Regulatory, Integrative and Comparative Physiology

Influence of gestational age and fetal iron status on IRP activity and iron transporter protein expression in third-trimester human placenta

Jenni Bradley, Elizabeth A. Leibold, Z. Leah Harris, Jane D. Wobken, Stephen Clarke, Kimberly B. Zumbrennen, Richard S. Eisenstein, Michael K. Georgieff


Placental iron transport during the last trimester of pregnancy determines the iron endowment of the neonate. Iron transport is a function of the major iron transport proteins: transferrin receptor-1 (TfR-1) and ferroportin-1 (FPN-1). The mRNAs for TfR-1 and, potentially, FPN-1 are posttranscriptionally regulated by iron regulatory protein (IRP)-1 and IRP-2. We assessed the effect of gestational age and fetal iron status on IRP-1- and IRP-2-binding activity and on the localization and protein expression of TfR-1 and FPN-1 protein at 24–40 wk of gestation in 21 placentas obtained from iron-sufficient nonanemic mothers. Gestational age had no effect on cord serum ferritin concentration, IRP-2 RNA-binding activity, transporter protein location, and TfR-1 or FPN-1 protein expression. IRP-1 activity remained constant until full term, when it decreased (P = 0.01). Placental ferritin (r = 0.76, P < 0.001) and FPN-1 (r = 0.44, P < 0.05) expression increased with gestational age. Fetal iron status, as indexed by cord serum ferritin concentration, was inversely related to placental IRP-1 (r = −0.66, P < 0.001) and IRP-2 (r = −0.42, P = 0.05) activities. Placental ferritin protein expression correlated better with IRP-1 (r = −0.45, P = 0.04) than with IRP-2 (r = −0.35, P = 0.10) activity. Placental TfR-1 and FPN-1 protein expression was independent of fetal or placental iron status and IRP activities. Iron status had no effect on transport protein localization. We conclude that, toward the end of the third trimester of iron-sufficient human pregnancy, the placenta accumulates ferritin and potentially increases placental-fetal iron delivery through increased FPN-1 expression. IRP-1 may have a more dominant role than IRP-2 activity in regulating ferritin expression.

  • ferroportin
  • ferritin
  • transferrin receptor
  • syncytiotrophoblast
  • pregnancy

iron is an essential nutrient during early human development (26). Its therapeutic range is narrow; therefore, its transport must be tightly regulated to avoid deficiency or excess states. Iron deficiency and excess significantly affect red blood cell, brain, heart, and liver development and function (5, 8, 22, 30). Fetal and neonatal organs are arguably at higher risk than organs of older children for injury from iron deficiency because of their rapid growth rates and organ development and from iron excess because of their low iron-binding capability (9) and poor antioxidant defenses (7). Perinatal iron deficiency occurs commonly in gestations complicated by maternal iron deficiency, intrauterine growth retardation, and maternal diabetes mellitus (10, 19, 20, 37), whereas iron overload occurs predominantly in the setting of congenital hemochromatosis (11).

Iron delivery to the fetus likely involves regulation of the placental expression of the proteins known to mediate vectoral iron transport across single-cell barriers, such as the duodenal epithelium, mammary gland, and blood-brain barrier. Because the syncytiotrophoblast takes up ferric iron bound to transferrin at the apical membrane, the likely transporter of interest at that site is transferrin receptor-1 (TfR-1) (6, 21, 38, 43). At the basal membrane, where iron is transported to the fetus, the putative transporters are ferroportin (FPN)-1 (13) and TfR-1 (43), both of which localize to this membrane at full term. These transporters, in turn, are postulated to be posttranscriptionally regulated by iron regulatory proteins (IRPs), which bind iron-responsive elements (IREs) found in the untranslated regions of the respective mRNAs (14). Although the responsiveness of the IREs on TfR mRNA is unquestioned, it remains unclear whether the IREs found on FPN-1 mRNA are active (31). Furthermore, which IRP is responsible for mRNA stabilization remains unclear (14). Genetic knockout of IRP-2 results in a mouse with significant iron abnormalities (27), whereas the IRP-1 knockout mouse has yet to be characterized.

