Apoptosis is a process of programmed cell death and is regulated by various proteins, including membrane-associated proteins, such as Fas (CD95), Fas ligand (CD95L), tumor necrosis factor (TNF) a, and TNF receptor, and cytoplasmic/nuclear proteins, such as Bcl-2, Bak, Bax, and caspases. Apoptosis takes part in both embryonic tissue morphogenesis and adult tissue regulation by balancing homeostasis. The most evident morphological signs of ap-optosis are cellular shrinkage, membrane blebbing, nuclear condensation, and fragmentation, which also are the final steps of consequential signaling cascades. Yui et al. suggested that a physiological role of TNFa and interferon-7 expression in the placental villi may induce apoptotic death of cytotrophoblasts.

Vasculogenesis is the process of new blood vessel formation from undifferentiated mesenchymal cells, whereas angiogenesis is the process of new blood vessel formation from preexisting vessels. Both processes occur in direct response to tissue demands and are crucial not only for the pregnancy-associated changes in the reproductive tract but also for embryonic and extraembryonic tissue (i.e., placenta) development. Endometrium, decidua, and placenta are rich sources of angiogenic growth factors. In a recent study, Demir et al. showed that the origin and stages of placental angiogenesis differ from those of the postnatal angiogenesis in terms of cell types and angiogenic factors.

Placental vasculogenesis starts just after the invasion of allantoic mesoderm. Thus, the first structure of tertiary villi emerge around 21 days postconception (dpc). Vasculogenesis consists of three major steps: differentiation of undifferentiated mesenchymal cells to hemangioblasts and then to angioblasts, assembly of primordial vessels setting up a primitive vascular network, and transition from vas-culogenesis to angiogenesis. Two forms of angiogenesis have been described: sprouting angiogenesis, and nonsprouting angiogenesis. During the vasculogenesis, first roundish or cord-like masses of hemangioblastic cells can be distinguished, followed by a clearly defined lumen around 28 dpc. However, Demir et al. showed that the first signs of primitive lumen formation start around 23 dpc.

As described above, placental vasculogenesis includes several steps, and collection of very early (22-48 dpc) placental samples to analyze these steps is quite difficult. Moreover, placental angiogenesis is different from conventional angiogenesis as described above, suggesting the existence of additional steps in the process. In the present study, we hypothesized that during placental vasculogenesis and angiogenesis, differentiating endothelial and mesenchymal cells undergo apoptosis during the lumen formation, angiogenic cell cord connection, and vascular branching steps. To our knowledge, no studies have described any apoptotic signs through placental vasculogenesis and/or an-giogenesis. Therefore, our aim was to analyze molecular and morphological markers of apoptosis using immunohis-tochemistry, TUNEL, histochemistry, and electron microscopy during very early stages of placental vasculogenesis.