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Keywords:

  • anemia;
  • iron deficiency;
  • iron metabolism;
  • placenta;
  • pregnancy

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CONCLUSION
  5. Acknowledgments
  6. REFERENCES

During pregnancy, the developing fetus is entirely dependent on its mother for nutrition and waste disposal, which take place almost exclusively across the placenta. The placenta is, however, not just a passive mediator; it can also regulate the rates and amounts of nutrient transported. This is particularly true for iron transport. This review examines how the fetus, and not the mother, regulates iron uptake and transfer across the placenta, how these processes interact, and how the fetus can modulate iron metabolism in the mother.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CONCLUSION
  5. Acknowledgments
  6. REFERENCES

Iron is an essential micronutrient, required for many biological processes.1 Apart from its role in hemoglobin, it is central to many redox processes throughout the body. However, in addition to being essential, iron has the capacity to generate free radicals, which, in turn, can cause severe cellular damage.2

During in utero fetal development, the mother and her child have a complex series of equations to balance. At birth, the baby has a total of about 1 g of iron in its body. All of that has come from the mother and all of it has been transferred across the placenta. About 600 mg comes from maternal dietary intake and from the cessation of menses. However, about 400 mg has to come from maternal stores.3 Questions then arise regarding how the fetus can mobilize iron from the mother's stores and how the mother can balance her own and her baby's requirements. This balancing is essential.

Several studies have shown that maternal iron deficiency has serious consequences for offspring, and others have shown that maternal iron supplementation increases the size of the infant at birth and decreases both the risk of prematurity and the risk of problems at birth.4–6 Good data also exist that suggest decreased iron status is associated with increased risk of developmental delay and increased incidence of mental problems in adulthood.7,8 In a series of dietary experiments, our group examined fetal development and pregnancy outcome in relation to maternal and fetal iron status in rats. The experiments showed that maternal iron deficiency results in fetal growth retardation, altered organ size,9 and, intriguingly, the development of hypertension in offspring, even though the offsprings' iron status returned to normal following weaning.10 Whether this occurs in humans as well as rodents is not certain, though data suggesting a relationship between poor iron status early in pregnancy and smaller size of offspring at birth has been presented. Iron overload due to iron supplementation in non-anemic women during pregnancy has also been associated with negative pregnancy outcomes, including low birth weight,11 and even with the development of gestational diabetes.5

Taken together, these results argue that regulation of iron transfer during pregnancy is very important. Not surprisingly, therefore, the body has evolved a complex series of checks and balances to ensure that it keeps a sufficient amount of iron while simultaneously keeping levels below potentially toxic amounts.

This review considers the mechanisms involved in iron transfer from the mother to her fetus, how the process is regulated, and how this process adapts to iron deficiency during pregnancy, which is a much more common problem than iron overload. The primary focus is on the roles played by the fetus and the placenta in the regulation and transport processes, and areas where questions remain unanswered are highlighted.

The mechanisms of iron uptake and transfer in the placenta have been investigated extensively over many years. The placenta plays the central role in transfer, and its complex structure is important when considering the process of transport. In mammals, there are several different types of placenta.12 Humans have a hemomonochorial placenta. This constitutes a single layer of syncytial cells, the syncytiotrophoblast, which is derived from trophoblast cells that form a discontinuous layer beneath the syncytium. The maternal-facing membrane of the syncytium folds into microvilli, greatly increasing the surface area of the placenta.13

Once a nutrient crosses the syncytium, it has to transfer across the barrier of the fetal capillary layers. Since there are very limited data on this process for any nutrient, and none presently exist for iron, the process will not be considered further in this review; it is recognized, however, that the process may be very important. Before we can begin to understand how the placenta regulates iron transfer, it is essential to think about the mechanisms involved in transfer. This is reviewed briefly below. An overall view of the mechanism of iron transfer across the placenta is shown in Figure 1.

image

Figure 1. Mechanism of iron transfer across the placenta. This diagram shows the pathways involved in iron transfer across the placenta as we understand them currently. The steps involved are explained in detail in the text.