TfR-1 and FPN-1 expression and IRP-1 RNA-binding activity have been demonstrated in full-term placenta (6, 13, 21, 38, 43). A preliminary study failed to demonstrate IRP-2 activity at full term (18). TfR mRNA and protein expression, as well as IRP-1 RNA-binding activity, appears to be responsive to fetal iron deficiency when maternal iron status is normal (18, 38). However, the normal developmental trajectory of the expression and regulation of these proteins throughout the third trimester of human pregnancies has not been studied. The specific objectives of this study were 1) to determine the developmental trajectory of TfR-1 and FPN-1 concentration and localization in human placenta during the third trimester and 2) to assess how third-trimester placental IRP-1 and IRP-2 activities relate to gestational age, calculated rate of placental iron transport, fetal/placental iron status, and transport protein expression.


Study population.

The study was approved by the Institutional Review Board of the University of Minnesota. Informed consent was obtained from all participating subjects. The research subjects were pregnant women delivering at United Hospital (St. Paul, MN). Twenty-one placental samples designed to fill the five gestational age groups were collected from consecutively consenting subjects. The recruitment was unbiased with respect to maternal age, parity, and race. Gestational age was determined by maternal dates when reliable, first- or second-trimester ultrasound when dates were unreliable, and physical examination for gestational age when the first two indicators were not present. Infants were weighed and measured, and the data were plotted on a standard growth curve to determine appropriateness of intrauterine growth and to rule out intrauterine growth retardation (4). Infants with birth weight z scores less than −1.5 or greater than +1.5 were excluded. Maternal charts were screened for the presence of confounding factors for fetal iron status. Pregnancies complicated by medical conditions that have been shown to affect fetal iron status, including maternal iron deficiency, small-for-gestational age infant at birth, maternal hypertension with intrauterine growth retardation, maternal diabetes mellitus, and neonatal infection, were excluded (10, 16). Seventy-one percent of mothers took prenatal iron supplements, and none had indexes of iron deficiency at delivery. Fifteen mothers delivered before 37 wk of gestation. The causes for premature deliveries are presented in Table 1.

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Table 1.

Clinical characteristics of the study population

Placental tissue and serum collection.

The collection was targeted to provide a minimum of four placentas at five gestational ages: 24–27, 28–30, 31–33, 34–37, and 38–40 wk. The placentas were obtained within 5 min of delivery via RNase-free technique and frozen at −80°C until assayed. Six 1-in.3 samples were obtained from the center of each cotylydon. Cord serum samples were collected at the time of delivery and frozen at −80°C until assayed for ferritin and erythropoietin concentrations.

IRP-1 and IRP-2 RNA-binding activity assays.

IRP-1 and IRP-2 activities on placental specimens were assessed by RNA band shift assay as previously described (18). Approximately 100 mg of tissue were excised from each placental sample and homogenized in 3× weight (0.1 mg = 0.1 ml) lysis buffer (10 mM HEPES, pH 7.6, 10 mM KCl, 0.25% NP-40, 1.5 mM MgCl2, 0.5 mM DTT, and 1 mM EDTA) containing 1 mM PMSF and 1 mM diisopropylfluorophosphate. The extracts were centrifuged for 20 min at 13,000 g at 4°C. Supernatants were assayed for total protein concentration using the Coomassie Plus protein assay reagent (Pierce Chemical, Rockford, IL). On the day of homogenization, 12 μg of total protein in 20 μl of binding buffer (10 mM HEPES, pH 7.6, 40 mM KCl, 5% glycerol, and 3 mM MgCl2) were incubated with an excess of 32P-labeled rat ferritin L-IRE RNA and subsequently treated with RNase T1 (20 U) and heparin (75 μg) (28). Samples were then incubated with rabbit anti-rat IRP-2 antibodies (23) for 20 min to supershift IRP-2 RNA complexes. Samples were analyzed by a 5% nondenaturing polyacrylamide gel and exposed to film or phosphor imaging for analysis. IRP-1 or IRP-2 band intensity was quantified using ImageQuant software. Three separate experiments were performed using fresh extracts.

Western blot analysis.

For IRP-1 and IRP-2, total protein (50 μg) of placental extracts was separated by an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were probed with chicken anti-rat IRP-1 antibodies or rabbit anti-rat IRP-2 antibodies (23). Membranes were probed with horseradish peroxidase-conjugated goat anti-chicken or goat anti-rabbit secondary antibodies for IRP-1 or IRP-2, respectively, and developed using Western Lighting Chemiluminescence Reagent Plus (Perkin-Elmer, Boston, MA).

