Comparative Medicine Vol 63, No 2

Copyright 2013 April 2013

by the American Association for Laboratory Animal Science Pages 127135

Original Research

Effect of Dietary Iron on Fetal Growth in

Pregnant Mice

Andrea C Hubbard,1,* Sheila Bandyopadhyay,2 Boguslaw S Wojczyk,2 Steven L Spitalnik,2 Eldad A Hod,2 and Kevin A Prestia1

Iron deficiency is the most common nutritional disorder. Children and pregnant women are at highest risk for developing iron
deficiency because of their increased iron requirements. Iron-deficiency anemia during pregnancy is associated with adverse effects
on fetal development, including low birth weight, growth retardation, hypertension, intrauterine fetal death, neurologic impair-
ment, and premature birth. We hypothesized that pregnant mice fed an iron-deficient diet would have a similar outcome regarding
fetal growth to that of humans. To this end, we randomly assigned female C57BL/6 mice to consume 1 of 4 diets (high-ironlow-
bioavailability, high-ironhigh-bioavailability, iron-replete, and iron-deficient) for 4 wk before breeding, followed by euthanasia
on day 17 to 18 of gestation. Compared with all other groups, dams fed the high-ironhigh-bioavailability diet had significantly
higher liver iron. Hct and Hgb levels in dams fed the iron-deficient diet were decreased by at least 2.5 g/dL as compared with those
of all other groups. In addition, the percentage of viable pups among dams fed the iron-deficient diet was lower than that of all
other groups. Finally, compared with all other groups, fetuses from dams fed the iron-deficient diet had lower fetal brain iron levels,
shorter crownrump lengths, and lower weights. In summary, mice fed an iron-deficient diet had similar hematologic values and
fetal outcomes as those of iron-deficient humans, making this a useful model for studying iron-deficiency anemia during pregnancy.

Abbreviations: GD, gestational day; RDW, RBC distribution width.

Iron deficiency is the most common nutritional disorder of hu-
mans worldwide, with an estimated prevalence of 24.8%.69 Iron-
deficiency anemia accounts for approximately 50% of anemia
cases worldwide; children and pregnant women are at greatest
risk because of their high iron requirements.17 During pregnancy,
the daily requirement for dietary iron absorption increases from
0.8 mg in the first trimester10 to 7.5 mg in the third trimester.53 In
addition, during pregnancy, the blood volume expands by ap-
proximately 35%, and growth of the fetus, placenta, and other
maternal tissues increase maternal iron demand.13 The World
Health Organization estimates that the global prevalence of ane-
mia in pregnant women is 41.8%, with the highest prevalence in
the third trimester.17 Although iron-deficiency anemia is thought
to be a serious nutritional deficiency in developing countries,
even industrialized nations are affected. The Centers for Disease
Control and Preventions Pediatric and Pregnancy Nutrition
Surveillance System for women participating in public health
programs in the United States reports a prevalence of 33.5% for
iron-deficiency anemia in pregnant women in their third trimes-
ter.60 Iron-deficiency anemia during pregnancy is associated with
adverse effects on the fetus, including low birth weight, growth
retardation, hypertension, intrauterine fetal death, neurologic
impairment, compromised immune system development, and
premature birth.1,30,69,73,76 Compared with nonanemic controls,
Received: 17 Sep 2012. Revision requested: 23 Oct 2012. Accepted: 08 Nov 2012.
1
Columbia University Institute of Comparative Medicine and 2Laboratory of Transfusion
Biology, Department of Pathology and Cell Biology, Columbia University College of
Physicians and Surgeons, New York, New York.
*
Corresponding author. Email: ah2911@columbia.edu
pregnant women with iron-deficiency anemia during the first
2 trimesters have a 2-fold higher risk of preterm delivery and a
3-fold higher risk of delivering a low-birthweight baby.13
Iron-deficiency anemia during pregnancy can have negative ef-
fects on mothers also. For example, anemia is associated with 20%
of maternal deaths worldwide.81 Therefore, iron supplementation
is recommended during pregnancy; however, controversy exists
because of conflicting data regarding whether this practice im-
proves maternal or fetal outcomes. For example, untargeted iron
supplementation in areas of high malaria prevalence is associated
with an increased risk of mortality in children.65 Furthermore,
iron deficiency is associated with a decreased incidence of placen-
tal malaria, especially in primigravidae.24,39,68 Although few stud-
ies examining the effects of iron supplementation on pregnancy
outcome in malaria-endemic regions are available, some,12,55,56 but
not all,51 suggest that iron supplementation increases the risk of
malaria. This potentially increased risk led one study to conclude
that there is a priority to establish if reversing iron deficiency
through iron supplementation programs either prior to or during
pregnancy enhances malaria risk.68 Therefore, our goal was to
establish a mouse model of iron-deficiency during pregnancy to
begin studying this issue.
Although several animal models of maternal iron deficiency
have been established, very few have used a mouse model to in-
vestigate the effects on the fetus during gestation. Several non-
human primate studies described the effects of iron deficiency
during pregnancy.31-34 Similar to humans, nonhuman primates
complete embryogenesis in the first trimester and have an ex-
tended period of postembryonic development. In contrast, mice

