Iron, copper and fetal development

Abstract

Pregnancy is a period of rapid growth and cell differentiation for both the mother and fetus. Consequently, it is a period when both are vulnerable to changes in dietary supply, especially of those nutrients that are marginal under normal circumstances. In developed countries this vulnerability applies mainly to micronutrients. Even now, Fe deficiency is a common disorder, especially in pregnancy. Similarly, Cu intake in the UK population is rarely above adequate levels, which is a matter of some concern, both in terms of public health and possible clinical consequences. In early studies it was shown that lambs born to mothers on Cu-deficient pastures develop 'swayback,' with neurological and muscular symptoms that cannot be reversed by postnatal supplementation. More recently, rat studies have shown that responses such as the 'startle' response are lost in offspring of Cu-deficient mothers. Data have shown that prenatal Fe deficiency results in increased postnatal blood pressure, even though the offspring have normal dietary Fe levels from birth. These observations emphasise the importance of Fe and Cu in growth and development. In the present review the importance of these metals and the consequences, both short term and long term, of deficiency will be discussed and some possible mechanisms whereby these effects may be generated will be considered.

Full text

Iron, copper and fetal development
Lorraine Gambling* and Harry J. McArdle
Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK
Pregnancy is a period of rapid growth and cell differentiation for both the mother and fetus.
Consequently, it is a period when both are vulnerable to changes in dietary supply, especially
of those nutrients that are marginal under normal circumstances. In developed countries this
vulnerability applies mainly to micronutrients. Even now, Fe deficiency is a common disorder,
especially in pregnancy. Similarly, Cu intake in the UK population is rarely above adequate
levels, which is a matter of some concern, both in terms of public health and possible clinical
consequences. In early studies it was shown that lambs born to mothers on Cu-deficient
pastures develop ‘swayback,’ with neurological and muscular symptoms that cannot be re-
versed by postnatal supplementation. More recently, rat studies have shown that responses such
as the ‘startle’ response are lost in offspring of Cu-deficient mothers. Data have shown that
prenatal Fe deficiency results in increased postnatal blood pressure, even though the offspring
have normal dietary Fe levels from birth. These observations emphasise the importance of Fe
and Cu in growth and development. In the present review the importance of these metals and
the consequences, both short term and long term, of deficiency will be discussed and some
possible mechanisms whereby these effects may be generated will be considered.
Iron: Copper: Pregnancy
The impact of inappropriate maternal nutrition on preg-
nancy outcome has been known for decades. The now
classic study by Ebbs et al. (1941) showed an association
between the consumption of a diet low in protein, Ca,
fruit and vegetables, i.e. a ‘poor’ diet, and an increased risk
of pregnancy complications when compared with women
who eat a ‘good’ diet. Now, >70 years later, it is clear that
nutrition in utero affects not only fetal and neonatal health
but also adult well-being. Many studies have demonstrated
a link between fetal nutrition, low birth weight and CHD,
hypertension and impaired glucose tolerance in adults
(Barker et al. 1993a; Ravelli et al. 1998; Law et al. 2000).
It has been proposed that adaptations made by the fetus to
cope with inappropriate nutrition may lead to morphologi-
cal and physiological changes that persist into postnatal
life. These changes, although ensuring fetal survival, may
have detrimental affects in later life (Barker, 1995).
It is unlikely that the differences between good and poor
diets with regard to fetal development can be attributed to
a single nutrient. Deficiencies in several essential micro-
nutrients, such as folate, vitamins K and A, I, Mn, Zn, Cu
and Fe (Hurley, 1981; Castillo-Duran & Cassoria, 1999;
McArdle & Ashworth, 1999; Black, 2001), have been
implicated in problems with development and with birth
defects. Clearly, discussing the impact of all these micro-
nutrients on fetal development is beyond the scope of one
review, hence only the role of Cu and Fe will be examined.
The consequences, both short term and long term, of
deficiency in utero will be discussed and some possible
mechanisms whereby these effects may be generated will
be considered.
Copper in fetal development
Enzootic ataxia
In the early 1930s it was reported that Cu, as well as Fe,
needed to be added to a milk diet in order for pregnancy
to be successfully established in female rats (Keil &
Nelson, 1931). Evidence for the importance of Cu in fetal
development arose from studies of sheep that grazed on
Cu-deficient pasture. Lambs were born with a disease
termed enzootic ataxia, more commonly known as sway-
back (Bennetts & Chaoman, 1937). This disease is charac-
terised by spastic paralysis, especially of the hind limbs,
severe incoordination, convulsions and blindness. Other
Abbreviation: BP, systolic blood pressure.