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In serum, iron is almost exclusively bound to transferrin. Under very exceptional circumstances, of quite severe pathology, non-transferrin-bound iron can be detected in plasma, but that is not of present concern. Transferrin binds two atoms of iron per molecule and, under normal circumstances, is about 30% saturated. This number is variable and it changes during pregnancy. Transferrin itself is a glycoprotein, but the function of the sugar residues is not well understood and, interestingly, their profile changes in pregnancy.14,15 Why this occurs is not known, but it may have a hitherto undiscovered role in iron transfer.16

The first step in the transfer of iron across the placenta involves transferrin binding to its receptor, the transferrin receptor (TfR), on the apical membrane. At pH 7.4, diferric transferrin has a high affinity for the receptor.17 Apo-transferrin, in contrast, has a low binding affinity, so even though apo-transferrin may be present at higher concentrations in serum, the diferric form will bind preferentially.

Following binding, the complex is internalized in coated pits, and the endosomes are acidified, probably through the action of an H-ATPase. At this lower pH, the binding affinities reverse; apo-transferrin has a higher affinity than diferric transferrin. Also, iron binding to transferrin is greatly reduced. Consequently, the iron is released from transferrin into the vesicle, while the protein remains bound to the receptor. The iron is then released from the vesicle into the cytosol. Under most circumstances, and in most cell types, it is assumed that this takes place through a divalent metal channel, DMT1. In the placenta, however, there must be an additional mechanism involved, because DMT1 knockout mice can still deliver iron from mother to fetus.18

How iron gets from the cytosol in the region of the vesicle to the basolateral region of the cell is completely unknown. In the case of copper, “chaperones” have been identified that bind the metal to reduce its oxido-reductive capacity and prevent free radical formation.19 Currently, there is very little evidence that equivalents exist for iron.

Once iron reaches the basolateral membrane, it is released into the fetal circulation through ferroportin (FPN1).20 It passes through this channel as Fe(II) but binds to fetal transferrin as Fe(III). It must, therefore, be oxidized prior to binding. In the gut, this is carried out by a copper-dependent ferroxidase called hephaestin. In the placenta, a homologue has been identified, which we have called zyklopen.21 We first noted it as a ceruloplasmin-like protein, which also had oxidase activity similar to ceruloplasmin, but which was differently affected by anti-ceruloplasmin antibodies.22 It was slightly larger than ceruloplasmin and was regulated by both iron and copper. When the human genome was published, we noted the existence of a hephaestin-like protein. We examined expression patterns in the placenta, showed it had ferroxidase activity and that siRNA to the sequence could downregulate the activity. We now hypothesize that zyklopen is the ferroxidase that oxidizes Fe(II) to Fe(III) in the placenta prior to incorporation into fetal transferring.21

Associated with ferroportin is a protein called HFE. This is a product of the hemochromatosis gene and is thought to regulate iron efflux from cells through interaction with ferroportin.23 Additionally, there is a second form of transferrin receptor, TfR2; this is involved in the regulation of iron uptake and its processing,24 but its role in the placenta is not defined. The levels of TfR and ferritin within the cell are regulated by iron regulatory proteins 1 and 2. Their role is complex, involving the binding of iron in an iron-sufficient situation, which prevents them from attaching to either the 5′ or the 3′ end of the mRNA for TfR or ferritin. This results in the degradation of TfR mRNA as well as the induction of translation in the case of ferritin mRNA. The reverse happens in the absence of iron, such that IRP binds to mRNA, stabilizing TfR mRNA and preventing translation of ferritin mRNA.25 We will not be considering their function further in this review, since we believe it is more specifically related to intracellular regulation rather than trans-placental transport. However, this may prove to be overly simplistic and it may turn out that the role of the iron regulatory proteins in this process may be significant and may have to be re-examined.

