Histological features of uterus
Morphological changes in the uterus of ewes during pregnancy suggest that the uterine wall facilitates embryonic development. The uterine wall of both the gravid and non-gravid uterus consisted of three layers
viz. the endometrium, myometrium and perimetrium (Fig 1). Similar uterine structure also occurs in cows
(Wimsatt, 1950; Espejel and Medrano, 2017).
Endometrium
On histomorphological identification, the lamina epithelialis mucosa was lined by the simple columnar epithelium. Similar observations were reported in other mammals including sows and ruminants
(Eurell and Frappier, 2006); camel
(Porjoosh et al., 2010). The height of the epithelium and endometrial thickness were measured from the different parts of the uterus and the data is presented in Table 1.
During pregnancy the height of the epithelium was maximum in the gravid uterus which is the major modification to the structure of the uterine wall (Fig 2). These changes indicate that in pregnancy the increased surface area is meant for proper attachment of placenta and exchange between mother and offspring. Comparatively, the entire endometrial thickness was more in the middle horn of the gravid uterus. It helps in interdigitation between placental cotyledons and endometrial caruncles form placentomes, which serve a primary role in foetal-maternal gas exchange and derivation of nutrients by the placenta for hemotrophic nutrition of the foetus
(Spencer, 2014). In non-pregnant ewes the endometrial thickness was more in middle horn as compared to the other part of the uterus (Fig 3). Similar results observed in non-luteal groups of cows and ewes
(Shaham- Albalancy, 1997; Benbia et al., 2013).
Comparatively, the lamina propria submucosa of gravid uterus was enriched with vascular loose connective tissue with many more fibrocytes, lymphocytes, plasma cells, macrophages and giant cells than the lamina propria submucosa of non-gravid uterus (Fig 4 and 5). Similar findings were also observed in sow and ruminants
(Eurell and Frappier, 2006; Espejel and Medrano, 2017).
The endometrium was divided into raised aglandular caruncular areas and intensely glandular inter caruncular areas. Identically,
Spencer (2014) identified the similar results in adult ruminants (cattle, goats and sheep). Grossly, the caruncles were concave disc shaped structure and their numbers as well as size were more in the gravid uterus (Fig 6). Comparatively in the gravid uterus, the caruncles were rich in fibrocytes, lymphocytes and plasma cells with extensive capillary networks than non-gravid uterus as earlier reported by
Eurell and Frappier (2006). This extensive capillary network is responsible for the potential exchange between mother and embryo after implantation.
Huckabee et al., (1961) described that in the non-pregnant uterus the blood flow is approximately 25 ml/min. It increases to approximately 200 ml/min. at the 80
th day of gestation and exceeds 1000 ml/min. near the end of the 150-day gestation period of the small ruminants like sheep and goat.
The simple coiled, branched tubular glands were found within the propria submucosal region of intercrancular areas but these were absent in the caruncular area (Fig 7). The glands were lined by simple columnar glandular epithelium which showed variation in the height in non-gravid and gravid uterus. This was in accordance with the results of
Benbia et al., (2013) and
Qureshi et al., (2015). Of note, the endometrial glands were hyperplastic in the gravid uterus but branched more frequently in non-gravid uterus due to high estrogen level (Fig 8) and
Spencer (2014) found these similar results. The cells of the luminal and glandular epithelium were PAS-AB positive. There was a strong alcianophilic reaction in the glandular epithelium of gravid uterus whereas in luminal epithelium, the reaction was faint (Fig 8). This result was in accordance with
Restall (1966). It suggests that during pregnancy, activity of uterine glands increases and secrete more PAS alcian blue positive mucous substances.
The unique structures for pregnancy development were found in the gravid uterus. These were the placental attachments with the caruncles, the placentomes. Simple villi which were developed from the areas of chorionic sac of foetal membrane in contact with elevated cup shaped structure of maternal endothelium (caruncles) was noticed in gravid uterus (Fig 9). The simple chorionic villi ramified to form branching cotyledons and they consisted of a vascular mesenchyme covered with a simple layer of trophoblastic cells. The trophoblast included columnar or irregular shaped mono nucleated cells and large binucleated cells (Fig 10).
Myometrium
The myometrium was composed of inner circular and outer longitudinal layers of smooth muscle fibers and a vascular layer was in between them. Similar observation was found by
Porjoosh et al., (2010) in camels,
Qureshi et al., (2015) in teddy goats and
Espejel and Medrano (2017) in dairy cows. Thickness of myometrium was measured and data is presented in the Table 1 and that showing the thickness of the myometrium, was maximum at the middle of the horn and minimum towards the tip of the horn in both the groups. The overall myometrial thickness was more in the gravid uterus (Fig 3). This difference might be due to hyperplasia and hypertrophy occurring during gestation presumably to provide histotrophic support for conceptus growth and development
(Eurell and Frappier, 2006; Gray et al., 2001). In between the inner circular and outer longitudinal layers, a separate vascular layer with abundant blood vessels within the connective tissue materials was visualized in all types of uterus. In the case of gravid uterus, this layer was packed with more blood vessels as compared to non-gravid uterus (Fig 11). It indicates profuse supply of blood during pregnancy.