*Corresponding author: Dr Lorraine Gambling, fax +44 1224 716622, email L.Gambling@rowett.ac.uk
Proceedings of the Nutrition Society (2004), 63, 553–562 DOI:10.1079/PNS2004385
g
The Authors 2004

abnormalities include aneurysms of the aortic arch, anaemia,
osteoporosis and other skeletal lesions, and abnormalities
of the skin and hair. These studies have been expanded to
identify neonatal ataxia in other species (Bennetts et al.
1941; Joyce, 1955; Wilkie, 1959; O’Sullivan, 1977). Sup-
plementing the animals postnatally does not reverse the
neurological and muscular symptoms (Bennetts & Chaoman,
1937), but it has been demonstrated that Cu supplementa-
tion of the mothers during gestation can prevent this
disease (Allcroft et al. 1959). Importantly, whilst the cause
of enzootic ataxia is clearly embryonic–fetal Cu defi-
ciency, the pregnant animals often appear quite healthy
and show no obvious signs of Cu deficiency (Bennetts et al.
1948).
Copper deficiency in man
In man nutritionally-induced clinical Cu deficiency is rare;
however, moderate or mild Cu deficiency may occur more
widely than is currently appreciated. It is estimated that
the average intake of Cu by women of childbearing age is
lower than the current estimated safe and adequate daily
intake for adults, which is 0
.
9–1
.
2 mg Cu/d (Department
of Health, 1991; Food and Nutrition Board and Institute of
Medicine, 2001). Cu deficiency can also occur as a second-
ary deficiency even if the dietary content is adequate; for
example, by interaction with drugs or other nutrients. Cu
metabolism can also be altered in disease states such as
diabetes, and it has been suggested that these alterations
may contribute to diabetes-associated teratogenicity (Uriu-
Hare et al. 1985; Jankowski et al. 1993).
The most devastating impact of Cu status on human
fetal development is seen in the X-linked disorder Menkes
disease (Danks et al. 1972). Menkes disease originates
in utero and manifests full symptoms during the perinatal
period. These symptoms include hypothermia, neuronal
degeneration, abnormalities of the hair, skin and connective
tissue, bone fractures and widespread vascular abnormal-
ities. Menkes disease is caused by a mutation in the gene
encoding for the Cu ATPase, ATP7A; mutations in this
gene lead to defective cellular export of Cu (Vulpe et al.
1993). Although this disease has been recognised to be a
disorder of Cu metabolism for >20 years, the prognosis for
infants with this disorder is still poor, with death typically
occurring by 3 years of age (Menkes, 1999).
Effect of maternal copper deficiency on
fetal development
The extent to which Cu deficiency affects pregnancy
outcome is very much dependent on both the severity and
timing of the deficiency. If deficiency occurs before mating
it may lead to reproductive failure and early embryonic
death (Dutt & Mills, 1960; Hall & Howell, 1969). Mater-
nal Cu deficiency during pregnancy has been shown to be
teratogenic in many species, including man, cattle, sheep
and rats (Hurley, 1981). Fetuses are found to suffer from
gross structural abnormalities, including skeletal, pulmo-
nary and cardiovascular defects (Fields et al. 1990;
Jankowski et al. 1993; Sarricolea et al. 1993; Keen et al.
1998; Table 1); symptoms that clearly mirror those
described for Menkes disease.
Short-term consequences of maternal copper deficiency
The effect of maternal Cu deficiency on embryo develop-
ment has been studied through the use of post-implantation
embryo culture systems. In these systems embryos are
removed at approximately 8–10 d gestation from rats or
mice fed a control or Cu-deficient diet. They are then
cultured for £ 48 h in serum obtained from control or Cu-
deficient animals. Embryos from Cu-deficient mothers
cultured in Cu-deficient serum have reduced crown–rump
lengths, head:crown–rump length and protein content
(Mieden et al. 1986). These embryos also have a marked
increase in brain and cardiac abnormalities (Hawk et al.
1998).
In contrast, moderate Cu deficiency, resulting in a 30
and 68 % drop in maternal and neonatal Cu levels re-
spectively, has little effect on either the number of live
births or neonatal weight (Masters et al. 1983; Ebesh et al.
