Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast

Objective: This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. Study design: Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n = 12) and term placental tissues (n = 12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-alpha/IFN-gamma or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means +/- SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. Results: Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-alpha and IFN-gamma or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. Conclusion: These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy. (c) 2012 Elsevier Ireland Ltd. All rights reserved.