For ferritin, TfR-1, and FPN-1, frozen placenta was pulverized in liquid nitrogen to a fine powder on ice and immediately resuspended in protein lysis buffer (25 mM HEPES, pH 7.5, 40 mM KCl, 5% glycerol, 1 mM DTT, 0.5% NP-40, and 1 mM PMSF). The extracts were subsequently centrifuged for 10 min at 13,000 g at 4°C, and the supernatants were assayed for total protein concentration by the protein assay method (Bio-Rad, Richmond, CA). A total of 50 μg of placental protein was loaded per lane and electrophoretically separated on a 7.5% SDS-polyacrylamide gel. Protein was then transferred onto enhanced chemiluminescence (ECL) nitrocellulose membranes via semidry electrophoresis using a Tris-glycine transfer buffer. Membranes were probed for ferritin using a rabbit anti-human ferritin (H-type) antibody (Boehringer Mannheim, Indianapolis, IN). A horseradish peroxidase-conjugated secondary antibody was used. TfR-1 was detected using an anti-TfR-1 primary antibody (Chemicon International, Temecula, CA) at a 1:100 dilution, and FPN was detected using a primary antibody (Alpha Diagnostics, San Antonio, TX) at a 1:500 dilution. A 1:3,000 dilution of ECL donkey anti-rabbit secondary antibody was applied to each, and protein visualization was accomplished using the ECL Western blotting analysis system (Amersham Pharmacia, Buckinghamshire, UK). The relative optical density of the protein was standardized as a function of actin expression.


Serial 12-μm sections were obtained using a cryostat (model CM1900, Leica Instruments, Nussloch, Germany) at −25 to −27°C, mounted on poly-l-lysine-coated slides, and stored at −80°C until immunohistochemical analysis for TfR-1 was performed as previously described (38). The same primary mouse anti-human TfR-1 antibody (Chemicon) used in the Western blot analysis was used at a 1:100 dilution. For FPN, a rabbit antibody against human FPN-1 was generated against amino acids 223–230 fused to glutathione S-transferase. The FPN-1 antibody was used at a dilution of 1:400. For FPN-1, a preimmune serum control was also utilized and demonstrated no reactivity. In experiments for all three proteins, the primary antibody was omitted from the control slides, which did not demonstrate background staining.

After incubation, slides were treated for 30 min in a biotinylated secondary antibody to match the species of the primary antibody and then with streptavidin-peroxidase complex (Vector Laboratory, Burlingame, CA). The bound peroxidase was revealed by incubation of the sections in 3,3′-diaminobenzidine (Vector Laboratory). Slides were visualized under a light microscope through a ×100 objective (Eclipse E600, Nikon, Melville, NY) and digitized to a computer by a Nikon DMX1200 digital charge-coupled device camera. The images were stored on a Dell Dimension 8200 computer in Adobe Photoshop (version 6) and assessed using ACT-1 software (version 2.20, Nikon).

TfR-1 and FPN-1 protein localization was assessed in each gestational age group by visual inspection. TfR-1 was assessed on the apical and basal trophoblastic membranes, and FPN-1 was assessed on the basal membrane in the location previously described by Donovan et al. (13).

Biochemical assays.

Cord serum ferritin concentrations were determined in duplicate by chemiluminescent immunoassay (Access Immunoassay System, Beckman-Coulter, Brea, CA) as previously described (18). Cord serum erythropoietin concentrations were graciously assayed by Dr. John Widness as previously described (19). Placental nonheme iron concentration was determined by atomic absorptiometry after digestion with nitric-perchloric acid as previously described (18, 38). Placental tissue was thoroughly rinsed with normal saline before assay.

Study design and statistical analysis.