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and rats complete organogenesis at gestational day (GD) 15 to
16 and are born shortly afterward (for mice, at GD 19 to 21). Al-
though the reproductive physiology of nonhuman primates is
more similar to that of humans than is that of rodents, the use of
nonhuman primates is limited by cost, regulatory requirements,
and ethical considerations.
Rodents are the most commonly used animal models for mod-
eling human development.33 Advantages of mice include their
small size and short generation time. The wide use of transgenic,
gene replacement, and knockout technologies in mice offers the
opportunity to study the effects of specific genes involved in iron
metabolism. Mice have also been used to study the effects of
iron deficiency on behavior in neonates.43,45 Mice have been used
extensively to study malaria.3,47,61,71,82 Although no single mouse
model replicates all of the pathologies that occur in human ma-
laria, different mouse models may reflect the diversity of patholo-
gies seen in humans.46 Previous studies of resistance to malaria in
mice with diet-induced iron-deficiency have used C57BL/6 mice
infected with either Plasmodium yoelii or P. berghei.41,49,82 For this
reason, we decided to use C57BL/6 mice to develop a model of
iron status during pregnancy.
To this end, we assigned cohorts of weanling female mice to
receive iron-deficient (5 ppm FeSO4), iron-replete (45 ppm Fe-
SO4), high-ironhigh-bioavailability (220 ppm iron as FeSO4), or
high-ironlow-bioavailability (220 ppm iron in nonstandardized
forms) diet for 4 wk, after which they were mated with male
mice. Pregnant mice were euthanized on GD 17 to 18 to measure
various parameters in the dam and the litter. We designed this
model to facilitate future explorations of the effects of iron sup-
plementation on iron-deficiency anemia during pregnancy. We
hypothesized that the fecundity of iron-deficient dams would be
decreased and that fetuses from iron-deficient dams would weigh
less and have shorter crownrump lengths than those derived
from dams fed iron-replete diet or a high-iron diet with high iron
bioavailability.
Materials and Methods
Animals. Female C57BL/6 mice (Charles River, Cambridge,
MA) were obtained at 3 wk of age. Male C57BL/6 mice for breed-
ing were obtained from the same source. Mice were housed at a
maximum of 5 mice per cage on autoclaved bedding (ALPHA-
dri/Cob Blend, Shepherd Specialty Papers, WF Fisher and Son,
Somerville, NJ) in static polysulfone microisolator cages. Mice
had access to acidified water (pH 2.5 to 3.0) and irradiated pel-
leted diet ad libitum. Mice were free of Sendai virus, pneumonia
virus of mice, murine hepatitis virus, minute virus of mice, mouse
parvovirus, Theiler mouse encephalomyelitis virus, reovirus type
3, epizootic diarrhea of infant mice virus, lymphocytic chorio-
meningitis virus, ectromelia virus, mouse adenovirus, K virus,
polyoma virus, Mycoplasma pulmonis, and ecto- and endopara-
sites. Mice were maintained in accordance with the Guide for the
Care and Use of Laboratory Animals37 in an AAALAC-accredited
facility. All procedures outlined in the study were approved by
Columbia University Medical Centers IACUC.
Diets. Upon arrival, female mice were assigned to experimental
groups and fed their assigned diet for 4 wk before mating; diets
included the facilitys standard diet for mice (high iron content
but low iron bioavailability; PicoLab 5053, PMI, St Louis, MO; 220
ppm iron; n = 10), purified AIN93M iron-supplemented (high iron
content and bioavailability; 220 ppm iron; n = 10) diet, AIN93M
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cm12000119.indd 128
