Int. J. Morphol., 39(1):38-44, 2021. Expression of ADAMTS13 and PCNA in the Placentas of Gestational Diabetic Mothers Expresión de ADAMTS13 y PCNA en las Placentas de Madres Diabéticas Gestacionales Süleyman Cemil Oglak1 & Mehmet Obut1 OGLAK, S. C. & OBUT, M. Expression of ADAMTS13 and PCNA in the placentas of gestational diabetic mothers. Int. J. Morphol., 39(1):38-44, 2021. SUMMARY: GDM is linked with overexpression of inflammatory cytokines and increased oxidative stress, leading to endothelial dysfunction and vascular disorder. Weaimed to examine the expression of ADAMTS13 and PCNA in the placentas of gestational diabe- tes mellitus (GDM) patients to investigate the effects of hypoxia, induced by GDM, on proliferation and extracellular matrix formation in the maternal and fetal placenta cells. A total of 60 placentas were collected from pregnant women admitted to the obstetrics clinic. Thirty of them were diagnosed with GDM, and 30 of them were diagnosed with non-GDM patients. Samples were fixed in 10 % formaldehyde, after routine follow-up, embedded in paraffin wax. Sections of 5 µm were cut stained with Mayer Hematoxylin-Eosin, examined under a light microscope. Sections for immunohistochemical analysis were cut and processed for antigen retrievalin citrate solution. Sections were incubated with ADAMTS13 and PCNA primary antibodies, counterstained with hematoxylin, and evaluate under a light microscope. In histopathological examination, the non-diabetic placentas showed that decidua cells in the maternal region were polygonal with oval nuclei and organized in groups. In the GDM group, there were pyknosis and apoptotic changes in decidua cell nuclei. Vacuolar areas were observed in large cavities in maternal connective tissue. Inflammation and dilatation with congestion were observed in the blood vessels of the villus. In the GDM group, positive ADAMTS13 expression was observed in the decidua cells vascular endothelial cells, and surrounding connective tissue fibroblast cells. In the GDM group, a significant increase in PCNA expression was observed in decidua cells, connective tissue cells and endothelial cells. Functional changes in ADAMTS13 proteases and PCNA were thought to induce maternal and fetal complications by stimulating extracellular matrix development. KEY WORDS: Gestational diabetes mellitus, ADAMTS13, PCNA INTRODUCTION Gestational diabetes mellitus (GDM) is one of the traumatic vaginal delivery, insufficient nursing, maternal/ most critical gestation complications affecting 7 % of all fetal hyperglycemia (Crowther et al., 2005). Morphological pregnancies. GDM is defined as any glucose intolerance with examinations of GDM placentas have shown structural the onset or first recognition during gestation can be alterations in the syncytiotrophoblasts, cytotrophoblasts, diagnosed with a glucose tolerance test during gestation. trophoblastic basement membrane, and fetal vessels (Hon- Hyperglycemia is characterized by several degrees of ma- da et al., 1992). ternal (cesarean delivery, future diabetes, maternal death) and fetal (premature birth, macrosomia, neonatal morbidity, A Disintegrin and Metalloproteinase with perinatal mortality) complications (Shaat & Groop, 2007). Thrombospondin motifs (ADAMTS) proteases are expressed in many tissues beginning from the development of the GDM is linked with overexpression of inflammatory embryo. They are involved in many processes, including cytokines and increased oxidative stress, leading to restructuring the extracellular matrix, remodeling tissue, endothelial dysfunction and vascular disorder (Di Fulvio et angiogenesis, organogenesis, and invasion of tumor cells al., 2014). GDM pregnancies carry high risk and require (Kelwick et al., 2015). Disintegrin or cysteine-rich domain close maternal and fetal follow-up. GDM can cause in ADAMTS family members have been shown to regulate morbidity and mortality such as polyhydramnios, intrauterine migration and cell adhesion. The active metalloproteinase growth restriction, fetal hypoxia, fetal macrosomia with domain has been reported to disrupt extracellular matrix 1 Department of Obstetrics and Gynecology, Health Sciences University, Diyarbakır Gazi Yasargil Training and Research Hospital, Diyarbakır, Turkey . 