, impaired myometrium contractility, lymphatic or cervical drainage, atrophy of endometrial folds and disturbed mucociliary clearance. It has been postulated that this decreased physical clearance may increase endometrial periglandular fibrosis [34] and result in decreased pregnancy rates in the mare. There is consensus amongst authorities that there is an initial insult to the endometrium, whether bacteriologic in nature or not, that triggers the start of a complex pathophysiologic process, ending in endometriosis [35–39]. Nonetheless, there are some underlying risk factors to the development of endometriosis. In the initial stages of endometriosis, stromal cells synthesize collagen fibers and differentiate into myofibroblasts, which in turn are responsible for extracellular matrix deposition, eventually ending in endometrial periglandular fibrosis [40]. Periglandular-accentuated mononuclear cell infiltrates (PAMC) have also been suggested as a possible triggering event to endometriosis development [41].

Once established, the endometrium is characterized by abundant fibrosis, ulcer-like holes present on the surface of the epithelium; the cells lack cilia, have few organelles and increased degenerative cell structures [42]. In spite of extensive tissue damage seen in endometriosis, cyclic vascular and non-vascular tissue growth occurs in a coordinated manner as in non-affected mares [43]. Different types of endometriosis are a reflection of different stages of fibrotic process [44]. These areas (endometriosis-affected) of the endometrium exhibit specific differentiation dynamics and become independent from normal uterine control mechanism [45]. Nonetheless, the cardinal feature of endometriosis according to Walter [46] is the deposition of extracellular matrix by myofibroblasts located around the endometrial glands. The epithelial differentiation of the fibrotic uterine glands can be divided into a cycle synchronous, asynchronous and intermediate forms [47]. Initially an epithelial hypertrophy with subsequent epithelial degeneration and glandular dilatation with congestion of secretions is observed, as well as epithelial cell atrophy; there are marked differences in several epithelial enzymatic secretion pathways in the fibrotic foci compared to unaffected glands [48]. Hoffman [49] found that there is a temporary activation of fibrotic stromal cells which were observed after experimentally induced bacterial endometritis likely mediated via profibrotic growth factors and cytokines released by inflammatory cells.

The precise mechanism of endometriosis in the mare is not yet fully understood despite many decades of research. It appears that there is an initial insult, whether non-infectious such as deposition of semen within the uterus or bacterial in origin, results in neutrophil (PMN) migration into the uterine lumen [50]. PMN are the first line of defense (part of the innate immune defense system) against invading microorganisms [51].

It should be noted that endometriosis does not appear to impair PMN functionality [52]. In susceptible mares (in relation to endometritis), those that have a IIB and III endometrial score of biopsy (**Table 1**) have a significant influx of PMN two to twenty-four hours post insult to the endometrium. This PMN hyperactivation and its role in causing severe inflammation and their role in the progression of endometriosis is not fully unknown [54]. PMN appear to cast off the DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) [55]. These consist of DNA associated molecule complexes which transport nucleic and cytoplasmic


**Table 1.**

*Categorization of endometrial score proposed by Kenny and Doig [53] and modified by Schoon et al. [12].*

proteins, which appear to have immunomodulating properties [56]. PMN play a positive role in infection control, however, they can also have a deleterious effect by their content releasing molecules, which can alter the endometrium potentially contributing to the formation of fibrosis on the endometrium architecture [57].

In inflamed tissues, an intricate network of pro-fibrotic cytokines interacts with extracellular matrix, fibroblasts and other cells to regulate collagen deposition and tissue fibrosis [58]. It is known that in the mare with endometriosis, those that are susceptible to endometritis, there is a release of pro-inflammatory cytokines, including interleukins (IL-1B) and TNFα - both of which have high mRNA expression [59]. However, early in the post insemination period a lower mRNA expression of IL-6 and IL-10 were detected in susceptible PMIE (post mating induced endometritis) mares [60]. Included in that study were mares diagnosed with endometriosis. In a further study, it was shown that *E. coli* induced endometritis showed a sustained expression of pro-inflammatory cytokines (IL-1 and IL-8) within the endometrium [61]. Thus it can be suggested that these disturbed expressions of endometrial inflammatory cytokines may play a role in the pathogenesis of endometriosis [62]. Researchers have found that expression levels of IL1RA (an interleukin receptor antagonist) were lower in endometriosis affected endometria compared with non-affected endometria, suggesting a protective role with the endometrium [63].