iron-replete diet (45 ppm iron; n = 10), and AIN93M iron-deficient
diet (5 ppm; n = 11). AIN diets were prepared by Harlan Teklad
(Madison, WI) by using ferrous sulfate as the sole iron source; all
other components were consistent between the AIN diets. The
diet with high iron bioavailability was formulated to contain a
similar amount of iron as that in the standard diet at our facility.
The National Research Council proposes an iron requirement of
35 mg/kg of diet (equivalent to 35 ppm) for mice but do not have
a recommended iron requirement for pregnant mice.38 In com-
parison, the National Research Council recommends 35 ppm iron
for growth and 75 ppm for maintenance of nonpregnant rats and
75 ppm for pregnant rats.38
Timed pregnancy. To produce pregnancy, a single male and a
single female mouse were housed together for as long as 48 h.
Female mice were checked for vaginal plugs each morning and
evening. When a vaginal plug was observed, the female mouse
was separated from the male mouse and weighed frequently to
confirm pregnancy by weight gain. In particular, body weights of
mice were recorded once weekly during the first 4 wk and 3 times
each week once the mice were mated.
GD 0 was defined as the day on which a vaginal plug was ob-
served. A few mice became pregnant but lacked vaginal plugs;
for this reason, our GD range encompasses 2 d. According to one
study,67 pregnancy in BALB/c mice is associated with a weight
gain on GD 8 of 8.09% to 9.39% (95% confidence interval). Be-
cause of the likelihood of interstrain differences, we modified
this estimate to be more conservative. Therefore, when female
C57BL/6 mice did not gain at least 10% in weight over 2 wk, mat-
ing was repeated. Female mice were bred until 6 mo of age and,
if no pregnancy occurred, were euthanized by carbon dioxide
asphyxiation.
Tissue collection. On GD 17 to 18, female mice were anesthe-
tized with isoflurane and cervically dislocated. The uterus was
removed and the number of implantation sites were counted
and expressed as the numbers of resorbed or nonviable fetuses
and viable fetuses. A fetus was considered nonviable when it
was pale white, had petechiae, or showed areas of discoloration.
Necrotic fetuses having a crownrump length of less than 1 cm
were considered to be resorbed; similarly, when a placenta had
no associated fetuses, they were considered to be resorbed. Each
fetus was weighed and measured from crown to rump by using
calipers. Viable and nonviable fetuses were decapitated, and the
heads were weighed; this tissue was used to determine brain iron
content. Livers from dams were collected, weighed, and used to
measure iron content. Nonheme iron in organs was determined
as described previously.75 Briefly, the wet weight of each organ at
necropsy was recorded; an approximately 100-mg portion was
dried at 65 C for 24 h and digested in an acid mixture at 65 C for
24 h. The iron content of the centrifuged acidified sample was de-
termined by using a colorometric method (bathophenanthroline).
The absorbance of samples and iron standards at 535 nm was
measured in duplicate, and mean values were used for calculat-
ing total organ iron.
Hematologic measures. Immediately after euthanasia of mice, a
25-gauge needle with heparinized syringe was used to collect car-
diac blood (at least 500 L) from each dam. Aliquots (100 L) were
removed for CBC analysis; the remainder was centrifuged for 5
min at 1000 g. The plasma supernatant was removed, stored
at 80 C, and subsequently used to measure ferritin by using a
mouse ferritin ELISA (Kamiya Biomedical, Seattle, WA) according
4/1/2013 9:58:49 AM
 Mouse model of iron deficiency

