**2. Results**

### *2.1. General Characteristics and Pregnancy Outcomes:*

At gestational day (GD) 19, dams (*n* = 5 per group) subjected to placental ischemia had reduced body weight (266.5 ± 13.5 g) compared to sham-operated (control) pregnan<sup>t</sup> rats (303.1 ± 9.1 g; *p* = 0.027; Figure 1A).

**Figure 1.** General characteristics of dams and fetuses subjected to placental ischemia. Dams had (**A**) reduced body weight, (**B**) reduced numbers of live fetuses, and ( **C**) increased fetal demise at gestational day (GD) 19 compared to the sham controls. Fetuses subjected to placental ischemia had (**D**) increased hematocrits and (**E**) no change in brain water content. Values for individual rats (*n* = 5 dams per group) are shown along with the Mean ± SEM. Fetal hematocrit and pup brain water content represent the mean of 1–2 pups/dam (*n* = 5 dams). Di fferences between groups were analyzed using an unpaired *t*-test.

A key characteristic of placental ischemia, induced using the RUPP procedure, is fetal demise in the form of increased fetal resorptions [19]. We therefore counted the number of live versus resorbed fetuses present at GD 19. Dams subjected to placental ischemia had a trend for fewer live fetuses (7 ± 1 in RUPP group versus 9 ± 1 in the sham control; *p* = 0.073; Figure 1B) and more fetal resorptions (6 ± 2 in RUPP group versus 1 ± 0 in the sham control; *p* = 0.021; Figure 1C) compared to the sham controls. This demonstrates that we successfully induced placental ischemia in the dams. We then assessed the effect of placental ischemia on the developing fetus. Because placental ischemia leads to reduced blood

flow to the fetal-placental unit, we hypothesized that fetuses would have evidence of systemic hypoxia. We, therefore, measured pups' hematocrits and found an increase in the hematocrits (36.7% ± 3.0% versus 29.3% ± 2.1% in sham) of fetuses exposed to placental ischemia (*p* = 0.040; Figure 1D). There was no di fference in fetal brain water content between the groups (87.73% ± 0.04% in sham versus 87.73% ± 0.07% in RUPP-exposed; *p* = 0.485; Figure 1E), suggesting no cerebral edema. Additionally, in this cohort, we found no di fferences in maternal blood pressure between the groups (98 ± 4 mmHg in sham versus 99 ± 5 mmHg in RUPP; *p* = 0.405). Thus, our findings are due to utero-placental ischemia, independent of elevated blood pressure.
