Synergy between Iron Deficiency and Inflammation Adversely Affects Pregnancy

Iron is essential for maternal and fetal health during pregnancy. In many developing countries, nutritional deficiencies are common, with anemia affecting upwards of 60% of pregnancies. In addition, infection and inflammation, which are known to be detrimental to pregnancy, are also very prevalent in these regions. Even in high-resource countries, approximately 22% of pregnant women are anemic, and obesity and diabetes are common causes of maternal inflammation. Thus, iron deficiency and inflammation commonly co-exist. In our studies, we noted in mice a striking synergistic adverse interaction between maternal iron deficiency and inflammation that resulted in embryotoxicity, not observed in either condition alone. Considering the high global prevalence of concomitant iron deficiency and inflammation in pregnant women, the issue has outstanding translational importance. We induced iron deficiency in mice by feeding females an iron-poor diet (4ppm) for 1-3 weeks prior to mating and for the duration of pregnancy. This resulted in development of iron deficiency and anemia in both mothers and embryos by term [embryonic day (E)18.5] and placental iron concentrations decreased by half, compared to pregnancies fed control diet (100ppm iron). To model acute systemic maternal inflammation, pregnant females received a single moderate dose of LPS (E. coli serotype O55:B5, 0.5μg/g bodyweight subcutaneously), either in the “2nd” (E8.5) or “3rd” (E15.5) trimester. Embryos and placentas from the E8.5 LPS treatment were collected at E18.5, while those from the E15.5 LPS treatment were collected after 24h. Consistent with other reports, LPS injection alone resulted in some adverse pregnancy outcomes: injection on E8.5 in iron replete dams caused abortion in 22% of dams (total n=9 dams) and at E15.5 caused preterm birth in 50% of dams (n=8) but no embryo malformations or resorption were noted. In contrast, when pregnant dams were both iron-depleted and LPS-injected, we observed a striking synergistic effect resulting in embryo death and resorption in all E8.5 treated pregnancies (n=3 dams). In E15.5-treated pregnancies, 60% of embryos were dead and resorbing (n=42 embryos from 5 dams). This dramatic interaction between iron deficiency and inflammation has not been reported previously. Our data suggest the mechanism appears to be, at least in part, related to activation of the TNFα/TNF-R1 pathway and apoptosis of placental endothelial cells. To test the contribution of the TNFα/TNF-R1 pathway to adverse synergy between iron deficiency and inflammation, we generated endothelial TNF-R1 knockout (KO) mice using two different Cre drivers ( Tnfsfr1 f/f;VEC-Cre + or Tnfsfr1 f/f;Tie2-Cre +). Tnfsfr1 f/f and KO females were iron-depleted and mated with Tnfsfr1 f/fmales. Pregnant dams received a single subcutaneous dose of LPS at E15.5 and pregnancy outcomes were assessed after 24h. Embryos from KO dams were protected from LPS-induced embryotoxicity (30% death, n=7 dams) compared to embryos from WT dams (70% death, n=3 dams). Surprisingly, embryo genotype did not play a role in adverse synergy. Our data suggest that maternal TNFR signaling facilitates adverse interactions between iron deficiency and inflammation during pregnancy, which results in embryo death. These studies are ongoing and our results could provide a mechanistic explanation for known adverse outcomes in complicated human pregnancies, and eventually help inform interventions to optimize outcomes in such pregnancies