Disruption of Hephaestin in the Placenta and Fetus Leads to Fetal Anemia

Background: The efficient transfer of iron across the placenta is crucial for fetal development, however, the molecular mechanisms are incompletely understood. The ferroxidase zyklopen was previously thought to play a role this process by facilitating the export of iron from the placenta to the fetal circulation, however, we recently showed that fetuses from mice lacking zyklopen showed no difference in iron status when compared to littermate controls. While two other ferroxidases, hephaestin and ceruloplasmin, increase the efficiency of iron export from several other tissues, whether they also function in the placenta is not known. Therefore, the aim of the current study was to examine the role of these ferroxidases in placental iron transfer.

Methods: To achieve this aim, we time mated female mice heterozygous for either ceruloplasmin or hephaestin knockout alleles with their respective homozygous knockout males to produce litters containing both heterozygous and homozygous knockout fetuses. This also causes disruption of the genes in the nutrient transporting layer of the placenta. Mice were euthanized at day 18.5 of gestation and placental and fetal tissues taken for analysis. Hematological parameters in the fetuses were examined using a Coulter AcT diff Hematology Analyzer. Total fetal iron levels, which represent the total amount of iron transported across the placenta, were determined by inductively coupled plasma mass spectrometry. The expression of iron transport and storage molecules was examined by western blotting and qPCR in both placenta and fetal liver. Placental iron transfer was directly measured using the radionuclide ⁵⁵Fe.

Results: We found no difference in the amount of iron transferred to the fetus when ceruloplasmin was knocked out compared to littermate controls, nor did we detect a difference in any other iron parameter in these mice. In contrast, the absence of hephaestin resulted in a significant reduction in fetal hemoglobin, red blood cell count, mean cell volume and hematocrit. However, there was no difference in the total amount of iron transferred to the fetus, which implies that the anemia seen is due to a redistribution of iron rather than a defect in placental iron export. In support of this, we observed a significant increase in the iron storage protein ferritin in livers from hephaestin knockout fetuses when compared to littermate controls, suggesting higher levels of iron in this organ. To investigate this further, we gavaged pregnant dams with ⁵⁵Fe to examine placental iron transfer directly. Thirty minutes after gavage, the level of the isotope was increased in the livers of the hephaestin knockout fetuses when compared to littermate controls.

Conclusions: Our data indicate that, unlike many body tissues, none of the known ferroxidases are required for the efficient export of iron from the placenta, as there was no change in fetal iron in any of our knockout models. Despite this, a lack of hephaestin led to fetal anemia. We propose that, in the absence of placental hephaestin, the iron exiting the placenta is not efficiently oxidised to the ferric form, leading to inefficient binding to the iron carrier protein transferrin in the fetal circulation. The resultant non-transferrin bound iron cannot be effectively utilised by developing red blood cells and so fetal anaemia ensues. Non-transferrin bound iron is also rapidly removed from the circulation by hepatocytes, explaining the increased iron levels in the livers of hephaestin knockout fetuses. These studies reveal a novel aspect of placental function and suggest that, unlike many body tissues, a ferroxidase is not required for the efficient export of iron by the placenta, but is necessary for the correct distribution of iron in the fetus.

Frazer:Pharmanutra S.p.a.: Consultancy, Other: Contract Research.