PROJECT SUMMARY Excess redox-active iron, and consequent oxidative stress, contribute to the morbidity and mortality associated with β-thalassemia. Iron overload develops due to low hepcidin and excessive intestinal iron absorption, and from blood transfusions. Anemia and tissue hypoxia, due to ineffective erythropoiesis, also typify β-thalassemia. Pregnancy in β-thalassemia is becoming more common but is considered high risk. The relative contributions of iron overload/oxidative stress and anemia/hypoxia to adverse fetal outcomes are, however, unknown. Given this gap in clinical knowledge, investigation in this area of scientific pursuit is warranted. We utilized a pre-clinical model of β-thalassemia, Th3/+ mice, to test the hypothesis that thalassemic pregnancy disrupts maternal, placental, and fetal oxygen and iron balance. Indeed, several pathophysiological perturbations were observed in Th3/+ dams (iron overload, anemia), placentas (placentomegaly, iron loading, hypoxia), and fetuses (growth restriction, iron loading, oxidative stress, hypoxia and global hypomethylation of DNA in brain), all as compared to WT pregnancies. Notably, abnormalities were seen even in WT fetuses (and pups) from Th3/+ dams. Elucidating how these in utero physiological disturbances impact fetal and postnatal development is an overarching goal of this proposal. In three specific aims, we will expand our experimental analyses of thalassemic pregnancies to define molecular pathways mediating adverse fetal and postnatal effects. In Aim 1, we will test the hypothesis that iron loading exacerbates pathological fetal outcomes in Th3/+ dams. We postulate that high fetal iron increases the risk for oxidative stress, which can cause molecular, cellular, tissue and organ damage. The rationale is that Th3/+ dams have elevated plasma iron throughout pregnancy, which contributes to iron loading of Th3/+ and WT fetuses. We also predict that exposure of developing tissues to oxidative stress in utero increases the risk for postnatal pathologies. In Aim 2, the hypothesis that hypoxia exacerbates pathological fetal outcomes in Th3/+ dams will be tested. We postulate that hypoxia plays an important role in the development of postnatal pathologies in mice born to Th3/+ dams, either by itself or in synergy with iron loading/oxidative stress. The rationale is that ineffective erythropoiesis and anemia lead to fetal tissue hypoxia, which would be predicted to lead to a shift in energy metabolism and cause other metabolic disturbances. Aim 3 will test the hypothesis that lowering plasma iron in Th3/+ dams will prevent fetal iron overload. The rationale is that hyperferremia in Th3/+ dams precipitates fetal iron loading. The approach entails in vivo inhibition of intestinal iron absorption and iron release from stores. The overarching goal of this investigation is to define the pathophysiological perturbations associated with thalassemic pregnancy, which may reveal novel molecular pathways ...