The first signs of endometriosis on histopathomorphology are atypical morphology and functional differentiation of periglandular stromal cells [64]. The first stage of fibrosis is characterized by large polygonal periglandular stromal cells (type 1) that synthesize collagen fibers [65]. In advanced fibrosis, metabolic active or inactive stromal cells (type II) without signs of collagen synthesis, as well as myofibroblasts, predominate [66]. In the latter, the contractability appears to be affected and may lead to a constriction of uterine glands resulting in glandular dilation [67]. Additionally myofibroblasts may be able to affect the composition and amount of extracellular matrix by secreting different mediators [68]. Typically the endometrium undergoes cyclic changes with typical histomorphological, ezymethistochemical and immunohistocehmical glandular patterns in response to steroid levels [69]. The changes observed in endometriosis on histopathology are independent of the stage of the estrous cycle.

A number of researchers have examined the alteration in secretion pattern of a number of uterine enzymes, namely uteroglobin, uterocalin, calbindin, uteroferrin, and their potential role in the pathogenesis of endometriosis [70–76]. Uteroclain

*Endometriosis in Mare; What the Mare Can Teach Us When Dealing with Endometriosis... DOI: http://dx.doi.org/10.5772/intechopen.100515*

is the most prominent progesterone dependent uterine derived protein, which is abundantly expressed during pregnancy [77]. Uteroglobin, also progesterone dependent and of uterine origin, is thought to mask the trophoblasts from the mare's immune system, allowing pregnancy to establish [78]. CalbindinD9K is a small cytosolic protein expressed by the endometrium, and has a role in the transport of calcium from glandular epithelia and from the blood supply to the uterine lumen [79]. Uteroferrin again is a endometrial derived progesterone dependent protein that is involved with iron transport during pregnancy [80]. Hoffman [81] showed a synchronous expression pattern: uterocallin and uteroglobin expression were decreased, whereas uteroferrin expression was increased in affected glandular epithelia. Lehman [82] found that affected endometria showed decreased fibrotic stromal cell expression of both estrogen and progesterone receptors compared to unaffected endometria. Normal cyclic dependent expression of these proteins were not observed in affected endometrial tissue, however what is observed is within fibrotic foci, there is a cycle asynchronous patchy protein secretion [83]. Hoffman [84] observed a decrease in uterocallin expression in mares suffering from both destructive and non-destructive fibrosis of their endometrium. Hoffman [85] found that there was decreased expression of CalbindinD9K in fibrotic glands of the endometrium; however, this was not observed by Lehman [86] and therefore is an unreliable marker for endometriosis in the mare. Lehman [87] concluded that uterogoblin and uterocallin should be utilized when trying to refine biopsy classification of endometrium biopsy for our mare population.

Two enzymes that are secreted in abundance in the process of fibrosis in the endometrium, that are capable of degrading collagen IV and laminin, are Tissue Transglutaminase (TG2) and Matrix Metalloproteinase (MMP2) [88]. These enzymes have also been implicated in causing dilation of uterine glands [89]. However, although these enzymes have been implicated in fibrosis, there is contradictory evidence to suggest that there is no correlation between the secretion of these enzymes and the development of endometriosis in the mare [90]. MMP-9 has been shown to degrade collagen IV, the main component of basement membranes [91]. In humans MMP-9 has been reported to be expressed in inflammatory cells as well as glandular and periglandular stromal cells in the endometrium [92].

In a two year experiment where mares were subjected to induced and repeated endometritis researchers found that these endometritis events had no significant exacerbation of the endometriosis observed [93]. Hoffman [94] found in the equine endometrium distinctly reduced ER (estrogen receptors) and PR (progesterone receptors) expression; in fibrotic stromal cells as compared to the unaltered stroma and these markers according to Hoffman [95] are the hallmarks of equine endometriosis. Studies in human patients with endometriosis have found similar results. On the microscopic level it has been reported that the hallmark of endometriosis in the mare is the appearance of concentric arrangement of stromal cells and/or collagen fibers around affected glands [96]. The degree of periglandular fibrosis is determined by the number of periglandular layers of stromal cells and the number of fibrotic nests of glands [97].

