In cattle, good reproductive performance is essential and it is considered as a vital factor in determining the efficiency of animal production. However, a number of problems can arise with the reproductive process which can lead to a reduced level of reproductive efficiency and infertility¹. Infertility in bovines was attributed to a number of conditions affecting the genital tract like hormonal disturbances, infectious diseases, nutritional causes, congenital and other pathological conditions. Among the most possible causes, uterine aberrations were identified as one of the important causes of infertility in cows²-³.

Even though several years back the uterus was considered as one of the body organs most frequently affected with malignancy in cattle, but the reports on uterine tumours in cows are very few and the general opinion is that uterine carcinomas are rare in cows⁴-⁵. The uterine carcinomas are not easily diagnosed in the cow and are probably not recognized until the animal comes to slaughter⁶. It was pointed out that small tumours might be overlooked in abattoirs, where the uterus receives only cursory inspection⁴. Further studies revealed that uterine carcinomas are usually asymptomatic⁷ with minimal gross lesions that remain undetected even at slaughter³.

Information regarding the techniques including cytopathology, histopathology and immuno- histochemistry are of value to detect the different tumours in the uterus. But the research till date, did not lay much emphasis on different histological types of uterine carcinomas as the cause of infertility. Keeping these in view, the present study was aimed to study the gross, cytological, histopathological and immuno-histochemical features of uterine adenocarcinomas in cattle.

A total of 150 cattle genitalia were collected and examined from various slaughter houses during the period from 2016 to 2018. Detailed examination of the uteri was carried out for the presence of any gross abnormalities. Impression smears were taken for cytological examination and the slides were fixed in methanol and stained with Leishman stain. Irrespective of gross lesions, tissue pieces were collected from the different areas and fixed in 10 per cent neutral buffered formalin for histopathological examination. The fixed tissues were processed by routine paraffin embedding technique. Sections of 5–6 μ thickness were made and stained with routine Haematoxylin and Eosin (H7E) method⁸.

Out of 150 uteri examined, 29 were positive for adenocarcinomas based on histopathological examination. In majority of the cases, external appearance of the uterus was noticed with the features including firm thickening of the endometrium with fine granularity and yellowish discoloration (Fig. 1, 2). Multiple small greyish white areas in the myometrium were also noticed. In few cases, the endometrium was uniformly thickened with lack of appearance of prominent caruncles. Impression smears taken from the different parts of the uterus revealed presence of clusters of glandular epithelial cells with round to oval hyperchromatic nuclei forming acini (Fig. 3) with more number of nucleoli.

Histopathologically, three patterns of adenocarcinomas were identified namely acinar, tubular and solid patterns. Acinar pattern of adenocarcinomas were noticed in 24 cases (82.75%), four cases (13.79%) revealed tubular pattern and one case (3.44%) was identified as solid pattern.

In acinar pattern, histopathological sections showed scanty stroma compared to glandular tissue. Proliferation of numerous glands lined by irregular single and multi-layered cuboidal to columnar epithelial cells with acidophilic material in the lumen was noticed in acinar pattern of adenocarcinomas. The cells had abundant acidophilic cytoplasm and large vesicular nuclei with prominent nucleoli. Clonal proliferation of small nests and cords of glandular epithelial cells infiltrated from the discontinuous basement membrane of the glands into the dense fibrous endometrial stroma resembling focal solid tumour growth pattern without acini formation was also noticed (Fig. 4). Characteristic features of the proliferated cells in the endometrial stroma were small, polygonal and oval to round with basophilic cytoplasm and had high mitotic index. In addition, the malignant endometrial glands were found to be separated by thin fibrous stroma. Subepithelially, numerous microvessels were found along with infiltrated chronic inflammatory cells including lymphocytes, plasma cells and macrophages.

Endometrial stroma consisting of collagenous connective tissue was penetrated deeply along the neoplastic glands into the myometrium indicated the condition of internal endometriosis (Fig. 5). In few cases, the lumen of the glands found in the myometrium was completely occluded with undifferentiated proliferated epithelial cells with marked anisocytic and anisokaryotic appearance. In some cases, invasion of neoplastic glands was found to the extent of perimetrium from the endometrium (Fig. 6).

In four cases (13.79%) neoplastic glands were arranged in tubular pattern in the endometrial stroma (Fig. 7). The lumen of the glands was filled with the proliferated anisocytic glandular epithelial cells. These glands were surrounded by the fibrous connective tissue and endothelial cells. In one case, neoplastic glands surrounded by myxomatous tissue with angiogenesis were noticed. Subepithelial infiltration of neoplastic glandular epithelial cells was found with multiple microvessels.

Solid proliferation of exclusively glandular epithelial cells occupying the entire endometrial stroma without the presence of architectural details of glands (Fig. 8) was noticed as a rare finding in one case (3.44%). These proliferating tumour cells in the form of solid sheets were partially or completely separated by the fibrous connective tissue septae.

