HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL FEATURES OF DIFFERENTIATED TROPHOBLAST IN CHORIONIC VILLI OF THE PLACENTA IN PRETERM LABOR

Abstract

The importance of studying the histochemical and immunohistochemical features of the diff erentiated trophoblast in chorionic villi of the placenta in preterm labor is determined by the threatening nature of this condition for both mother and fetus, as well as its high prevalence and serious consequences for the health of both. The understanding of the damage to the trophoblast of chorionic villi in the placenta can be expanded by histochemical and immunohistochemical methods, which allow the assessment of the concentration of specifi c marker molecules in one way or another. Objective. To determine certain histochemical and immunohistochemical characteristics of proteins in the trophoblast of the intermediate and terminal chorionic villi of the placenta in preterm labor. Material and Methods. The obtained material (30 placentas from preterm deliveries and 30 placentas from normal pregnancies) was fi xed for 20-24 hours in 10 % neutral formalin solution buff ered in Lilly’s phosphate buff er. After tissue removal, the placental tissue was dehydrated in an ascending ethanol series and embedded in paraffi n at a temperature of approximately 58 °C. Serial histologic sections were cut at 5.0 μm thickness using an MS-2 sliding microtome. After deparaffi nization, histological sections were stained with hematoxylin and eosin, histochemical methods for total protein with bromophenol blue according to Bonhéme, and immunohistochemical techniques according to the manufacturer’s protocols (Dako, Denmark). In particular, immunohistochemical reactions were performed with monoclonal antibodies against trophoblast hormone- placental lactogen and placental alkaline phosphatase. Visualization of primary antibodies was performed using the Dako polymer visualization system with diaminobenzidine as chromogen (resulting in brown staining of the sites of studied antigens). In addition to the descriptive method of histopathologic research, computer morphometry of digital microphotographs of histologic sections was performed using a Delta Optical Evolution 100 microscope and an Olympus SP550UZ digital camera. Digital copies of the images were processed using a legitimate copy of the ImageJ v1.52f computer program developed for histometric studies (National Institutes of Health, USA). Specifi cally, the evaluation of staining intensity (optical density) was performed on digital microphotographs using the method of computer microdensitometry. For this purpose, a microprobe method was used to obtain a computer brightness value in an 8-bit analysis system with 256 gray levels – from black (0) to white (255). The obtained values were then transformed into relative optical density values (r.OD) by logarithmic transformation (natural logarithm method). The relative optical density value ranges from 0 (absolute transparency of the object) to 1 (absolute opacity of the object). The obtained digital data were processed using statistical analysis methods. A legitimate copy of the statistical analysis computer program PAST v4.14 was used, with a preliminary check for normal distribution using the Shapiro- Wilk test. Since, according to this test, the hypothesis of normal distribution was not rejected for the statistical samples studied (at p=0.05), parametric methods of statistical analysis were applied: calculation of the mean and its standard error, Student’s t-test (two-tailed, unpaired). In addition, the non-parametric Mann- Whitney test was used for the reliability of the conclusions. The research was conducted as part of the scientifi c research project «Preservation and Restoration of Reproductive Health in Women and Girls with Obstetric and Gynecological Pathology» at the Department of Obstetrics and Gynecology of Bukovinian State Medical University (state registration number 0121U110020, duration 2021-2025). Results. Microscopic examination of histological sections stained with hematoxylin and eosin did not reveal any diff erences in the structure of chorionic villus trophoblast in the placenta of preterm labor compared to normal pregnancy. However, histochemical and immunohistochemical methods of investigation revealed a number of features in the trophoblast of chorionic villi, where fundamental events related to substance exchange occur – intermediate immature, intermediate mature, terminal villi, including terminal «specialized villi». Conclusions. According to the obtained histochemical and immunohistochemical data, in preterm labor, compared to normal pregnancy, no changes are observed in the trophoblast of intermediate immature villi, while in intermediate mature and terminal villi, there is a decrease in histochemical staining for total protein and immunohistochemical staining for specifi c trophoblast proteins – placental lactogen hormone and placental alkaline phosphatase enzyme.

Keywords

• Preterm Labor; Chorionic Villi of the Placenta; Diff erentiated Trophoblast