Trophoblast invasion of the uterine extracellular matrix, a critical process for human implantation and uteroplacental vascular development, is a striking example of controlled invasiveness. To examine cellular behavior relevant to this process, human trophoblasts were cultured on (a) Millicell filters prelayered with Matrigel and (it) coverslips precoated with a gentle slope of Matrigel (Matribeach). Histologic sections of the Millicell system demonstrated significant invasion. However, on Matribeach the cells exhibited markedly different characteristics depending on the thickness of the Matrigel. On zone 1 (1-4 ,um thick), flat aggregates and syncytia were seen. In contrast, cells on zone 2 (4-14 pzm) formed rounded aggregates with intercellular processes. In this zone, prominent degradation of pericellular Matrigel proteins was assessed by both light microscopy and scanning electron microscopy. Treatment with 8-bromo-cAMP inhibited this proteolytic process. On zone 3 (14-60 pmm), unicellular trophoblasts or small aggregates caused minimal matrix degradation. JEG-3 human choriocarcinoma cells exhibited similar morphologic and degradative properties on Matribeach, but zone 2 proteolysis was not affected by 8-bromo-cAMP. Our results suggest that extracellular matrix thickness has profound effects on cellular morphology and proteolytic activity. Furthermore, while both normal and malignant human trophoblasts can degrade extracellular matrix proteins, only normal trophoblast extracellular matrix degradation is inhibited by 8-bromo-cAMP.During human gestation, trophoblasts invade the uterine extracellular matrix (ECM), remodel the maternal vasculature, and line vascular channels in the placental bed (1). The biological repertoire necessary for these unique trophoblast-ECM interactions is unknown. Although trophoblast behavior has been compared to that of tumor cells with invasive activity, trophoblasts generally exhibit tightly regulated growth and invasion in vivo.Certain features of cytotrophoblast differentiation in vivo can be recapitulated in vitro when human cytotrophoblasts are purified and cultured from term placentae (2). Once isolated from the placental villi, these mononuclear cells undergo morphologic as well as biochemical differentiation in vitro. Cytotrophoblasts form multinucleated syncytia and carry out de novo secretion of human chorionic gonadotropin (hCG), human placental lactogen, and progesterone (2-4). During this differentiation process cytotrophoblasts also attach to ECM proteins (5) and synthesize fibronectin (6) and laminin (7). Urokinase-type plasminogen activator (u-PA) is secreted by cytotrophoblasts in culture (8), as are the plasminogen activator inhibitor (PAI) types 1 and 2 (9). cAMP has profound effects on cytotrophoblasts in culture by affecting their intracellular morphology and synthesis of hCG, fibronectin, and u-PA (4,6,8).Since cytotrophoblasts in culture are capable of exhibiting multiple facets of the trophoblast differentiation process, we wished to determine whet...