Patients who were hospitalized for delivery between July 2019-2020 at Pamukkale University Medical Faculty Hospital Gynecology and Obstetrics Clinic were included in the study. Women who did not have diabetes, vaginal infection or other systemic disease in the PE group, whose blood pressure was higher than 140/90 mm Hg when measured at different times (6-hour interval), and protein more than 300 mg in 24-hour urine were included (n=20). The control group had 16 normotensive women who had no health problems. The placentas in our study were collected within one hour after cesarean or vaginal delivery. The fetal and maternal surfaces of the placenta were cleaned and examined. Two samples were taken from both surfaces, the margin region of the placenta and the middle region between the center and the margin. They were followed by immunohistochemical staining. In order to determine the serum CD97 level of the pregnant women, 5 milliliter blood samples were taken from each patient after obtaining the consent of the patients. The Pamukkale University Scientific Research Projects Coordination Unit funded our study after ethical approval (project number. 2019HZDP021).
Preparation and Immunohistochemical Staining of Tissue Sections
10% Neutral buffered formalin (NBF) fixed parrafin embedded (FFPE) placenta tissues cut at 5 µm thickness by rotary microtome. Parrafin disolved from the tissues by xylen. After deparaffinization, xylene is removed with 100% ethanol and the tissues are rehydrated through a descending series of alcohol to water. With sodium citrate buffer, the heat-induced epitope retrieval method was utilized (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Incubating the tissue with 3% hydrogen peroxide (H2O2) for 10 minutes inhibits endogenous peroxidase activity. The tissue slides were incubated overnight at 4 °C with polyclonal antibodies against E-cadherine (Bioasssay Tech. Lab. BT-AP02809, Dilution rate: 1/100, Birmingham, United Kingdom), N-cadherine (Bioasssay Tech. Lab. BT-AP05798, Dilution rate: 1/200, Birmingham, United Kingdom), Integrin 4 (Fine Test, Fine Biotech. Co., Ltd, FNab04351, Dilution rate: 1/100, Wuhan, China) and CD97 (Fine Test, Fine Biotech. Co., Ltd, FNab01510, Dilution rate: 1/100, Wuhan, China). DAB (3,3′-Diaminobenzidine) used for chromogen staining and hematoxylen utilized for counterstaining.
Elisa Method
The indirect elisa method was used to quantitatively detect CD97 serum levels. Each serum sample of the individuals and the standard reagents of the CD97 elisa kit were analysed in duplicate with the CD97 ELISA kit (Fine Test, Fine Biotech Co. Ltd., EH7229, Wuhan, China) as per the guidelines provided by the manufacturers. Each assay's absorbance was measured using a plate reader (Promega, GloMax®-Multi Detection System, USA) at a wavelength of 450 nm.
Semi-Quantitative Assessment
All the immunostaining slides were examined in a blinded approach by three independent observers. Using the ImageJ technique, staining intensity and the proportion of positively stained cells were determined by capturing 15 picture samples from each sample under the microscope (X40). The percentage of positive cells was graded as 0 (˂10%), 1 (11%–50%), 2 (51%–75%), and 3 (>76%), whilst the intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The intensity x percentage staining score formula was used to transform the immunohistochemical staining expressions of E-cadherin, N-cadherin, Integrin beta-4, and CD97 that were included in the statistics. Semi-quantitative analysis resulted in a final score that varied from 0 to 9 [16,30-32].
Statistical Analysis
Software SPSS 21.0 (SPSS Inc., Chicago, IL) was used for the statistical analysis. Quantitative data were expressed as mean value±Standard Deviation (SD). Semi-quantification of immunohistochemical staining scores was analysed by a Mann Whitney U Test. The comparison between E-cadherin, N-cadherin, integrin beta-4 and CD97 expression and clinicopathological features was analyzed using the Pearson test. Statistical significance was designated as p<0.05.