The primary independent variables of the study were gestational age and fetal iron status as determined by cord serum ferritin concentration. Secondary variables included maternal hemoglobin and placental ferritin protein expression. The dependent variables of the study were IRP RNA-binding activities and iron transport protein concentrations. They were assessed as functions of gestational age group, iron status, and calculated maternal-fetoplacental iron transport rate by ANOVA with Scheffé's post hoc test. The mean gestational ages for the five groups were 26, 29, 33, 35, and 39 wk. Estimated weekly iron transport from the mother to the fetoplacental unit was calculated on the basis of the expected weekly iron accretion of the infant and placenta. The fetal iron status was calculated from the measured weight at birth with use of an assumed fetal body iron content of 75 mg/kg (36). The placental nonheme iron status was calculated from the average placental weight for gestational age (34) and the measured placental nonheme iron contents from the collected placenta. Actual placental weights were not obtained from the collected specimens. The fetal and placental calculated values were summed. The difference in fetoplacental iron content between two consecutive gestational age groups was divided by the number of weeks separating the two groups to generate the weekly iron transport value, which was expressed as milligrams of iron per kilogram of fetoplacental weight per week. The relations between cord serum ferritin, placental ferritin, and IRP activity and between IRP activity and transporter concentration were assessed by linear regression analysis. The effect of fetal iron status was also assessed by comparing the IRP activities and transporter/storage protein expressions of the placentas from the highest tertile of cord ferritin with those from the lowest tertile. Values are means ± SE. Statistical significance was set at P < 0.05.


Table 1 demonstrates the gestational age group characteristics of the sample and the reasons for premature birth. Birth weight z scores, maternal hemoglobin concentration, and cord erythropoietin concentrations were not different among the groups. Cord erythropoietin concentrations were low (19) and did not correlate with gestational age (r = 0.16), suggesting that none of the fetuses were hypoxic and that the infants delivered prematurely were not more hypoxic than the full-term infants. In addition, we found no significant relation between maternal hemoglobin and cord hemoglobin (r = 0.005) or natural logarithm of erythropoietin (r = 0.33) or ferritin concentrations (r = −0.28).

Gestational age group did not have a significant effect on serum ferritin concentrations (Table 2, Fig. 1A). These findings are consistent with previously published data (40) and with the working model that the fetus maintains a relatively stable amount of storage iron per kilogram of body weight throughout the third trimester (36). In contrast, placental ferritin protein expression was highly positively correlated with gestational age (Fig. 1B), implying that the placenta stores a large amount of iron toward the end of gestation, potentially serving as an iron reservoir for the fetus. Serum ferritin concentration and placental ferritin protein expression were correlated (r = 0.59, P < 0.01). Cord serum ferritin concentration and placental ferritin protein expression were not positively related to maternal hemoglobin concentration.

Fig. 1.

Serum ferritin concentrations (A) and placental ferritin protein expression normalized to actin protein (B) as functions of gestational age. For serum ferritin, n = 21, r = 0.31, P = not significant (NS). For placental ferritin, n = 21, r = 0.76, P < 0.001. OD, optical density.

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Table 2.

Fetal and placental iron parameters

IRP-1 and IRP-2 activity was present throughout the third trimester, in contrast to our previous study where IRP-2 activity was not detected in full-term placentas (18) (Fig. 2). IRP-1 activity remained constant until the end of gestation, at which point it decreased significantly (Table 2). IRP-2 activity did not change with gestational age. The IRP-1 activities of placentas from the lower tertile of cord serum ferritins were significantly higher than those from the upper tertile (Table 3). Furthermore, the cord serum ferritin concentration correlated inversely with IRP-1 (Fig. 3A) and IRP-2 (Fig. 3B) activities, suggesting that both proteins respond to the fetal iron status. Placental ferritin protein expression correlated better with IRP-1 (r = −0.46, P = 0.04) than with IRP-2 (r = −0.35, P = 0.10) activity.

Fig. 2.

Iron regulatory protein (IRP)-1 and IRP-2 RNA-binding activity in full-term and preterm placentas. Placental protein was incubated with a 32P-labeled ferritin iron-responsive element (IRE) probe, and the IRP-IRE complexes were analyzed on a 5% native polyacrylamide gel. Lane 1, extract from human embryonal kidney 293 cell line that has high expression of IRP-2; lanes 2–10, extracts from full-term (C) and preterm (P) placentas demonstrating IRP-1 and IRP-2 activity.

Fig. 3.

IRP-1 (A) and IRP-2 (B) activity as a function of cord serum ferritin concentration. For IRP-1, r = −0.66, P < 0.001. For IRP-2, r = −0.42, P = 0.05.

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Table 3.