38 OGLAK, S. C. & OBUT, M. Expression of ADAMTS13 and PCNA in the placentas of gestational diabetic mothers. Int. J. Morphol., 39(1):38-44, 2021. components, thereby promoting cell proliferation, cell test was performed after a fasting period of 8 to 14 hours in migration, and angiogenesis by inducing the secretion of the morning. Patients experienced the OGTT after three days cytokines and growth factors (Martin et al., 2015; Ota et al., of unlimited physical activity and free diet (>150 grams 2016). ADAMTS13 performs an essential role in regulating carbohydrates per day). One or more of the test’s values must thrombosis and hemostasis by cleaving the von Willebrand be equaled or exceeded for the diagnosis of GDM (Committee factor (VWF) (Dong et al., 2002). ADAMTS13 mRNA was on Practice Bulletins—Obstetrics, 2018). Patients with health detected in human placentas (Plaimauer et al., 2002). Once conditions related to intrauterine hypoxia (smoking, a cell undergoes apoptosis, its chromatin begins to conden- preeclampsia, maternal obesity, anemia, advanced maternal se, and its size begins to shrink. Later the cell divides into age, inflammatory diseases during pregnancy, infections, and smaller apoptotic bodies. Fragmented nuclei and all asthma) were excluded. structures of the cell are eliminated. Histopathological examination. Placental parts were fixed Proliferative cell nuclear antigen (PCNA) is a marker in 10 % formaldehyde, followed by washing, dehydration, for the cell cycle. Although PCNA is an accessory protein clearing in xylene and incubated at 58 °C in paraffin wax. required for DNA synthesis, its deficiency prevents cell Five mm sections were taken from paraffin blocks, and division (Jónsson & Hübscher, 1997). Besides DNA sections were transported to distilled water through replication, PCNA is also related tochromatin modification, deparaffinization and descending alcohol series. We stained DNA repair, sister-chromatid adhesion, and cell cycle con- the sections with Mayer Hematoxylin-Eosin and mounted trol (Maga & Hubscher, 2003). In their study, Acar et al. with Entellan™. Zeiss Imager A-2 Axio (Germany) light (2008) reported that PCNA immunopositive cell frequency microscope was used for the evaluation of the tissue sections. decreased in diabetic rat placentas and the control group. In another study, it was observed that the number of PCNA Immunohistochemical examination. Five µm sections immunopositive cells in the placenta decreased in parallel were taken from paraffin-embedded placenta blocks, then with the gestational age. These findings support that the deparaffinized passed through the alcohol series and brought placenta loses its proliferative character as it approaches term to distilled water. Sections were held in the microwave oven (Maruo et al., 2001). for 3 x 5 min in citrate solution for antigen retrieval. After allowing to cool for 10 min at room temperature, we washed In this study, we aimed to examine the expression of the sections with a phosphate buffer solution (PBS). They ADAMTS13 and PCNA in the placentas of gestational dia- were then treated with 3 % hydrogen peroxide (H2O2) for betes mellitus (GDM) patients to investigate the effects of 10 minutes. Samples were rinsed in distilled water and hypoxia, induced by GDM, on proliferation and extracellular washed with PBS (pH 7.6). Then, we incubated the sections matrix formation in the maternal and fetal placenta cells. with mouse monoclonal anti-CD44 antibody (Santa Cruz, 1:100) and mouse monoclonal anti-PCNA antibody (Santa Cruz, 1:100). Washing three times in PBS, secondary MATERIAL AND METHOD antibody solution (Biotinylated Goat Anti-Mouse, LabVision) was dropped on slides for 10 minutes. The streptavidin peroxidase solution (Streptavidin Peroxidase, The study protocol was approved by the Health LabVision) was used in the sections for 15 minutes. After Sciences University Diyarbakır Gazi Yas¸argil Training and washing with PBS, a 3-amino-9-ethylcarbazole (AEC) Research Hospital Ethical Committee, and all patients signed chromogen solution was applied to slides for 8 minutes. The an informed consent form. A total of 60 placentas were sections were washed with distilled water and counterstained collected from pregnant women who were admitted to the with hematoxylin for 2 minutes to evaluated under a light obstetrics clinic. Only placentas between 28-38 weeks of microscope (Nikon). gestation were included. These placental tissues were obtained from non-diabetic and diabetic pregnancies Statistical analysis. Statistical analyses of the data were immediately after cesarean section. conducted using IBM SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Measured variables were presented as The groups were assigned 30placentas of GDM mean±standard deviation. Shapiro-Wilk tests were used to patients and 30placentas of non-diabetic patients. A fasting determine whether the numerical data matched the normality plasma glucose >126mg/dl is diagnostic of overt diabetes. distribution. The independent t-test was used to compare the GDM was diagnosed with 75 g oral glucose tolerance test normally distributed data. We used the Mann-Whitney U test (OGTT) venous plasma/serum threshold values as follows: to compare the non-normally distributed data.
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