Effects of maternal iron status on placental and fetal iron homeostasis

Abstract

Iron deficiency is common worldwide and is associated with adverse pregnancy outcomes. The increasing prevalence of indiscriminate iron supplementation during pregnancy also raises concerns about the potential adverse effects of iron excess. We examined how maternal iron status affects the delivery of iron to the placenta and fetus. Using mouse models, we documented maternal homeostatic mechanisms that protect the placenta and fetus from maternal iron excess. We determined that under physiological conditions or in iron deficiency, fetal and placental hepcidin did not regulate fetal iron endowment. With maternal iron deficiency, critical transporters mediating placental iron uptake (transferrin receptor 1 [TFR1]) and export (ferroportin [FPN]) were strongly regulated. In mice, not only was TFR1 increased, but FPN was surprisingly decreased to preserve placental iron in the face of fetal iron deficiency. In human placentas from pregnancies with mild iron deficiency, TFR1 was increased, but there was no change in FPN. However, induction of more severe iron deficiency in human trophoblast in vitro resulted in the regulation of both TFR1 and FPN, similar to what was observed in the mouse model. This placental adaptation that prioritizes placental iron is mediated by iron regulatory protein 1 (IRP1) and is important for the maintenance of mitochondrial respiration, thus ultimately protecting the fetus from the potentially dire consequences of generalized placental dysfunction.

Keywords: Hematology; Homeostasis; Obstetrics/gynecology; Reproductive Biology.

Figure 1. Maternal hepcidin and serum iron levels determine embryo and placental iron status.

The iron status of WT C57BL/6 female mice was altered using diet or iron dextran injections. (A) Adult females were fed standard chow (185 ppm iron) or a low-iron diet (4 ppm iron) ad libitum 2 weeks prior to and throughout pregnancy, or were injected with 20 mg iron dextran at the time of mating. Pregnant females were analyzed at E12.5, E15.5, and E18.5. Nonpregnant (Non-P) females were subjected to an equivalent iron treatment. (B–F) Maternal measurements of (B) hepcidin (Hamp) mRNA and (C) serum hepcidin. (D) Liver nonheme iron. (E) Serum iron concentration. (F) Hb concentration. Statistical differences between groups was determined by 1-way ANOVA for normally distributed values, or otherwise by 1-way ANOVA on ranks (indicated by a single asterisk after the P value). (G–I) Embryo measurements at E18.5 for (G) serum iron and (H) liver nonheme iron. (I) Hb concentration. (J) Placental nonheme iron levels at E12.5, E15.5, and E18.5. (K) Placental weight at E15.5 and E18.5 (we did not obtain whole placentas at E12.5). Statistical differences between groups was determined by 1-way ANOVA for normally distributed values followed by the Holm-Sidak method for multiple comparisons versus the iron-replete control group (^###P < 0.001) or 1-way ANOVA on ranks followed by Dunn’s method for multiple comparisons versus the iron-replete control group (^#P < 0.05). The numbers of animals are indicated in the x axes of the box and whisker plots.

Figure 2. Placental iron transporters respond to changes in maternal iron status.

Immunofluorescence staining of human (A) and mouse (B) placentas for TFR1 (red) and FPN (green) . Original magnification, ×100. Nuclei are blue. M, maternal circulation; F, fetal circulation. Mouse placentas from Figure 1 were analyzed by Western blotting to determine the protein concentration of TFR1 (C) and FPN (D). β-Actin was used as a loading control. Four representative placentas are shown in the Western blots, and a total of 8 placentas (from 3 to 4 different dams per group) were used for quantitation. Data are presented as the mean ± SEM. Statistical differences between groups were determined by 1-way ANOVA for normally distributed values followed by the Holm-Sidak method for multiple comparisons versus the iron-replete control group. (E) The PIDI is the ratio of expression of placental FPN protein to placental TFR1 protein and reflects iron export to the fetus relative to iron import into the placenta from the maternal circulation. Statistical differences were determined using a 2-tailed Student’s t test. (F–H) Correlation of nonheme iron with the PIDI at E12.5, E15.5, and E18.5. The numbers of animals are indicated in the x axes of the box and whisker plots.

Figure 3. ⁵⁸Fe transport across the placenta.

(A) WT C57BL/6 female mice were maintained on a standard chow diet or placed on an iron-deficient diet 2 weeks prior to mating and were maintained on an iron-deficient diet throughout gestation. At E17.5, the dams received a single i.v. injection of ⁵⁸Fe-Tf. The dams were sacrificed 6 hours post injection (p.i.), and placental and embryonic tissues were collected and analyzed using ICP-MS. (B) Placental TFR1 and FPN protein expression was assessed by Western blotting, and quantitation of protein expression relative to β-actin was performed. Total ⁵⁸Fe content in (C) placentas and (D) fetal livers. Total ⁵⁶Fe content in (E) placentas and (F) fetal livers. (B–F) Statistical analysis was performed by 2-tailed Student’s t test for normally distributed values and otherwise by Mann-Whitney U rank-sum test (denoted by an asterisk after the P value). The numbers of animals are indicated in the x axes of the box and whisker plots.