1999). However, when born the offspring of the Cu-
deficient dams do suffer from gross structural abnormal-
ities. Cu-deficient neonates are typically characterised by
severe connective tissue abnormalities. Cardiac haem-
orrhages are a frequent finding in Cu-deficient sheep, rats,
guinea-pigs and mice (Tinker & Rucker, 1985; Rucker
et al. 1998). Carotid and cerebral arteries of Cu-deficient
neonates tend to have sparse poorly-developed elastin that
lacks the concise fibril arrangements seen in control
animals. Skeletal defects also often occur as a result of
prenatal Cu deficiency. Lambs with enzootic ataxia may
have poorly-developed light brittle bones and frequent
fractures. Lung abnormalities are also a frequent conse-
quence of prenatal Cu deficiency (Abdel-Mageed et al.
1994). After birth 35% of the newborn Cu-deficient rats
show respiratory distress syndrome (Sarricolea et al.
1993). Brain neurochemistry is also affected; offspring of
Cu-deficient dams show reduced noradrenaline levels
in specific brain regions and there is also an increase in
regional dopamine levels. (Prohaska & Bailey, 1994).
The timing of the onset of Cu deficiency is critical to the
survival of the offspring. In their established murine
model Prohaska & Brokate (2002) have shown that if Cu
deficiency is induced 8 d before birth there is no effect
on pregnancy outcome or birth weight, but none of the
offspring survives beyond postnatal day 13. When the defi-
ciency is induced 2 d before birth all offspring survive
through to weaning.
Table 1. Consequences of maternal copper deficiency during
pregnancy for the offspring
Short term Long term
Connective tissue
abnormalities
Cardiac ultrastructure
abnormalities
Pulmonary insufficiency Immune suppression
Neuronal degeneration Impaired cognitive and
Skeletal defects behavioural function
Lower survival rate
554 L. Gambling and H. J. McArdle

Long-term consequences of maternal copper deficiency
The effect of maternal Cu deficiency on postnatal gene
expression and function has been assessed in both rat and
murine models. Effects have been noted in both brain and
liver gene expression. In the mouse model higher brain
Cu–Zn superoxide dismutase activity and higher levels
of brain thiobarbituric acid-reactive substances (an index
of lipid peroxidation) are seen in response to oxidative
stress (Arce & Keen, 1992). In addition, the restoration of
liver Cu levels as a result of being weaned onto control
diets does not lead to a recovery in metallothionein ex-
pression, indicating that prenatal Cu deficiency could have
permanently altered the expression of proteins involved in
Cu metabolism (Arce & Keen, 1992).
Marginal-Cu-deficient diets beginning during the peri-
natal period and continued following weaning have been
shown to lead to long-term abnormalities in cardiac ultra-
structure and a suppression in the response of immune
cells to in vitro stimuli at 6 months of age (Hopkins &
Failla, 1995; Wildman et al. 1995; Table 2).
The clearest evidence for long-term functional conse-
quences of maternal Cu deficiency in utero and during
lactation comes from studies of the Sprague Dawley rat.
The rats were fed a Cu-deficient diet from mid pregnancy
and through lactation, the offspring were weaned onto a
control diet and the long-term effects on brain function
were investigated. Whilst the offspring show normal
responses to tactile startle, the auditory startle response of
those offspring born to Cu-deficient dams is markedly
decreased (Prohaska & Hoffman, 1996). These neurobehav-
ioural abnormalities occur in both genders, and there is no
difference in the Cu status of the control and experimental
groups at the time of testing.
Possible mechanisms of action
One major area for discussion is the means by which these
deficiencies exert their effects. Are the effects seen in
micronutrient deficiencies the result of a direct effect on
the embryo–fetus or a result of indirect effects via altera-
tions in maternal metabolism? In the case of Cu deficiency
in utero there is substantial evidence to indicate that the
developmental abnormalities, both pre- and postnatal, are
a result of direct effects. Mieden et al. (1986), using
the embryo culture system, have reported that embryos
cultured in Cu-deficient serum show developmental
abnormalities. However, when Cu is added to the same
serum all embryos subsequently cultured develop nor-
mally. In addition, the clinical features of both enzootic
ataxia and Menkes disease can be explained by deficien-
cies in the activities of Cu-containing enzymes.
