Morpho-functional studies regarding the fertility prognosis of mares suffering from equine endometrosis

Abstract

The aim of the present study was to characterize the morpho-functional features of endometrosis in barren and foaling mares, using both conventional histopathological and immunohistochemical methods. Endometrial biopsy samples were collected during the physiological breeding season from 159 estrous, clinically healthy mares (mean age 12 years), and the quality and degree of endometrosis was histomorphologically defined. The mares were bred and those that foaled were put in the foaling group whereas those that did not foal were placed in the barren group. Foaling mares were then compared with barren mares. Sixty-four percent (101/159) of uterine samples showed varying degrees of endometrosis and were used for this study. The sample population consisted of 51 barren and 50 foaling mares suffering from endometrosis. Expression of steroid hormone receptors (estrogen receptor, progesterone receptor) and endometrial protein secretion patterns (uteroglobin [UG], uterocalin [UC], calbindin_D9k [CAL], uteroferrin [UF]) was evaluated by immunohistochemistry (barren mares N = 51, foaling mares N = 31). In comparison with unaffected glands, fibrotic glands generally showed a cycle-asynchronous, partially patchy protein expression pattern which is interpreted as a sign of endometrial maldifferentiation within fibrotic areas. In barren mares (N = 51) more than half of biopsy samples (27/51) showed a destructive mostly moderate (20/27) type of endometrosis. In affected glands, staining for UG (17/21) was decreased (P < 0.001). Foaling mares (N = 50) frequently showed a mild, nondestructive endometrosis (35/50). Compared with barren mares, foaling mares had statistically (P < 0.05) more often a cycle-synchronous or increased UG expression pattern within fibrotic glands. Obvious deviations of either UG or UC rarely occurred. Within fibrotic foci, UF often demonstrated a cycle-synchronous or more intense expression pattern in both foaling (28/31) and barren mares (41/51), compared with healthy glands. Mares of both groups showed a cycle-asynchronous staining for estrogen receptor and progesterone receptor in the stromal cells in areas of periglandular fibrosis and the glandular epithelia. These findings indicate that affected areas become independent of the uterine control mechanisms and exhibit specific differentiation dynamics. Immunohistochemical investigations showed that the secretory patterns differ between barren and foaling mares. The findings in this study should be considered as a useful addition to the “classical” Kenney categorization.

Introduction

Equine endometrosis is an age-associated, degenerative alteration of the uterine glands and the surrounding stroma, directly related to fertility problems in mares [1]. The degree of endometrosis increases with age of the mare, but a correlation with the number of previous foalings does not exist [2]. Cyclic and seasonal endocrine changes seem not to have an influence on the progress of the disease [3]. To date the etiology of endometrosis remains unclear and no effective treatment is available. 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 nondestructive with biopsies displaying more than 75% of the fibrotic foci of one of these groups being termed “active” or “inactive” fibrosis respectively [3]. Biopsies with approximately equal ratios of active and inactive periglandular stromal cells are termed “mixed” endometrosis. Endometrial fibrosis leads to both epithelial and stromal alterations resulting in degeneration, dilatation, and atypical differentiation of affected glands [4].

In mares, the preimplantation period is particularly long [5], and therefore the endometrial secretory products play a significant role in nutritional support of the conceptus. The equine endometrium with endometrosis seems unable to produce sufficient histotrophe, possibly resulting in embryonic loss in mares [6].

Using immunohistochemistry, equine endometrosis is characterized by abnormal steroid hormone receptor and secretory protein expression patterns [3].

Uterocalin (UC), is the most prominent progesterone-dependent uterine protein, and is maximally expressed during both early pregnancy and the period of the endometrial cup reaction [7]. It acts as a carrier protein and transports small hydrophobic molecules from the mare to the conceptus [8], [9]. Another major progesterone dependent protein of the horse uterus is uteroglobin (UG). Uteroglobin may support the pregnancy by masking trophoblastic cells from the immune defense system of the mother [10], as well as having anti-inflammatory and antichemotactic activities [10], [11] and performing carrier functions for highly specific lipophilic ligands [12], which may influence endometrial receptivity. The calcium-binding protein calbindin_D9k (CAL) is a small cytosolic protein which is expressed by the uterus [13]. It transports calcium from the basolateral site to the apex of the glandular epithelia and from the blood to the lumen of the uterus [14]. There are species-specific variations in expression of steroid hormone receptors. Uteroferrin (UF), another endometrial progesterone-induced protein, has also been partially characterized in uterine secretions from the pregnant mare [7], [15]. It appears to play an important role as an iron transporter from the maternal endometrium to the conceptus [16] and as a hematopoietic growth factor in the developing embryo [17]. The secretion is induced by progesterone and synergistic effects of estrogen, released by the equine conceptus [18].

Hoffmann [3] established histochemical staining procedures and immunohistochemical methods to characterize the secretory patterns of selected endometrial proteins in mares suffering from endometrosis. Furthermore, Hoffmann [3] showed an asynchronous, mostly decreased expression pattern of UC and UG and an increased expression of UF by affected as opposed to unaffected glandular epithelia. This suggests that the deviation of the protein pattern may be a possible cause for the reduced fertility in barren mares suffering from endometrosis.

The categorization of Kenney and Doig [19] to summarize and classify the different histopathological findings in endometrial biopsies has been accepted for years. Since 1986, many additional histopathological findings (i.e., endometrial maldifferentiation, degenerative and inflammatory angiopathies) relevant for breeding prognosis have been described in equine gynecopathology. These findings and the reversibility of lesions as well as the age of the mare are not considered in the current categorization system. Consequently, the “classical” Kenney categorization requires a number of modifications [20]. To date, the categorization of equine endometrosis [19] is only based on the degree of fibrosis. This investigation attempted to ascertain whether the quality of fibrosis and any obvious deviations of the endometrial protein patterns should be considered as additions to the classification system for a more precise fertility prognosis.