Figure 4. Placental response to maternal iron deficiency in human pregnancy.

Placentas from uncomplicated human pregnancies were analyzed by Western blotting to determine protein expression of TFR1 and FPN, normalized to β-actin. qPCR was performed to determine TFRC mRNA expression, normalized to HPRT. (A and B) TFR1 protein levels, (C and D) TFRC mRNA levels, and (E and F) FPN protein levels according to maternal ferritin during weeks 32–34 or at delivery. (G and H) The PIDI was calculated as the ratio of expression of placental FPN to TFR1 protein, with a lower PIDI reflecting pregnancies at increased risk of fetal iron deficiency. The PIDI was lower in pregnant women with serum ferritin levels below 10 ng/mL than in those with ferritin levels above 10 ng/mL, regardless of whether ferritin was measured at 32–34 weeks of pregnancy or at delivery. No difference between <10 ng/mL and >10 ng/mL ferritin groups was observed for (I and J) placental nonheme iron concentrations, (K) maternal Hb, (L and M) cord blood Hb, or (N and O) cord blood ferritin. (P) PHTs were treated with 100 μM DFO, apo-Tf or holo-Tf for 24 hours. TFR1, FPN, ferritin heavy chain (HC), and β-actin expression was assessed by Western blotting. Statistical differences between groups was determined by 2-tailed Student’s t test or Mann-Whitney U rank-sum test for non-normally distributed values (denoted by an asterisk after the P value). The numbers of animals are indicated above the box and whisker plots.

Figure 5. IRP1 mediates placental iron homeostatic responses during maternal iron deficiency.

(A) Activity of IRP1/2 in placentas from iron-deficient, iron-replete, and iron-loaded pregnancies was analyzed by EMSA. Statistical differences between groups was determined by 1-way ANOVA on ranks followed by Dunn’s method for multiple comparisons versus the iron-replete control group (^#P < 0.05). (B) Irp1^+/– females were mated with Irp1^+/– males, and pregnant females were fed an iron-deficient diet from E7.5 until E18.5. Placentas and embryos were harvested on E18.5. (C) Placental TFR1 and FPN protein expression was assessed by Western blotting, and quantitation of protein relative to β-actin was performed. (D) Placental Tfrc mRNA expression. (E) Placental Reg1 mRNA expression. (F) IRP-IRE binding as determined by EMSA in Irp1^+/+ and Irp1^–/– placentas. (G) PIDI for Irp1^+/+ and Irp1^–/– placentas. (H) Placental and (I) fetal liver nonheme iron concentrations. (C–I) Statistical analysis was performed by 2-tailed Student’s t test for normally distributed values and otherwise by Mann-Whitney U rank-sum test (denoted by an asterisk after the P value). The numbers of animals are indicated in the x axes of the box and whisker plots.

Figure 6. Placental and embryonic hepcidin does not regulate placental iron homeostasis under iron-replete or iron-deficient conditions.

Hamp^+/– female mice were mated with Hamp^+/– male mice. Females were placed on an iron-replete or iron-deficient diet 1 week prior to mating. Placentas and embryos were analyzed on E18.5. (A) Fetal liver iron and (B) fetal serum iron concentrations. (C) Placental iron concentration, (D) Hamp expression, (E) Tfrc expression, and (F) FPN protein levels. Statistical differences between groups were determined by 2-way ANOVA (non-normally distributed values are indicated by an asterisk). The P value shown is for variation by diet. There were no statistical differences between genotypes for any of the measured parameters. The numbers of animals are indicated in the x axes of the box and whisker plots. E, embryos; M, mothers; Het, heterozygous.

Figure 7. Iron deficiency impairs oxidative phosphorylation in PHTs.

PHTs were treated for 24 hours with 100 μM DFO, apo-Tf, or holo-Tf. (A) Western blotting for OXPHOS complexes CI–CV. β-Actin was used as a loading control. (B) Mitochondrial respiration under basal conditions following injection of oligomycin, the uncoupler FCCP, or the electron transport inhibitors antimycin A and rotenone (AA/ROT). (C) Quantitation of basal respiration, ATP-linked respiration, maximal respiratory capacity, and spare respiratory capacity normalized to total cells per well. Statistical differences between groups were determined by 1-way ANOVA for normally distributed values followed by an all-pairwise multiple comparison (Holm-Sidak method) (^###P < 0.001) or 1-way ANOVA on ranks for non-normally distributed values followed by an all-pairwise multiple comparison (Tukey’s test) (^#P < 0.05). Lowercase letters indicate a statistical difference compared with DFO (“d”), Apo-Tf (“a”), or Holo-Tf (“h”) group. n = 6 technical replicates. (D) ECAR. (E) Basal OCR versus basal ECAR.