Cytochrome c oxidase
Cytochrome c oxidase is an inner-mitochondrial-mem-
brane protein complex that catalyses the reduction of O
2
to water and utilises the free energy of this reaction to
generate a transmembrane proton gradient during respira-
tion. A decrease in the activity of this enzyme would affect
the offspring’s ability to carry out oxidative phospho-
rylation, which may in turn lead to lower levels of ATP.
When brain tissue from lambs suffering from enzootic
ataxia is analysed for cytochrome c oxidase activity, there
is a marked reduction in activity compared with that of
normal lambs (Howell & Davison, 1959). Subsequent
studies have supported this finding, indicating that enzootic
ataxia is caused by low brain Cu concentrations, leading
to a deficiency in cytochrome c oxidase activity in the
motor neurons and aplasia of the myelin surrounding
these neurons (Mills & Williams, 1962; Fell et al. 1965).
The effect of maternal Cu deficiency on cytochrome c
activity has also been confirmed in the murine model
(Prohaska & Bailey, 1993).
Dopamine-b-monooxygenase
Murine models have also established that the effect of
maternal Cu deficiency on the activity of Cu-containing
enzymes in the brain may not be restricted to cytochrome c
oxidase. When compared with normal offspring of the
same age the offspring of Cu-deficient mothers have
altered brain catecholamines pools. A reduction in nor-
adrenaline levels in the brain occurs in parallel with an
increase in dopamine levels, indicating that the activity
of dopamine-b-monooxygenase, which is responsible for
conversion of dopamine to noradrenaline in noradrenergic
neurons, is reduced in the offspring of Cu-deficient
mothers (Prohaska & Bailey, 1993).
Lysyl oxidase
In addition to the effect on neuronal development,
abnormalities in both connective tissue and lung develop-
ment (O’Dell et al. 1978; Tinker et al. 1985; Harris, 1986;
Abdel-Mageed et al. 1994; Rucker et al. 1998) can be
linked to impairment of the activity of one particular
enzyme, lysyl oxidase. Lysyl oxidase is an extracellular
Cu-containing enzyme that is responsible for the formation
of lysine-derived cross-links in connective tissue, particu-
larly in collagen and elastin. It is this cross-linking that is
required for connective tissue stability. Studies on rat heart
(Farquharson & Robins, 1991) and chick lung (Harris,
1986) show that Cu deficiency adversely influences the
production of mature elastin and collagen. Carotid and
cerebral arteries of Cu-deficient offspring have sparse
poorly-developed elastin, and a reduction in the activity of
lysyl oxidase has been implicated (Tinker & Rucker, 1985;
Rucker et al. 1998). The impairment in lysyl oxidase
activity is also thought to be the cause of the increased
bone fragility observed in Cu-deficient neonates (Tinker &
Rucker, 1985). The impact of maternal Cu deficiency on
the activity of lysyl oxidase has been directly examined
in the lungs of offspring born to Cu-deficient rabbits
Table 2. Consequences of maternal iron deficiency during
pregnancy for the offspring
Short term Long term
Reduced fetal weight Increased blood pressure
Asymmetric organ growth Increased glucose tolerance
Fe deficiency Impaired cognitive and behavioural
Lower survival rate function
Micronutrient interactions and public health 555

(Abdel-Mageed et al. 1994). Offspring born to the Cu-
deficient mothers have a markedly reduced 24 h survival
rate. Analysis of the activity of lysyl oxidase in the lungs
of these Cu-deficient offspring indicates that it is half that
of control animals (Abdel-Mageed et al. 1994).
Superoxide dismutase
Superoxide dismutase activity in embryos cultured in Cu-
deficient serum is markedly reduced when compared with
that of their control counterparts (Hawk et al. 1998).
Embryo abnormalities are reduced on the addition of re-
active oxygen scavengers to the culture medium, clearly
supporting a functional role for the reduction in superoxide
dismutase activity (Hawk et al. 1998). These results
suggest that free radical-induced damage is involved in
the induction of embryo abnormalities. Further to this
finding, the embryo culture system has been used to estab-
lish that not only are superoxide anion concentrations
higher in the Cu-deficient embryos, but that these anions
are localised in areas of the embryos that are characterised
by abnormalities, e.g. forebrain and heart (Hawk et al.
2003).
