Apoptosis in placental vessels from pregnancies complicated by diabetes mellitus type I

Introduction: Diabetes mellitus type I (DM type I) in pregnancy is a complication that has an impact on the placental morphology. Due to this pathological condition the placental vasculature also displays structural changes. There are reports on increased level of angiogenesis in placentas from pregnancies complicated by DM type I. While neovessel formation is manifested by an increased proliferation rate, parts of microvasculature might be also eliminated via apoptosis. There are several immunohistochemical markers for detection of apoptosis. The execution phase of apoptosis is performed by activated proteases of caspase family. The detection of activated caspases including caspase-3 was repeatedly used for visualization and quantification of apoptotic processes in tissues.

Aim: We decided to quantify the number of apoptotic cells in placentas from normal pregnancies and pregnancies complicated by DM type I. The main goal of this quantification was to determine whether increased angiogenesis in placentas from pregnancies of DM type I mothers is associated with an increased level of apoptosis in the placental vasculature.

Methods: The placentas from normal pregnancies (n=8) and placentas from mothers with DM 1 (n=18) were obtained at the time of delivery. There were 11 diabetic mothers who had an elevated level of glycated haemoglobin (>4.0 g/l) and remaining 7 diabetic mothers with normal glycated haemoglobin (<4.0 g/l). The specimens were collected using unbiased systematic random sampling, fixed with 4% formaldehyde and embedded in paraffin. Immunohistochemical detection of activated caspase-3 was performed according to a three-step immunoperoxidase method including antigen retrieval in the microwave oven, incubations with anti-active caspase-3 rabbit polyclonal antibody (Cell Signaling Technology) and detection using LSAB+ kit, peroxidase (DAKO). The number of active caspase-3 immunoreactive cells (apoptotic cells) was quantified and normalized to the cross-sectional surface of villi.

Results: In the diabetic group, an average number of apoptotic cells in vessels was 18,07±18,73/mm2. In placentas from normal pregnancies, this value was 8,30±5,75/mm2. The difference was not statistically significant. We also compared the number of apoptotic cells in vessels form pregnancies of mothers with good (normal glycated haemoglobin) and poor (elevated level of glycated haemoglobin) metabolic compensation. Those with good compensation had 17,0±15,2 apoptotic cells in vessels per mm2 of villous cross-sectional surface, while in those with poor compensation this value was 18,8±22,18/mm2. Also this difference was not statistically significant.

Discussion: We found that number of apoptotic cells in placental vessels from DM type I pregnancies as well as in normal pregnancies is rather variable. For this reason a significant difference between these groups was not determined. Regarding vascular changes previously observed in DM type I placentas it could be concluded that these arise as a consequence of neovessel formation, but without significantly increased apoptosis of cells within the vessel wall.