Comparison of placental iron transporters, iron storage proteins, and IRP activities from placentas with the lowest and highest tertiles of cord ferritin concentrations

TfR-1 and FPN-1 were detectable at 24 wk of gestation and were present throughout the third trimester. The primary location of the transport proteins did not change with gestational age. TfR-1 was found predominantly on the apical trophoblastic membrane but was also present on the basal membrane, consistent with previous reports (43). FPN-1 was localized exclusively to the basal membrane (Fig. 4). Regression analysis demonstrated that gestational age had no effect on the protein expression of TfR-1 (r = 0.05, P = not significant). FPN-1 expression increased with gestational age (r = 0.44, P < 0.05). Within the range of iron status tested, TfR and FPN-1 did not correlate significantly with cord serum ferritin (r = −0.29 for TfR and r = 0.20 for FPN-1) or IRP-1 or IRP-2 activity.

Fig. 4.

Top: placental transferrin receptor (TfR) localization and density at 24–27 wk (left) and 39 wk (full term, right) of gestation. Note strong apical membrane staining at placental-maternal interface and weaker basal membrane staining (arrows). Middle: divalent metal transporter-1 (DMT-1) localization and density at 27 wk (left) and 39 wk (right) of gestation. Bottom: ferroportin (FPN) localization and density at 24 wk (left) and 40 wk (right) of gestation. Note exclusive basal membrane staining (arrow). Control, antibody control image showing no positive staining; M, maternal space; F, fetal space.


The iron status of the newborn may in large part determine the risk of subsequent postnatal iron deficiency. Neonatal iron status is primarily a function of third-trimester maternal-placental-fetal iron transport. The present study provides novel information about the regulation of this transport. This study demonstrates that the major iron transporters, TfR-1 and FPN-1, as well as both mRNA-stabilizing IRPs, are present in the placenta throughout the third trimester. We were able to assess the effects of gestational age and fetal/placental iron status on iron transporter expression and regulation by studying an iron-sufficient maternal population that gave birth to infants with a range of fetal iron status. Gestational age had little effect on any of the measured parameters until near term, when IRP-1 activity decreased and placental ferritin and FPN-1 concentrations increased. The lack of change in cord serum ferritin concentrations, IRP activity, and transporter expression from 24 to 37 wk of gestation is consistent with a relatively constant delivery of iron on a per-weight basis to the fetus during this time period (36). The increases in placental ferritin and FPN-1 concentration at full term suggest increased iron storage by the placenta and a modest increase in placental-fetal iron transport at the end of gestation. These findings are consistent with the large increase in total iron content of the fetus associated with fetal growth near term (36).

This study also demonstrates that IRP-2 is present in the placenta throughout the third trimester. Both IRPs appear to respond to fetal iron status, with a tighter relation to IRP-1 than to IRP-2. The variance of IRP activity based on iron status suggests that the regulatory system is intact beginning at ≥24 wk of gestation. Two relevant iron transporters, TfR-1 and FPN-1, are expressed in their expected locations from the beginning of the trimester. Although the use of an iron-sufficient maternal population with no known risks for iron-deficient fetuses was imperative to determine gestational age effects, it constrained our ability to determine the role of maternal and fetal iron in regulating the system. However, given the gestational age template presented in this study, the regulatory factors that occur during iron-deficient and iron-overloaded gestations can now be studied. Even within the relatively narrow range, the effect of fetal iron status on IRP activity can be appreciated.

The vectoral movement of iron from one compartment to another across a single-cell layer occurs in the intestine, mammary gland, blood-brain barrier, blood-cerebrospinal fluid barrier, and placenta (for reviews see Refs. 41 and 44). All these systems appear to use some combination of the three characterized iron transporters, TfR-1, divalent metal transporter-1 (DMT-1), and FPN-1, based on the form of iron presented to the apical surface of the single cell layer and on the mechanism of basal surface transport. When iron is presented to the apical surface from a serum source (e.g., at the blood-brain barrier or to the trophoblast), it is in its ferric form bound to transferrin. Diferric transferrin has a high affinity for TfR, which incorporates the complex into the cell via an endosome. Subsequent processing includes acidification of the endosome and transport across the endosomal membrane of the resultant ferrous iron by DMT-1 (29). Iron presented to the apical surface in its ferrous form is imported via DMT-1 located on the apical surface membrane. This mechanism appears to be unique to the duodenal epithelial cell, because it is the only surface that receives non-serum-derived iron (3).

