Maternal Iron Deficiency Alters Trophoblast Differentiation and Placental Development in Rat Pregnancy

Abstract

Iron deficiency, which occurs when iron demands chronically exceed intake, is prevalent in pregnant women. Iron deficiency during pregnancy poses major risks for the baby, including fetal growth restriction and long-term health complications. The placenta serves as the interface between a pregnant mother and her baby, and it ensures adequate nutrient provisions for the fetus. Thus, maternal iron deficiency may impact fetal growth and development by altering placental function. We used a rat model of diet-induced iron deficiency to investigate changes in placental growth and development. Pregnant Sprague-Dawley rats were fed either a low-iron or iron-replete diet starting 2 weeks before mating. Compared with controls, both maternal and fetal hemoglobin were reduced in dams fed low-iron diets. Iron deficiency decreased fetal liver and body weight, but not brain, heart, or kidney weight. Placental weight was increased in iron deficiency, due primarily to expansion of the placental junctional zone. The stimulatory effect of iron deficiency on junctional zone development was recapitulated in vitro, as exposure of rat trophoblast stem cells to the iron chelator deferoxamine increased differentiation toward junctional zone trophoblast subtypes. Gene expression analysis revealed 464 transcripts changed at least 1.5-fold (P < 0.05) in placentas from iron-deficient dams, including altered expression of genes associated with oxygen transport and lipoprotein metabolism. Expression of genes associated with iron homeostasis was unchanged despite differences in levels of their encoded proteins. Our findings reveal robust changes in placentation during maternal iron deficiency, which could contribute to the increased risk of fetal distress in these pregnancies.

Keywords: anemia; iron deficiency; nutrient; placenta; pregnancy; trophoblast.

Figure 1.

Pregnancy outcomes, litter size, and Hb levels in dams fed iron-replete and iron-deficient diets. (a) Maternal cumulative food intake over gestation. (b) Maternal cumulative weight gain over initial body weight. (c) Litter size (mean number of live fetuses per litter on GD18.5). (d) Number of resorptions per litter. In panels (a)-(d), data are presented as mean ± SEM based on measurements from 8 control-fed and 10 iron-deficient (ID) dams sacrificed on GD18.5. (e) Maternal Hb, n ≥ 5 dams per group. (f) Fetal Hb on GD18.5 (control: n = 18 fetuses, ID: n = 25 fetuses, from at least 7 dams each group; each value represents the average of the measurements obtained per litter). Data are presented as mean ± SEM. In panel (e), P values reflect 2-way ANOVA outcomes; in all other panels, P values reflect Student’s t test outcomes. An asterisk (*) denotes statistical significance (P < 0.05).

Figure 2.

Fetal body weight and organ weight changes in dams fed iron-replete and iron-deficient diets. Dams were fed iron-replete (control) or iron-deficient (ID) diets starting 2 weeks before mating. Fetuses were collected on GD18.5. (a) Fetal body weight; (b) Relative brain weight; (c) Relative liver weight; (d) Relative kidney weight; (e) Relative heart weight. Measurements from fetuses collected from 8 control and 9 ID dams (≥ 28 control and ≥ 33 ID fetuses) were used. Data are presented as mean ± SEM, with each value representing the average of the measurements obtained per litter. P values reflect Student’s t test outcomes. An asterisk (*) denotes statistical significance (P < 0.05).

Figure 3.

Changes in placental weight and morphology following exposure of dams to iron-replete or iron-deficient diets. Dams were fed iron-replete (control) or iron-deficient (ID) diets starting 2 weeks before mating. Placentas and fetuses were collected on GD13.5 or GD18.5. (a) Placental weight on GD13.5; (b) Placental weight relative to fetal weight on GD13.5; (c) Placental weight on GD18.5; (d) placental weight relative to fetal weight on GD18.5. (e) Schematic representation of a GD18.5 placenta. (f) Representative images of GD18.5 placentas collected from control (left) and ID (right) dams following cytokeratin immunohistochemistry. Scale bar, 1000 µm. Cross-sectional areas were measured for GD13.5 placental (g) labyrinth and (h) junctional zones, as well as for GD18.5 placental (i) labyrinth and (j) junctional zones. Data are presented as mean ± SEM. In panels (g) and (h), data represent placental measurements from 8 control-fed dams and 6 ID dams, while in panels (i) and (j), data represent 5 control-fed dams and 8 ID dams. P values reflect Student’s t test outcomes. An asterisk (*) denotes statistical significance (P < 0.05).

Figure 4.

Gene expression analysis of GD18.5 placentas collected from dams fed iron-replete or iron-deficient diets. (a) Pie chart summarizing the number of transcripts unaltered, upregulated (≥1.5-fold; P < 0.05) and downregulated (≥1.5-fold; P < 0.05) in maternal iron-deficiency (ID) compared with placentas from dams fed control (iron-replete) diets. (b) Volcano plot showing the distribution of differentially expressed genes with cutoff criteria of absolute fold change ≥1.5 and P < 0.05. (c) Hierarchical clustering of the top 26 differentially expressed genes in maternal ID. (d) Microarray results were validated using qRT-PCR. Microarray fold change results are presented as means; results from qRT-PCR are presented as means ± SEM, with P values reflecting Student’s t test outcomes. An asterisk (*) denotes statistical significance (P < 0.05, n = 4 dams per group, averaged from 2 placentas per dam). (e) Representative Western blots showing protein levels of FTH1, FTL1, and TFRC in placentas from maternal ID compared with controls. GAPDH was used as a loading control. Each sample represents a placenta from a different dam. On the right, densitometric analysis of band intensity relative to GAPDH, and normalized to control placentas (red dashed line) is shown. (f) ALOX15 immunofluorescence (green) in the junctional zone of placentas collected from iron-replete (control, top panels) and ID dams (bottom panels) on GD18.5. The middle panels show nuclei, which were counterstained blue with DAPI. A merged image is shown in the right panels. Scale bar = 100 μm.

Figure 5.

Gene ontology of placental genes differentially expressed in maternal ID. Biological processes affected by (a) upregulated genes and (b) downregulated genes in maternal iron deficiency, as determined by DAVID. The gene ontology identification number and a list of differentially expressed genes included in each category are provided.

Figure 6.

Increased expression of junctional zone markers in rat TS cells cultured with an iron chelator, deferoxamine. Rat TS cells were incubated in the presence or absence of 25μM or 50μM deferoxamine (DFO) for 48 hours. (a) qRT-PCR analysis showing transcript levels of Cdx2, Rbp4, Mmp9, Mmp10, Prl3d1, and Tpbpa in rat TS cells cultured with and without DFO. (b) Immunofluorescence was used to detect TPBPA in rat TS cells exposed to 0, 25, or 50μM DFO. DAPI was used to stain nuclei. Results from qRT-PCR are presented as means ± SEM, with P values reflecting 1-way ANOVA with Tukey’s post hoc test. An asterisk (*) denotes statistical significance (P < 0.05, n = 3-4). Scale bar = 50 μm.