Twenty five(86.20%) cases of adenocarcinomas showed strong and diffuse staining of the VEGF in the cytoplasm of the neoplastic glandular epithelial cells and in the few stromal cells (Fig. 9). Moderate staining intensity was noticed in four (13.79%) cases of acinar pattern of adenocarcinomas. Pan Cytokeratin marker was applied to all the epithelial tumors. In all the histopathologically positive cases, membrane and cytoplasmic staining was noticed in the extensively proliferated neoplastic glands and in the nests of clonally proliferated cells found in the endometrial stroma. Staining intensity was moderate to strong (Fig. 10).

Grossly, the endometrium and serosal surfaces were appeared uninvolved with very minimal features like firm, yellowish and thickening of the endometrium with fine granularity. It was supported by previous reports⁷,⁹,¹⁰ who stated that endometrial adenocarcinomas were asymptomatic with minimal gross lesions. The thickening and granularity was due to an increase in the size and number of glands which are irregular in their distribution. Cytologically, shedding of clusters of pleomorphic glandular epithelial cells with multiple nucleoli were noticed. Similar findings were reportedin buffaloes¹⁰. The continuous shedding of epithelial cells from the uteri was observed both in normal and malignant tissues, but in case of malignant tissues, there was excessive shedding of the cells compared to normal tissues. The shed cells were highly abnormal showing malignant features which can be clearly noticed in impression smears¹¹.

Histopathologically, the normal endometrial glands had a regular glandular structure and were lined by single layer of columnar epithelial cells with basal nuclei. In the neoplastic tissue, the regular glandular architecture was lost and there was proliferation of irregularly shaped acini and tubules which varied considerably in size¹². In women and most of the animals, cystic endometrial hyperplasia was the cause for precancerous lesion by producing excess estrogen⁷. But in the present study on uterine adenocarcinomas, very few cases showed follicular cysts in the ovaries and majority of the cases were noticed without the involvement of ovaries. Though cystic endometrial glands were noticed along with the malignant epithelial cells proliferation, it might not be the cause for uterine carcinoma especially in cattle¹³. In few cases, in addition to neoplastic changes in endometrial glands, chronic endometritic changes by infiltration of chronic inflammatory cells including lymphocytes, plasma cells and macrophages were noticed. The most common cause of endometritis is infection. In women, chronic endometritis due to constant destruction of healthy cells by injuries was stated as one of the major factor responsible for cancer development in uterus¹⁰,¹⁴,¹⁵. On the other hand, neoplastic transformation might also derived from mutagenic reactive oxygen and nitrogen species released from macrophages, cytokines and growth factors which all have tumour inducing properties¹⁶,¹⁷,¹⁸. The presence of plasma cells might be due to the increased levels of proinflammatory mediators including IL-1, TNF-alpha, IL-6 which was produced from necrotising tissue or from infections associated with uterine tumours. IL-6 produces B-cells which will further differentiate into plasma cells¹⁹.

The abnormal epithelial cells were noticed as invaded into endometrial stroma as nests of malignant cells and into the inner circular muscle layers of the myometrium and into the perimetrium. This finding is similar to the report on endometrial adenocarcinoma in a mare²⁰. Stromal infiltration was occurred due to the discontinuity of the basement membrane of neoplastic glands²¹. The Stromal and myometrial invasion was one of the characteristic features of adenocarcinoma²². Numerous microvessels were noticed as a prominent feature when compared to normal tissues. Increased microvessel density was noticed during transition from endometrial hyperplasia to endometrial carcinomas²³.

Vascular endothelial growth factor (VEGF) is an essential and most specific growth factor for vascular endothelial cells. In addition to endothelial cells, VEGF and its receptors are expressed on numerous non-endothelial cells including tumour cells. Vascular endothelial growth factor (VEGF) secreted by tumour might interact with the VEGF receptors that are expressed on those tumour cells. VEGF secreted by tumour cells functions in an autocrine manner and promotes tumour growth²⁴. This autocrine signalling was necessary to sustain self-renewal of tumour cells. In the present study, VEGF positivity was noticed in the cytoplasm of the neoplastic glandular epithelial cells and few endometrial stromal cells also showed positive immunoreactivity along with the tumour cells²⁵-²⁶. The proliferated malignant glandular epithelial cells might have expressed high levels of VEGF gene by autocrine signalling which is necessary for formation of new blood vessels which will in turn supply essential nutrients and oxygen for continuous growth of carcinoma cells²⁴ and this might be a reason for strong expression of VEGF in the adenocarcinoma cases.

Besides histopathological examination, the pleomorphic neoplasms arising from the epithelial cells of the uterus can be further diagnosed by using the marker cytokeratin. Moderate expression of this marker was noticed in the malignantly transformed epithelial cells in the present study in endometrial adenocarcinomas. Our findings were in agreement with previous studies²⁷.

It was concluded that VEGF is responsible for progression of tumour cells and it can be used as best diagnostic marker to identify the presence of angiogenesis and malignancy in the uterus of cattle. These tumour cells may respond to therapy targeting VEGF genes and it can be established in future by further studies.

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