The mechanism of vectoral iron transport across a basal cell membrane (usually into a serum compartment) is controversial. The intestine is the most studied model and is likely most analogous to the placenta. TfR-1 and FPN-1 are expressed on the basolateral membrane in the duodenal epithelial cell and provide alternative (or complementary) mechanisms for iron efflux (3, 32, 41, 44). In the intestine, FPN-1 is hypothesized to transport ferrous iron at this surface by providing a site for donation of a positive charge from a copper-containing protein to reconvert the iron to its ferric state. Ceruloplasmin and hephaestin are the leading candidates for this copper-mediated mechanism (2). The resultant ferric iron is hypothesized to bind to serum transferrin before shuttling into the vascular space. TfR-1 may also play a direct role in uptake of transferrin from the serum adjoining the basal surface and directly obtaining iron intracellularly (32).

In the present study, placental FPN-1 expression was localized to the basal membrane of the syncytiotrophoblast and increased significantly with gestational age. TfR-1 was localized to the apical and basal membranes and did not change with gestational age. The increase in FPN-1 expression is potentially consistent with increased placental-fetal iron transport, because the fetus grows and the blood volume expands proportionately as the third trimester progresses. Nevertheless, FPN-1, DMT-1, and TfR-1 are localized to the region of the basal membrane (13, 17, 21, 43), and others have demonstrated that DMT-1 and TfR-1 expression is also responsive to iron status (17, 43). Although DMT-1 staining of the basal membrane is discontinuous (21), we confirmed in this study that FPN-1 staining is continuous (13). Our immunohistochemical techniques are not sensitive enough to detect whether the basal structures expressing these two ferrous iron transporters are identical. We also visualized TfR-1 on the basal membrane, as has been previously reported (43). At this time, the data point to the possibility that any of the transporter systems expressed on the basal membrane of the syncytiotrophoblast may be the primary one (13, 17, 43) and that redundancies may exist to provide more precise regulation of fetal iron delivery.

The regulation of the protein expression of the iron transporters remains under investigation. The most thoroughly characterized system is the coordinate posttranscriptional regulation of TfR-1 and ferritin mRNA (14). During iron-deficient states, IRPs bind the IREs in the 3′-untranslated region of TfR and prevent binding of RNases. The consequent preservation of TfR-1 mRNA produces more copies of TfR-1. Simultaneously, IRPs also bind IREs in the 5′-untranslated region of ferritin mRNAs, preventing ribosomal binding of the 40S ribosomal subunit and subsequent translation. The result is reduction in available ferritin for intracellular iron storage. Coupled with an increase in cellular iron uptake via TfR-1, iron homeostasis is restored. The present study demonstrates that the IRP system is active throughout the third trimester and that IRP-2 is present in addition to IRP-1, in contrast to our previous report that found no detectable IRP-2 activity in full-term placenta (18). At that time, we speculated that IRP-2 was absent in the placenta or was present in levels too low to be detected by our methods. Given the larger sample size in this study, it appears that methodological issues may have prevented us from detecting IRP-2 in the previous study. Because placental extracts were assayed on the day of preparation in the present study, we conclude that IRP-2 may be particularly susceptible to degradation in frozen extracts.

As would be predicted, reduced levels of fetal and placental ferritin throughout the third trimester were associated with higher levels of IRP-1 in particular. In contrast, there was not sufficient variance of TfR-1 expression in these iron-sufficient placentas to allow us to reach any conclusions about TfR-1 regulation by the IRPs. On the basis of our previous observations in iron-deficient full-term placenta (18, 38), we had hypothesized a direct relation between placental TfR-1 and IRP-1 to increase placental importation of iron in response to decreased fetal stores. However, Western blot analysis showed that this relation (although trending in the right direction; Table 3) did not reach statistical significance. Similarly, FPN-1 concentrations did not change in relation to the IRPs or to iron status. As with TfR-1, this may also be due to lack of a wide enough range of iron status in this study or may support the hypothesis that the IREs found on FPN-1 mRNA are not functional. Much less is known about the regulation of FPN-1 than TfR-1. mRNA of FPN-1 contains IRE or IRE-like elements, suggesting that they may also be posttranscriptionally regulated. However, the functionality of FPN-1's IREs is highly debated. Sequences within and flanking the IRE appear to contribute to differences in function of these RNA regulatory elements and may determine which IRP interacts with specific IRE-containing mRNA (42). Such factors include the distance of the IRE from the 5′ end of the mRNA, the secondary structure of the IRE stem region, and the presence or absence of flanking sequences that stabilize the IRE structure (12, 24, 25). Interestingly, distance of the IRE from the 5′ end of FPN-1 mRNA appears to vary in a specific manner, and this may contribute to the selective and divergent effects of iron on FPN-1 regulation (1).

