Pregnant rats were exposed to hypoxia (equivalent to 11% oxygen) for defined intervals during gestation (day 6.5 to day 8.5, panels B and C; day 6.5 to day 9.5, panels E and F; day 7.5 to day 9.5, panels H and I; day 8.5 to day 9.5, panels K and L) and then returned to normoxia until gestation day 13.5 when all animals were sacrificed. Gestationally matched pair-fed rats exposed to ambient conditions were used as controls (panels A, D, G, and J). Invasive trophoblast cells were monitored by cytokeratin immunostaining. Panels C, F, I, and L are high magnifications of areas highlighted in the boxes shown in panels B, E, H, and K, respectively. Chromogen: AEC; counterstain: hematoxylin; scale bars: panels A, B, D, E, G, H, J and K = 0.5 mm; panels C, F, I, and L = 0.25 mm. Panel M, quantification of the depth of cytokeratin positive cell penetration into the uterine mesometrial vasculature. Values are means ± the standard error of each mean (n=5 for each group). Asterisks indicate a significant difference between pair-fed normoxia controls and maternal hypoxia exposed placentation sites (day 6.5 to day 9.5, P<0.0003; day 7.5 to day 9.5, P<0.006; day 8.5 to day 9.5, P<0.003). Abbreviations: PF-N, pair-fed normoxia; HYP, maternal hypoxia; Invasion Index, see Materials and Methods or legend to Fig. 3. Please note that exposure to hypoxia from day 8.5 to day 9.5 of gestation is critical to activate the invasive trophoblast lineage.