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%.