Hoffman [98] observed that there is accumulation of fibronectin and proteoglycans particularly in active destructive fibrotic foci and is probably due to an increased number of secretively active myofibroblasts. Hoffman [99] suggests a pathogenesis of endometriosis which the authors describe as conceivable; there is an initial epithelial alteration and activation with a partial thickening of the affected parts of the basal lamina. Only an intact basal lamina is able to suppress the epithelial cell activation and the synthesis of profibrotic growth factors [100]. The early stages of fibrosis are characterized by slight basal lamina alterations with a focal accumulation of stromal cells which synthesize collagen fibers [101].

It has been described in equine endometriosis that there is stromal cell proliferation and their differentiation into myofibroblasts as well as their increased synthesis of extracellular matrix (ECM) occurs in response to a milieu of synergistically and autoinductively acting mediators which might result in a periglandular fibrosis [102].

With different grades of endometriosis, increasing amounts of collagen IV are deposited around the endometrial glands and fibrotic nests [103]. Aresu [104] hypothesized that MMP-9 production would increase with endometriosis; however their results showed no significant increase in this protein level with endometriosis affected mares and controls. They concluded that immunohistochemistry is not useful in clinical practice to evaluate fertility. Hoffman [105] has concluded that it does seem as though endometritis is the initiator to activate the process of endometriosis.

There is clear evidence between age and the degree of endometriosis observed in our equine patients; however the degree of inflammation and grade of endometriosis is poorly correlated [106]. Fibrosis continues to progress even after the inflammatory process has stopped [107]. Therefore this process becomes independent of the initial inflammatory event of which it arose from.

## **3. Role of prostaglandins in the pathogenesis of equine endometriosis**

It has been demonstrated that fibrosis of the lungs is often the result of low grade, chronic inflammation, and this may be true in the development of endometriosis [108]. In addition to the cytokines described above, the role of eicosanoids including prostaglandin E2 and F2ɑ could provide an alternative pathway to fibrogenesis [109].

The short half-life of prostaglandins would suggest that they act locally via specific receptors [110]. When PGE2 binds to its E prostanoid receptor it triggers numerous antifibrotic events within fibroblasts, epithelial cells and leukocytes. However when it binds to PGF2ɑ receptors (to which it has lower affinity) it induces fibrosis with lung tissue. In the woman, there is emerging evidence of the role of PG and the development of fibrosis, with PG receptors providing a signaling pathway for their enzymes to cause vasoconstriction, increase myometrial contractions and pain [111]. It has been shown in the mare's endometrium, those suffering with endometriosis, there is altered synthesis of PG and mRNA transcription of prostaglandin synthases [112]. In i*n vitro* studies, it has been shown that decreased PGE2 production coupled with an increase in mRNA type 1 collagen level due to sustained, low grade chronic infectious stimulus may establish a pathway to endometrial fibrosis [113].

NETs have been shown to decrease prostaglandin E2 that exerts an antifibrotic response after binding to the E prostanoid receptor [114]. It has been suggested that mares with underlying endometriosis predisposes the uterus to endometritis, rather than the other way around. This is based on observations that *in vitro* induced bacterial endometritis with subsequent treatments was not associated with the progression of endometriosis over a 2 year observation period in 90% of the mares. Whether endometritis is the initiator to endometriosis or vice versa, a mutual influence of endometritis and endometriosis has been postulated by a number of authors [115].

Hoffman [116] found that advanced dedifferentiation of the stromal cells within the fibrotic foci led to inadequate hormone receptor expression that are not able to react to cyclic endocrine changes, becoming independent of hormonal control mechanisms in the uterus.

The establishment of equine endometriosis is a dynamic and complicated process, involving PMN, their role in NETs production and the release of their contents, pro-fibrotic cytokines, interleukins and PG production.