to the manufacturers instructions. CBC analyses were performed

on an automated hematology analyzer (Forcyte Veterinary Hema-
tology Analyzer, Oxford Science, Oxford, CT)
Statistics. For comparisons among fetuses, the values for each
litter were averaged and considered as a single point, rather than
treating each fetus as a single point. All data were analyzed by
using Prism 5 software (GraphPad Software, San Diego, CA). Sta-
tistical significance was determined by one-way ANOVA with a
Bonferroni posttests to compare different diet groups. All data are
expressed as scatter plots showing the mean SEM. Data were
considered statistically significant at an level of P less than
0.05.

Results
Compared with all other groups, dams fed an iron-deficient
diet had significantly decreased Hct (26.9%; P < 0.0001; data not
shown) and Hgb (8.9 g/dL; P < 0.0001) levels (Figure 1 A). In
contrast, Hgb and Hct levels in mice on the high-ironlow-bio-
availability, iron-replete, and high-ironhigh-bioavailability diets
were within (or close to the lower limit of) the normal reference
ranges used by the diagnostic laboratory. Similar to iron-deficient
humans, mice fed the iron-deficient diet showed decreased MCV
(P < 0.0001), decreased MCH (P < 0.0001), and increased RBC dis-
tribution width (RDW; P < 0.0001) compared with those of all
other groups (Figure 2).In contrast, MCV, MCH, and RDW values
for all other groups were within the respective reference ranges.
Although the average platelet count for mice on the iron-deficient
diet was still within the reference range, it was significantly (P <
0.05) higher than that of all other diet groups (Figure 1 B) and was
consistent with findings from humans with iron deficiency.35,66
Finally, WBC counts were not different between the groups (data
not shown).
Liver iron content generally is considered to be the most ac-
curate measure of iron status.36 Maternal liver iron concentrations
were significantly (P < 0.01) higher in mice fed the high-ironhigh-
bioavailability diet as compared with all other diets (Figure 3 A). The
liver iron concentration in the group fed the iron-deficient diet
did not differ from that of dams on the iron-replete or high-iron
low-bioavailability diet. Compared with nonpregnant C57BL/6
mice, pregnant mice fed the iron-replete, high-ironlow-bioavail-
ability, or high-ironhigh-bioavailability diet had significantly Figure 1. Effect of maternal iron deficiency on maternal blood hemato-
(P < 0.0001) lower liver iron content (Figure 3 B). However, liver logic parameters measured at GD 17 to 18. The area between the dotted
iron content did not differ between pregnant and nonpregnant lines represents the normal reference range. (A) Hgb levels were sig-
mice that received the iron-deficient diet. Consistent with the liv- nificantly (, P < 0.0001) decreased in the mice that received the iron-
deficient diet compared with all other diets. (B) Platelet counts in the
er iron results, serum ferritin levels in dams fed the iron-deficient
iron-deficient group were significantly higher than those of mice fed the
diet were significantly (P < 0.001) lower than those of other groups high-ironlow-bioavailabilty (*, P < 0.05), high-ironhigh-bioavailability
(Figure 3 C). In addition, serum ferritin levels were significantly (*, P < 0.05), or iron-replete (, P < 0.001) diet.
(P < 0.05) higher in mice fed the high-ironhigh-bioavailability
diet than in the other groups.
Severe iron deficiency during pregnancy can lead to decreased though not statistically significant, the mean percentage maternal
maternal weight, fecundity, and fetal viability.74 However, ma- weight gain among dams fed the iron-deficient diet was 15.55%
ternal body weight at GD 0 to 1 or GD 17 to 18 did not differ less than that of dams fed the high-bioavailability diet and 17.5%
between any of the diet groups. The weight ranges for female less than that of mice fed the iron-replete diet.
mice at GD 0 to 1 were 18.4 to 22.9 g, 18.6 to 25.1 g, 19.8 to 25.0 g, The mean number of breeding attempts needed to achieve preg-
and 18.4 to 23.8 g for the low-bioavailability, high-bioavailability, nancy was 6.3, 3.7, 3.9, and 3.7 for the iron-deficient, iron-replete,
iron-replete, and iron-deficient diets, respectively (Figure 4 A). high-ironhigh-bioavailability, and high-ironlow-bioavailability
However, percentage maternal weight gain during pregnancy diet groups, respectively; differences between groups were not
(that is, [{GD 17 to 18} {GD 0 to 1}] / [GD 0 to 1]) for dams on statistically significant. In addition, the number of viable fetuses
the iron-deficient diet was significantly (P < 0.05) lower than that per litter was not statistically different among groups (Figure 5 A).
of mice fed the high-ironlow-bioavailability diet (Figure 4 B). Al- However, the number of resorbed or nonviable fetuses per litter

