**6. The Influence of Fluid Shear Stress on Trophoblast Motility**

Early after blastocyst implantation and initial development of primary placental villi, extravillous trophoblasts detach from villi and start to invade into the decidual stroma. Once these extravillous trophoblasts have eroded uterine spiral arteries (now referred to as endovascular trophoblasts [1]), they are suggested to migrate along the luminal surfaces of the vessels and remodel them by interdigitating between the endothelial cells [14]. Thereby, endothelial cells are increasingly replaced and most of the musculoelastic tissue in the vessel walls dissolve, resulting in low-resistance vessels to guarantee constant and maximal uteroplacental blood flow at the transition from the first to second trimester of pregnancy. Hence it is more than likely that fluidic flow not only influences physiology of the villous trophoblast, but also that of endovascular trophoblasts located in invaded spiral arteries. Importantly, with the erosion of the uterine vessel walls and the phenotypic switch from interstitial extravillous trophoblasts into endovascular trophoblasts, cells are exposed to a much higher fluid shear stress than the subpopulation of villous trophoblasts that are faced with rather low shear stress in the intervillous space. In the context of spiral artery remodeling, a vast majority of studies used co-cultures of extravillous trophoblast cell lines (Table 1) with endothelial cells, such as HUVEC [14,18,20,28] and human uterine microvascular endothelial cells (UtMVEC) [23,24]. On the basis of such co-cultures, as well as trophoblast monocultures, a growing body of evidence suggests that in particular motility of endovascular trophoblasts is controlled by flow. However, it should be noted here that used extravillous trophoblast cell lines (Table 1) seem to be the right choice for such studies, although it remains to be discussed to what extent they resemble the endovascular phenotype.

Initial experiments with early gestation macaque trophoblast cells exposed to flow, showed that cells exhibit clear migration in the direction of flow as well as shape changes that involve extension and retraction of filopodia at its leading edge (Table 1, [22]). In doing so, macaque trophoblast migration velocity and movements increased with the magnitude of the applied shear stress (from 7.5 and 15 up to 30 dyn/cm<sup>2</sup> ). These observations have been confirmed by others, showing that migration of the human first trimester trophoblasts occur generally in the direction of flow (up to 30 dyn/cm<sup>2</sup> ), with only a few cells migrating against the flow stream [19]. The enhanced motility under fluid shear stress seems to be accompanied by increased expression of integrin β1, which mediates adhesion of human and macaque trophoblast cells to endothelial cells in vitro [39]. This has been shown in previous migration experiments, suggesting that factors expressed on the surface of uterine endothelial cells and factors released by the endothelium regulate trophoblast migration under flow [23]. While the extent of migration against flow at higher and more physiological shear stress levels (15 and 30 dyn/cm<sup>2</sup> ) decreased significantly for macaque trophoblasts alone, migration against flow remained virtually unchanged for trophoblasts co-cultured on human uterine microvascular endothelial cells. Hence, migration behavior of trophoblasts cultured under flow clearly depends on the substrate they are seeded.

When SGHPL-4 trophoblasts were cultured on the extracellular matrix glycoprotein fibronectin or a layer of endothelial cells, they did not undergo directional migration in 0.5 and 2 dyn/cm<sup>2</sup> cultures. However, under conditions of 4 and 6 dyn/cm<sup>2</sup> , trophoblasts migrated with the direction of flow [14]. In contrast, another study suggests that the average migration velocity of human first trimester trophoblasts cultured on a type I rat collagen-coated surface increased almost linearly with increasing shear stress (from 7.5 up to 30 dyn/cm<sup>2</sup> ) (Table 1, [20]), whereas migration velocity remained almost unchanged at all levels of shear stress when cells were cultured on endothelial cells. Similar behavior was observed for the displacement of trophoblasts by flow. The absolute x-direction displacement increased with increasing shear stress, when trophoblasts were cultured on a collagen-coated surface. However, when cultured on top of an endothelial cell monolayer, first trimester trophoblasts showed a strong ability to withstand displacement by flow. Subsequent flow experiments with neutralizing antibodies suggested that integrin β1 regulated adhesion of trophoblasts to endothelial cells under shear stress [20]. However, another study using SGHPL-4 cells suggested that fluid shear stress did not affect expression of the adhesion molecules E-selectin and integrins α4, β1, and αVβ3 [14]. Different cell types and particularly varying fluid shear stress values exposed to cells may explain discrepancies in motility and expression of adhesion molecules. Accordingly, James et al. recently suggested a scenario that extravillous trophoblasts in early first trimester do not undergo directional migration under very low shear stress, whereas later on, as shear stress increases with complete dissolution of trophoblast plugs, the number of trophoblasts are stimulated to migrate in the direction of flow [14].