These data clearly support the hypothesis that maternal
Cu deficiency impacts on embryo and fetal development
through several direct mechanisms. Brain development
and function are directly affected by the reduction in the
activity of a number of key enzymes, and other teratogenic
effects occur via a compromised oxidant defence system
and extracellular matrix integrity.
Iron in fetal development
Iron deficiency during pregnancy
The World Health Organization (2003) considers Fe
deficiency to be the primary nutritional disorder in the
world, second only to tuberculosis as the world’s most
common and costly health problem. It is prevalent in
most of the developing world. In industrialised countries
the occurrence of Fe deficiency is highest among young
children and women of childbearing age, particularly preg-
nant women (see Fairweather-Tait, 2004).
In most species maternal blood volume increases and
the packed cell volume and Hb concentration fall during
pregnancy; this condition is known as the anaemia of
pregnancy. However, in a high percentage of women the
fall in Hb levels is greater than that regarded as both
physiological and safe (World Health Organization, 1992).
A high proportion of these types of anaemia arise as a
result of Fe deficiency (World Health Organization, 2003).
Several recently-completed studies indicate that currently
in Europe the level of maternal Fe deficiency during
pregnancy is a major cause for concern (Hercberg et al.
2001). These investigations have studied pregnant women
in Germany, Belgium and Scotland, and they indicate that
the incidence of Fe deficiency during pregnancy is in the
region of 20–40 % (Bergmann et al. 2002; Fosset et al.
2003; Massot & Vanderpas, 2003).
Inadequate intake of Fe related to diets poor in bio-
available Fe is thought to be responsible for the majority of
Fe deficiency both before and during pregnancy (Bothwell,
2000). In one recent survey carried out in France 93% of
women of childbearing age were reported to have dietary
Fe intakes lower than the recommended daily allowance
(Galan et al. 1998). In the UK > 40 % of women between
the ages of 19–34 years had total Fe intakes below the
lower reference nutrient intake level (Henderson et al.
2003). An increase of 50 % is recommended in the daily
dietary intake of Fe during pregnancy (Galan et al. 1998).
An additional risk factor for Fe deficiency during
pregnancy is multiparity, which can lead to subsequent
successive deficits without sufficient repletion of reserves
(Hindmarsh et al. 2000).
The consequences of maternal Fe deficiency are serious,
both for the mother and her developing fetus. Many studies
have shown that mothers suffering from Fe-deficiency
anaemia are at increased risk of mortality and morbidity
(for review, see Rush, 2000). Babies have an increased risk
of being born premature and/or smaller (Allen, 2000).
Several studies have shown that Fe deficiency during
pregnancy, both in man and in animal models, results in
both short- and long-term problems for the offspring.
Short-term consequences of maternal iron deficiency
In order to investigate the effects of maternal Fe deficiency
on fetal growth and development, as well as the long-term
health and well-being of the offspring, several rodent
models have been established (Crowe et al. 1995; Kwik-
Uribe et al. 1999; Lewis et al. 2001b; Gambling et al.
2002). As would be expected the fetuses from the Fe-
deficient dams are Fe deficient, with lower packed cell
volume and lower liver Fe levels (Gambling et al. 2002).
However, the level of Fe deficiency seen in the fetus is
markedly less than that seen in the mother. Maternal Fe
status also has a direct impact on the Fe stores for early
neonatal life; consistent with fetal findings, neonates are
also Fe deficient (Gambling et al. 2003). Early studies in
man have established a direct relationship between the
concentration, as well as the total content, of storage Fe in
the fetal liver and maternal plasma Fe levels, suggesting
that babies born to Fe-deficient mothers would have poor
Fe stores (Singla et al. 1985). More recent studies have
now established that Fe deficiency in the mother is a
risk factor for Fe deficiency in the young infant (Preziosi
et al. 1997; Singla et al. 1997; Allen, 2000; Halvorsen,
2000; Harthoorn-Lasthuizen et al. 2001), with consequent
more pronounced anaemia at approximately 10–12 weeks
of age.