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was significantly (P < 0.05; Figure 5 B) higher and the percentage

of viable fetuses per litter was significantly (P < 0.01; Figure 5 C)
lower in dams fed the iron-deficient diet compared with all other
diets. Of particular note, 5 of the 11 dams fed the iron-deficient
diet had no viable fetuses; dams on iron-replete, high-ironlow-
bioavailability, or high-ironhigh-bioavailability diet all had at
least 1 viable pup per pregnancy. The mean fetal weight per lit-
ter from dams fed the iron-deficient diet was at least 30% less (P
< 0.05) than those for all other groups Figure 6 A). In addition,
mean fetal crownrump length per litter was at least 15% less (P <
0.001) for mice fed the iron-deficient diet compared with all other
diets (Figure 6 B). Finally, fetuses from dams fed the iron-deficient
diet had the lowest (P < 0.001) brain iron levels among all groups
(Figure 6 C).

Discussion
The present study evaluated the effects of different iron-con-
taining diets on pregnant mice and their fetuses. Dams fed an
iron-deficient diet for 4 wk before mating had microcytic anemia
with decreased Hgb, Hct, MCV, MCH, and serum ferritin levels
and increased RDW; taken together, these results are consistent
with iron-deficiency anemia. The normal reference ranges for
Hgb and Hct in women are 12.0 to 16.0 g/dL and 36.0% to 46.0%,
respectively.42 Similarly, in normal female C57BL/6 mice, Hgb
and Hct are 16.3 to 17.7 g/dL and 43.1% to 55.2%, respectively.72
According to the Centers for Disease Control and Prevention,
pregnant women with a Hgb concentration of less than 9.0 g/dL
or a Hct of less than 27% should be referred for further evaluation
by a physician familiar with anemia during pregnancy.13 In the
current study, mean Hgb and Hct values in female mice fed an
iron-deficient diet were slightly below these recommended cutoff
values.
In the current study, platelet counts were increased in mice fed
an iron-deficient diet as compared with the other 3 diets. Throm-
bocytosis is associated with iron deficiency in humans and was
reported to occur in rats fed an iron-deficient diet. 14 Although
the cause of this reactive thrombocytosis is unknown, it may be
related to increased platelet production.16 Pregnant mice fed an
iron-deficient diet had more nonviable or resorbed fetuses, de-
creased fetal weight, and decreased fetal crownrump length. In
humans, iron deficiency during pregnancy results in an increased
risk of premature birth, smaller infants, intrauterine fetal death,
and developmental problems, as well as an increased risk of hem-
orrhage in the mother.1,11,29,30,64,69 The results of the current study
correlate well with those that occur in human iron deficiency dur-
ing pregnancy.
Studies similar to the current one examined rats provided with
various amounts of iron in the diet.26 Although severe iron defi-
ciency during pregnancy can lead to reduced maternal weight,
fertility, and fetal viability, mild iron deficiency had no significant
effect on fetal number or weight in rats.9,25,26,73,74 However, here we
found that fetuses from iron-deficient pregnant mice weighed less