In our rat model, maternal iron deficiency resulted in the birth of pups that were smaller, with larger hearts and higher systolic blood pressure. This phenomenon persisted into adulthood, even though the pups had normal iron status following weaning.10 Why this occurs is presently unclear and is currently being investigated.

Our experiments aimed at furthering understanding of the regulation of iron transfer across the placenta involved a complex series of dietary treatments.26 In these experiments, weanling female rats were fed a normal diet for 2 weeks. One group was then given an iron-deficient diet for 4 weeks before being mated with rats of the same strain. One group of the iron-deficient rats was then put back onto a normal diet with an iron supplement. A second group was given iron from the second half of pregnancy, while a third group was kept on the iron-deficient diet until day 21.5. The first intriguing observation was that maternal hematocrit levels in the iron-deficient animals did not show any change until after day 12.5 of pregnancy. By day 21.5, the hematocrit levels of the iron-deficient animals had decreased, as expected. Dietary supplementation with iron over the first half of pregnancy but not in the second half still resulted in decreased hematocrit levels.

When maternal iron levels were measured, we found that iron supplementation in either half of pregnancy did not restore liver stores to the levels seen in controls. However, it did have some effect, with iron stores being significantly higher in the supplemented group than in the animals kept on an iron-deficient diet throughout pregnancy. These data, together with our observation that maternal iron levels in deficient animals can be up to 30% lower than those of controls, whereas fetal iron levels are about 70% of those of controls, give us some idea of the hierarchy of iron status management. Maternal hematocrit is important and is maintained over maternal iron stores, but something else takes priority when iron is taken up from the diet. This is, of course, the developing fetus.

The pertinent results from the experiments described above can be summarized as follows. 1) Fetal hematocrit and iron levels were reduced in the iron-deficient animals compared to the controls. 2) Supplementation in the second half of pregnancy restored fetal iron levels and hematocrit levels to those of controls. 3) Supplementation solely in the first half of pregnancy did not have the same effect. 4) At day 21.5, iron levels in the two supplemented groups of animals were lower than in the controls, but they were still higher than in the animals that were deficient throughout pregnancy. These results suggest that the maternal liver accumulates iron during the first half of pregnancy and then donates that store to the fetus in order to keep fetal levels as high as possible. This information provides a priority list for where iron is directed during pregnancy, i.e., 1) the fetus, 2) maternal hematocrit, and as a very poor number 3) maternal iron stores. If these results can be extrapolated to humans, they have important implications, particularly for the nutrition of women between pregnancies. Following a first pregnancy, it seems very important to try to restore the woman's iron stores to her pre-pregnancy level; this should be done to reduce the risk of possible problems associated with iron deficiency, which would otherwise become greater with subsequent pregnancies.

With priorities established, the question then arises as to how these priorities are managed. In order to investigate this, we examined gene expression of the major regulators of iron status, TfR1 and TfR2, ferroportin, HFE and hepcidin, and the storage proteins ferritin H and L. In the maternal liver, TfR levels were higher in the deficient animals than in the controls or in either of the supplemented groups. The animals that received iron supplementation in either the first or second half of pregnancy had the same levels of TfR as the controls, despite the fact that iron levels had not returned to control levels. This was a surprising result, which began to suggest that perhaps iron levels in the maternal liver may not be the only factor regulating TfR expression – an idea that is explored further below.

In contrast to the TfR expression, hepcidin levels in the maternal liver follow the predicted pattern. Hepcidin is a negative regulator of iron absorption, and iron uptake across the gut increases as hepcidin levels decrease. Supplementation in the second half of pregnancy induced a decrease in TfR expression in the maternal liver, while levels remained high in the animals with low levels of iron in the maternal liver. None of the dietary treatments altered the expression of TfR2, ferritin, or HFE.

