Laboratory methods for determining the marker of proliferative activity of Ki-67 in the placenta of cows in complicated pregnancy and pathological childbirth

Abstract

In modern high-tech dairy enterprises, the problem of perinatal diseases in highly productive dairy cattle is acute. The current study presents the distribution of extracellular matrix in cows during physiological pregnancy ending in spontaneous labor (FB/SR) and complicated pregnancy with symptoms of preeclampsia/eclampsia ending in pathological labor (OB/PR) using histological, histochemical (HC) and immunohistochemical (IHC) research methods. Biopsy material of placentas was obtained from 10 cows during labor: from 5 cows with FB/SR and from 5 cows with OB/PR. Samples of placentas from cows with FB/SR and OB/PR were subjected to standard histological procedures. Paraffin sections of the placenta, 5 µm thick, were mounted on glasses treated with poly-L-lysine (Menzel). Using IHC studies, the immunolocalization of the Ki-67 biomarker was studied. A histochemical study in the placentas of cows with FB/SR revealed the presence of a large number of small capillaries both in the villi of the cotyledons and in the caruncular crypts, as well as an equilibrium ratio of mesenchymal and epithelial structures. The reaction to vimentin showed degradation of fibroblasts. In the lumen of the crypts, only structureless masses and a small number of macrophagelike cells were recorded. In addition, in the central part of the unchanged villi, a sharp decrease in the number of small capillaries was established, as well as a reduction in the terminal chorionic villi and a decrease in the vascularization of the remaining villi. Immunolocalization of the content of intermediate biomarkers showed the content of a large amount of Ki-67 in the cells of the crypt lining. During pregnancy OB/PR, a decrease in the vascularization of chorionic villi in the cotyledons and degradation of cytokeratin filaments involved in the formation of cellular contacts between the cytotrophoblasts of the chorionic villi of the cotyledons of the fetal part of the placenta and the epithelial cells of the crypts of the caruncles of the maternal part of the placenta have been demonstrated, which will make it possible in the future to develop an enzyme-linked immunosorbent assay (ELISA) as a rapid – test for diagnosing reproductive pathologies in dairy cattle. Pronounced expression of the Ki-67 protein was found in the cells lining the crypts of the caruncles, as well as in the cytoplasmic membrane of the villous cytotrophoblasts. In the placenta of cows whose pregnancy and childbirth were complicated by pathology, the proliferative activity of the cells lining the uterine crypts remained at a physiological level, while individual chorionic villi were completely reduced, and in those that remained, the Ki-67 expression index was 36.94%.