Figure 2. Effect of maternal iron deficiency on maternal blood hematologic

parameters measured at GD 17 to 18. The area between the dotted lines rep-
resents the normal reference range. (A) MCV and (B) MCH were decreased
significantly (, P < 0.0001) in mice fed the iron-deficient diet as compared
with all other diets. (C) RDW values were significantly (, P < 0.0001) high-
er in mice fed the iron-deficient diet as compared with all other diets.

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Figure 3. Iron stores in mice fed diets containing different amounts of
iron. (A) Mice fed a high-ironhigh-bioavailability diet had significantly
increased liver iron, as compared with those fed a high-ironlow-bioa-
vailability (, P < 0.001), iron-replete (, P < 0.01), or iron-deficient (, P <
0.0001) diet. There were no significant differences between any of the
other diets. (B) Except for mice fed the iron-deficient diet, nonpregnant
mice had significantly (, P < 0.0001) higher liver iron values than did
pregnant mice fed the same diet. (C) Serum ferritin levels in mice on the
iron-deficient diet were significantly (, P < 0.001) lower than those in
mice on the iron-replete, high-ironlow-bioavailability, and high-iron
high-bioavailability diets. Mice fed the high-ironhigh-bioavailability
diet had higher (*, P < 0.05) serum ferritin levels than those of mice fed
the high-ironlow-bioavailability or iron-replete diet.
and had a shorter crownrump length than did those in the other
diet groups. Crownrump length is commonly used to evaluate
teratogenic effects on fetal development. Although weight has
been used for comparing iron-deficient pups,4,18 to our knowl-
edge, crownrump length has not been reported in previous
cm12000119.indd 131
Mouse model of iron deficiency
Figure 4. Weights of dams on diets containing different amounts of iron.
(A) No differences in body weight were found at baseline (GD 0 to 1)
among groups of mice on the different diets. (B) Pregnant mice fed the
iron-deficient diet had a lower (*, P < 0.05) percentage weight gain than
did those fed the high-ironlow-bioavailability diet.
mouse studies of iron status. In addition, although we did not
observe a significant difference in maternal weight among the
groups of pregnant mice, the percentage maternal weight gain
during pregnancy was lower in those fed the iron-deficient diet
(5 ppm iron) compared with the standard diet for our facility
(220 ppm iron with low bioavailability). We also observed more
resorbed or nonviable fetuses in iron-deficient dams, a finding
similar to that of a study on the influence of chronic alcohol con-
sumption on reproductive performance in mice fed an iron-defi-
cient (2 ppm) or iron-adequate (30 ppm) diet.18
The iron status of the fetus is assumed to be independent
of maternal iron status during pregnancy, except in cases in
which infants are born to severely anemic women.1,26,40 A study
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Figure 5. Pup viability among dams fed diets containing different
amounts of iron. (A) The number of viable pups per dam did not differ
between dams fed different iron-containing diets. (B) The number of re-
sorbed or nonviable pups was significantly (*, P < 0.05) greater for dams
fed the iron-deficient diet as compared with all other groups. (C) The
percentage of viable pups per litter was significantly lower for dams
fed the iron-deficient diet as compared with those on the regular (, P
132
cm12000119.indd 132
in rats found that the maternal hematocrit is maintained at the
expense of reducing iron stores in the maternal liver until fetal
demands become too high; then the maternal hematocrit de-
creases to maintain fetal supply.27 Other studies have indicated
that when maternal iron status is poor, the number of placental
transferrin receptors increases so that iron will preferentially
be imported by the placenta for delivery to the fetus.1,28 As ma-
ternal iron stores are depleted, intestinal iron absorption is
increased to maintain adequate iron supply in both the mother