Debate has centered on the individual roles of the two IRPs in mammalian biology (14). IRP-1 is an iron-sulfur containing constitutively expressed cytoplasmic protein that assumes greater IRE-binding activity in its apo form than in its saturated form (14). The apo form predominates during intracellular iron-deficient states. In contrast, the concentration of IRP-2 varies with iron status, increasing during periods of iron deficiency (18). Both have been shown to bind active IREs in vitro. The role of IRP-1 in vivo has been questioned because of the apparent lack of a recognizable iron-overload phenotype in the homozygous IRP-1 knockout mouse. Deletion of IRP-2 in a mouse leads to significant iron overload of the intestine and brain (27). Our study suggests that IRP-1 activity is more closely related to fetal and placental iron parameters than IRP-2. Interestingly, a recent report suggests that deletion of IRP-1 in the mouse interferes with early developmental stages and, therefore, may disrupt early embryonic survival (15). The finding suggests that IRP-1 may play an important role in establishment and maintenance of pregnancy.

In a human-based study population such as this, there are a number of potential confounders to consider. Our assessment of pregnancies throughout the third trimester necessitated the analysis of premature births, which are by definition abnormal events. Subjects with risk factors known for fetal iron abnormalities [which can also result in prematurity (39)], including diabetes mellitus, intrauterine growth retardation, and infection, were excluded. However, it is impossible to completely rule out the possibility that other factors leading to premature birth may have altered fetal iron status or placental iron transport regulation. Examples would include multiple gestations or fetal hypoxia. Seven of the preterm deliveries were secondary to multiple gestation (i.e., twins and triplets). The independent effect of multiple gestation on iron transport and regulation has not been reported. However, unpublished data from our 1987 study showed no differences in neonatal iron status indicators between twins and controls. Fetal hypoxia, which in turn could alter iron distribution (10), may result from a failing placenta before delivery of a premature infant. The generally low and similar cord serum erythropoietin concentrations in preterm deliveries compared with full-term deliveries imply that prematurely delivered infants in this study were not more hypoxic as fetuses. The highest individual natural logarithm of erythropoietin concentration (4.2 IU/l) was well below the mean for the iron-deficient infants of uncontrolled diabetic mothers (19).

Another factor that we could not fully address in this study is the relative contribution of maternal vs. fetal regulation of placental iron transport. Multiple studies have demonstrated that relatively severe maternal iron deficiency compromises fetal iron stores (for review see Ref. 33). The precise maternal hemoglobin at which this relation becomes operational remains controversial. Increased maternal intestinal iron absorption with consequent increased iron delivery to the fetus appears to compensate for mild degrees of maternal iron deficiency (35). The rate of maternal iron deficiency of the degree expected to affect fetal iron status is low in our population. The predelivery maternal hemoglobin in our study was 91–131 g/l, a range in which no relation between maternal hemoglobin concentration and fetal ferritin concentration or placental ferritin expression existed. Placental IRP activities were not related to maternal hemoglobin concentrations. Although the role of maternal regulation could not be assessed in this study, controlling for maternal iron status in this manner, we were able to determine a role for fetal/placental iron in placental IRP-1 activity.

In summary, this study demonstrates the presence of a regulated iron transport system in the placenta during the third trimester in the human. The proteins that are likely to be involved in maternal-fetal iron transport localize to the same membrane surfaces at 24 wk and at full term. Furthermore, IRP-1 and IRP-2 activities are present throughout this part of gestation and respond in predictable manners to fetal and placental iron status. As it nears full term, the placenta stores large amounts of iron as ferritin under the direction of IRP-1, while the presence of increasing FPN-1 expression at the same time suggests an increase in placental-fetal iron transfer.


This study was supported by National Institutes of Health Grants HD-29421 (M. K. Georgieff), GM-45201 (E. A. Leibold), and DK-47219 (R. S. Eisenstein).


The authors thank Ron Goertz for subject recruitment and placental specimen collections and Dr. John A. Widness for performing serum erythropoietin assays.


  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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