Rosenfeld et al., (1974) observed that the uteroplacental blood flow progressively increased throughout the pregnancy and was maximum at the third trimester. “Ligamenta viva uteri” was found mainly within the myometrium of the body of the gravid uterus in the form of the larger and smaller groups. These were the thickenings of the collagen fibers on the wall of the blood vessels which occluded almost the whole of the lumen of the blood vessels (Fig 12). Similar findings were studied in sheep
(Katica et al., 2014) and in women with pre-eclampsia
(Ong et al., 2005).
The special histochemical staining of the uterus with Weigert’s method revealed the presence of elastic fibers around the blood vessels (Fig 13). The longitudinal sections stained with Masson’s Trichrome method showed presence of collagen fibers as a predominant fibers in the vascular layer of tunica muscularis and found in lamina propria submucosa also (Fig 14).
Perimetrium
The perimetrium was clearly separated from myometrium and no variation was noticed between the gravid and non-gravid uterus
(Eurell and Frappier, 2006; Katica et al., 2014). It consisted of loose connective tissue fibers and collagen fibers which were covered by peritoneal mesothelium.
Immunohistochemical studies
IHC is a technique for identifying cellular or tissue constituents (antigens) by means of antigen-antibody interactions
(Kabiraj et al., 2015). In the present study, the greatest number of T-lymphocytes was identified in the glandular and luminal epithelium and in the stroma immediately adjacent to the epithelium of the sheep endometrium. This result was in accordance with the result of
Gottshall and Hansen (1992), Gogolin
Ewens et al., (1989) and
Lee et al., (1988).
Immunohistochemically, CD8+ cells, subtype of T lymphocyte, were identified in different parts of the uterus and were distributed mostly in the stroma and subepithelial regions of both the caruncular and inter caruncular areas of the uterus in gravid and non-gravid ewes.
Meeusen et al., (1993) and
Cobb and Watson (1995) described that the cytotoxic (CD8+) T cells were located primarily adjacent to the epithelial layer of the uterus of sheep. Similar observation was documented in the uterus of the female goat
(Perez-Martinez et al., 2002) and in the uterus of sow
(Kaeoket et al., 2001). We observed more CD8+ cells in the stromal component than in the lining epithelium of endometrium (Fig 15 and 16). Although,
Majewski et al. (2001) reported that CD45R, g¶ T cells and CD8 positive cells were located predominantly in the luminal and glandular epithelium and only a few scattered lymphocytes were present in the stroma of the uterus of the sheep. In some areas within the caruncular stroma of the gravid uterus, CD8 positive cells appeared in the form of clusters (Fig 16). This is in accordance with the findings observed by
Lee et al., (1988). The epithelium of the endometrial glands and their ducts was frequently infiltrated by CD8+ T lymphocytes at the inter-caruncular areas (Fig 17) but the same was absent in the non-glandular carancular areas.
Lee et al., (1988) observed the similar results at all stages of the oestrous cycle inconsistently. However,
Gottshall and Hansen (1992) reported that in most sections, CD4+ and CD8+ cells were rarely observed or absent in the uterine glands of ewes.
The lymphocyte population was significantly more at the middle of the horn of the uterus as compared to the tip of the horn of the uterus in both the gravid and non-gravid ewes (Fig 15 and 16). In comparison between the gravid and non-gravid uterus, the population of CD8+ cells was more in the gravid uterus (Fig 16 and 18) but in the placentome of the gravid uterus, CD8+ cell was nearly absent. Present results indicate that these changes, rather than due to locally acting conceptus factors, are instead the result of hormonal signals of maternal or foetal origin that either act directly on endometrial lymphocytes or stimulate the uterine endometrium to induce synthesis of regulatory molecules that affect lymphocyte dynamics as earlier observed by
Majewski et al., (2001) in sheep. The role of immunocompetent cells was documented by
Bischof et al. (1995) who observed acute inflammation in the pig endometrium in response to fertile mating which included marked changes in the tissue and immune cell components of the endometrium. There appeared to be suppression and activation of various immune cell components in the uteri of pregnant pigs. This phenomenon was presumably in response to foetal or trophoblast antigens, suggesting that the local immune system was involved in the uterine response to pregnancy. In the gravid uterus, CD8+ cell was nearly absent in the placentomal region. A few CD8+ lymphocytes were observed among the stromal cells of the caruncular septa throughout pregnancy.
Gogolin-Ewens et al., (1989) reported that after Day 50 of pregnancy, CD8+ lymphocytes had also infiltrated into the interplacentomal uterine epithelium.