and fetus.50,74 One study suggests that once maternal iron stores
reach a minimal limit, no further iron is mobilized from the
maternal liver to the fetus.4 Given our finding that liver iron
stores are decreased in pregnancy as compared with those of
nonpregnant female mice on the same diets, with those on the
iron-deficient diet as the exception, we hypothesize that this
storage form of iron is mobilized preferentially to the fetus
and, only when depleted, does anemia manifest in the dam as
RBC iron is mobilized for the fetus. Although pregnant mice
fed the high-ironhigh-bioavailability diet had a decreased
liver iron concentration compared with that of the nonpreg-
nant cohort, these values were still significantly greater than
those of any other diet group during pregnancy. Nonetheless,
additional studies are needed to determine what effect this
increased liver iron may have on fetuses and on the outcome
of future pregnancies.
Ferritin is a protein complex responsible for intracellular
iron storage. It accounts for approximately 95% of the iron
stored in the liver.6,8 Ferritin is often used as a tool to assess
the size of the iron storage pool. For example, serum ferritin
levels of less than 15 ng/mL in anemic women are diagnostic
of iron-deficiency anemia.13 In nonsupplemented humans, as
the iron supply is diverted to the fetus, ferritin levels decrease
dramatically as pregnancy proceeds.22,50,77 We observed lower
serum ferritin levels in iron-deficient mice, consistent with iron
deficiency.
Dams on the high-ironhigh-bioavailability diet had the
highest liver iron stores among our test groups. Even though
the low- and high-bioavailability diets have the same iron con-
tent (that is, 220 ppm), we did not see comparable iron levels in
the dams fed these diets. This difference likely reflects the rela-
tive bioavailability of the iron in the 2 diets. Iron in the high-
bioavailability diet is present as ferrous sulfate; in contrast,
most of the iron in the low-bioavailability diet is in various
forms in the grain ingredients, such as ferrous carbonate and
ferric oxide, as contaminants of the calcium and phosphorus
salts. These forms of iron are less bioavailable than is ferrous
sulfate.23 Many foods high in iron are limited by the bioavail-
ability of that iron. For example, heme iron is 2- to 3-fold more
absorbable than is nonheme iron. 13 Furthermore, the pres-
ence of polyphenols, phytates, tannins, bicarbonate, calcium,
phosphates, fiber, and some food preservatives can reduce the
amount of absorbable iron.13,50,80
We determined fetal iron status by measuring brain iron
levels. Iron is vital for brain growth and function because it
< 0.01), high-ironhigh-bioavailability (, P < 0.01), or iron-replete (, P
< 0.001) diet.
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Figure 6. Effect of iron deficiency on fetal iron status and growth. (A)
Fetuses from dams fed the iron-deficient diet weighed significantly less
than did those from dams fed the high-ironlow-bioavailability (, P <
0.01), high-ironhigh-bioavailability (, P < 0.01), or iron-replete (*, P <
0.05) diet. (B) Fetuses from dams fed the iron-deficient diet had a shorter
cm12000119.indd 133
Mouse model of iron deficiency
supports myelination, neurotransmitter synthesis, and neu-
ronal and glial energy metabolism.7,15,19,46,58,84 In iron deficiency,
iron in RBC is preserved over brain iron, which is preserved at
the expense of heart and skeletal muscle iron.70 In the current
study, fetal brain iron was decreased in the group fed the iron-
deficient diet. Whether human infants have lower brain iron
levels when born to iron-deficient mothers is unknown, but
studies in rats and mice have shown decreased fetalneonatal
brain iron associated with maternal iron deficiency. 9,20,21,45,52
These studies followed the cognitive development and the
effects of iron supplementation on brain development. Iron
deficiency negatively affected the performance of mice and
rats in the Morris maze test, indicating effects on learning and
memory; in addition, disturbances in maze performance per-