Moving down the supply line, we next examined placental gene expression and iron levels. Iron levels, measured as both heme and non-heme fractions, were altered in a similar pattern to that observed in the maternal liver; however, the reduction in iron levels was less severe. It is clear that the placenta's function is so important that its nutrient levels are maintained, even in the face of nutritional stress.

There was, of course, a significant increase in TfR receptor expression levels in the placentas of iron-deficient dams. As with the maternal liver, there were no changes in TfR2 or ferritin levels. In many other tissues, iron storage and efflux are regulated through an efflux protein called ferroportin.27 In the placenta, however, there was no change in ferroportin levels. In the gut, there is a complex interplay among the proteins involved in the regulation of efflux. As mentioned above, hepcidin signals liver iron requirements. It binds to HFE, which, in turn, is associated with ferroportin. As iron requirements go down, ferroportin is internalized and broken down by ubiquitination. This does not appear to happen in the placenta. Instead, the accumulation and transfer of iron is regulated by the levels of TfR.

This is mediated by fetal liver iron and hepcidin levels. In the iron-deficient fetus, hepcidin levels are much lower, and when correlated with fetal iron levels, a very strong relationship is evident. Hepcidin expression was equally strongly related to the levels of TfR in the placenta, although a role for placental iron levels in the regulatory process cannot be excluded. This gives an indication of the regulatory steps involved in the transfer of iron from mother to fetus.

Iron in the fetal liver regulates the expression of hepcidin. As the levels rise, so too does hepcidin production. Hepcidin is then released into the fetal circulation and interacts with the placenta through a mechanism about which little is known. Communication between the basolateral side, where the hepcidin is located, and the microvillar side, where the TfRs are found, results in changing levels of receptor and modulated accumulation of iron. How this process is transduced is completely unknown, but it seems to be different from the processes in the gut, where regulation takes place at the basolateral, rather than the apical, side.

An even more interesting aspect of our data is their suggestion that the regulation of iron transfer across the placenta by the fetal liver does not stop there. As mentioned earlier, the TfR levels in the maternal liver do not correlate particularly well with the iron levels in that tissue. Instead, we found a “broken stick” relationship between the levels of iron in the fetal liver and TfR expression in the maternal liver. This is a complex interaction, as shown in Figure 2. In essence, it shows that, within limits, the levels of iron in the mother's liver are tightly regulated to make sure that iron transfer to the fetus is maximal. At a certain point, however, the maternal liver reasserts control and protects itself against further reductions in iron. Teleologically, we can assume this is the minimum level the mother's liver can tolerate.

image

Figure 2. Regulation of TtfR MRNA expression in maternal liver at gestational day 21.5. Solid lines represent predicted values using a broken stick model (P < 0.0001, R2 = 0.98). Results from Gambling et al.26

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There are some clues regarding how this signaling system operates. When fetal liver levels are below a critical point, somewhere around 1,300 µg/g dry weight, maternal hepcidin levels are very low, maximizing absorption. However, when the levels get close to control in the fetal liver, the call for iron from the maternal gut is reduced by increasing hepcidin (Figure 3).

image

Figure 3. Mean fetal liver iron levels plotted against maternal liver hepcidin mRNA expression. Dotted line represents the mean liver iron level of the control fetuses. Results from Gambling et al.26

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CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CONCLUSION
  5. Acknowledgments
  6. REFERENCES

This review has demonstrated the critical role of the fetus, specifically the fetal liver, in the regulation of maternal iron metabolism during pregnancy. It is clear that a hierarchy of demand is directed towards the maintenance of fetal iron stores. It is also clear that there are limits beyond which the systems restore the maternal priority. The interplay among all these systems is beginning to be elucidated, but there is still much to be learned.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CONCLUSION
  5. Acknowledgments
  6. REFERENCES

Funding.  The authors' work is supported by Scottish Government, the European Union (EARNEST and NuGO), and the International Copper Association.

Declaration of interest.  The authors have no relevant interests to declare.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CONCLUSION
  5. Acknowledgments
  6. REFERENCES
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