sisted despite iron supplementation at weaning.21,43 Similarly,
iron-deficient neonatal rats had poorer learning performance
in the spatial water maze test than did their iron-sufficient con-
trols.20 Several studies found that iron deficiency affected grip
strength, a measure of motor function, in neonatal mice and
rats.20,43,45 One study using chronic marginal iron deficiency
during fetal development identified persistent changes in do-
pamine metabolism and myelin composition in mice. 44 In ad-
dition, postnatal consumption of iron-adequate diets did not
fully reverse all of the observed biochemical disturbances. 44
Numerous controlled studies in humans similarly showed that
postnatal iron supplementation did not improve cognitive per-
formance in babies.69 Therefore, our mouse model of iron defi-
ciency can be used to study whether various methods of iron
supplementation improve outcomes in neonates.
Provision of routine iron supplementation for pregnant
women is a topic of debate.2,51,59,62,68,83,85 The World Health Or-
ganization recommends maximal supplementation with 60
mg iron daily for 6 mo during pregnancy, whereas the United
States and Canada recommend 30 mg and 16 mg, respectively,
daily throughout pregnancy. 13,30 The United Kingdom does
not currently recommend supplementation, unless anemia is
present.2,30 Iron supplements were reported to increase mater-
nal Hgb, MCV, and serum iron and ferritin levels during late
pregnancy. However for women who enter pregnancy with
low iron stores, iron supplementation often fails to prevent
iron deficiency.1 Furthermore, iron treatment is associated with
an increased risk of infections, including placental malaria68
and gram-negative bacterial sepsis in neonates.5 Because iron
is an essential nutrient for pathogen growth, limiting iron
availability is a mechanism by which mammals prevent infec-
tion. Mouse models suggest that increased iron availability en-
hances infection for various organisms (for example, Salmonella
typhiumurium,79 Mycobacteria,63 Plasmodia,49 and Trypanosomes57).
Therefore, the current mouse model of iron deficiency during
pregnancy could be used to study the effects of iron supple-
mentation on the mother and fetus, in conjunction with vari-
ous clinically relevant infectious diseases.
In summary, we found that pregnant mice fed an iron-
deficient diet were less fertile, had fewer viable pups, and
had pups with shorter crownrump length, decreased body
(, P < 0.001) crownrump length than did those in all other groups. (C)
Fetuses from dams fed the iron-deficient diet had significantly (, P <
0.001) decreased brain iron levels as compared with those from dams
fed all other diets.
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Comparative Medicine
April 2013

weight, and decreased brain iron. Iron deficiency also affected tation for perinatal iron-deficiency anemia in rats. Behav Brain Res
the pregnant dams, by decreasing liver iron stores and causing 171:261270.
anemia. In contrast, a high-iron diet with high iron bioavail- 21. Felt BT, Lozoff B. 1996. Brain iron and behavior of rats are not
normalized by treatment of iron deficiency anemia during early
ability improved iron stores in pregnant mice. Therefore, our
development. J Nutr 126:693701.
mouse model of iron deficiency during pregnancy can be used 22. Fenton V, Cavill I, Fisher J. 1977. Iron stores in pregnancy. Br J
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Acknowledgments IngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRAS-
We thank the animal care staff of the Institute of Comparative SubstancesSCOGSDatabase/ucm261298.htm.
Medicine for their care of the mice. We appreciate the support and 24. Friedman JF, Kurtis JD, Kabyemela ER, Fried M, Duffy PE. 2009.
encouragement of Dr David Ruble. This work was supported by NIH The iron trap: iron, malaria, and anemia at the motherchild interface.
grants U01-HD064827, K08-HL103756, and a Louis V Gerstner Scholars Microbes Infect 11:460466.
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