While maternal Fe deficiency has no effect on the
fertility and growth of the dams or the viability and
number of fetuses, it does markedly reduce fetal weight
(Gambling et al. 2002). In addition to changes in total fetal
weight, fetal liver weights (expressed as a proportion of
fetal size) are decreased, indicating disproportionate fetal
growth. The reduced fetal weight seen in the Rowett
Hooded Lister model (Gambling et al. 2002) is consistent
with that seen in other rat models (Tojyo, 1983; Crowe
et al. 1995). As with maternal Cu deficiency, the severity
of the Fe deficiency induced substantially affects the
outcomes. While milder maternal Fe deficiency has no
556 L. Gambling and H. J. McArdle

marked effect on fetal number, fetal weight or placental
weight (Sherman & Moran, 1984), severe Fe deficiency
during pregnancy can lead to a fall in maternal weight,
fertility and fetal viability (Tojyo, 1983).
Increased placental weight:birth weight is one of the
indicators for the development of adult diseases, such as
cardiovascular problems and diabetes (Barker et al. 1993b;
Phipps et al. 1993). In the early 1990s maternal anaemia
and Fe deficiency were linked to an increase in placental
weight:birth weight (Godfrey et al. 1991). An increase
in placental weight and placental weight :birth weight in
anaemic and Fe-deficient pregnancies has since been
confirmed in several studies (Williams et al. 1997; Lao &
Wong, 1997; Hindmarsh et al. 2000). An increase in
placental weight:fetal weight is also seen in rodents (Lewis
et al. 2001b; Gambling et al. 2002).
Using the Rowett rat model these studies have been
expanded to investigate the early postnatal period. The
survival rate of the offspring born to Fe-deficient mothers
is reduced, with 25% of the pups born to Fe-deficient
mothers dying within the first 24 h after birth (Gambling
et al. 2003). The pups that survive are smaller and have
larger hearts and smaller kidneys than their control
counterparts. Lower birth weight is a consistent finding in
several rodent models (Crowe et al. 1995; Lewis et al.
2001b) and, of course, man (Allen, 2000). The effects on
the offspring of maternal Fe deficiency during pregnancy
are summarised in Table 2.
Long-term consequences of maternal iron deficiency
Blood pressure
Long-term effects of maternal Fe deficiency on the health
and well-being of the offspring have been predicted by
animal models since the mid 1990s. Crowe et al. (1995)
have examined the effects of maternal Fe deficiency on the
systolic blood pressure (BP) of the offspring. Before
weaning the BP of the offspring of Fe-deficient mothers
is lower than that of controls. This reduction is followed by
a pronounced post-weaning rise in BP when compared
with controls. This raised BP has since been shown to
occur in both male and female offspring of Fe-deficient
mothers (Lewis et al. 2001c). The study into the effect of
maternal Fe deficiency on BP in the offspring has been
further expanded using the Rowett model (Gambling et al.
2003). At birth both the control and the Fe-deficient litters
were cross-fostered to control dams and the BP of the
offspring measured at 6, 10 and 16 weeks. Results show
that the BP of the male offspring born to Fe-deficient dams
is raised at all time points, while the BP of the corres-
ponding female offspring is lower at 6 weeks, but at 10
and 16 weeks it is higher than that of the controls. There
are no differences in growth rate, body or organ weight
between the two groups of offspring at any of the time
points. Measurement of the total liver Fe levels of the rats
at each of the time points shows that at no time is there
a difference between the levels for the offspring born to
control and Fe-deficient mothers. Thus, the alterations in
BP occur despite the offspring being of normal Fe status
(Gambling et al. 2003).
Glucose tolerance
As well as increased BP the Wistar rat model of maternal
Fe deficiency studied by the Hales group in Cambridge
(Lewis et al. 2001c) has also shown an effect on glucose
tolerance in offspring of Fe-deficient mothers. At 3 months
of age offspring of Fe-deficient mothers display an im-
provement in glucose tolerance. However, unlike the effect
on BP the effect of maternal Fe deficiency on glucose
tolerance in the offspring does not appear consistent across
time and models. When the experimental time point is
extended in the Wistar rat model to 14 months an increase
in BP in the offspring of Fe-deficient mothers is still
present; however, there is no longer a difference in glucose
tolerance between the offspring (Lewis et al. 2002).
Glucose tolerance and insulin levels have been investi-
gated in the Rowett model (Gambling et al. 2003).
However, in these rats there are no apparent differences
at 10 weeks of age between the offspring born to control or
Fe-deficient mothers.
Neuronal development
In man the clearest evidence for a long-term effect of
maternal Fe deficiency on postnatal development relates to
brain development and function (Deregnier et al. 2000;
Nelson et al. 2000; Tamura et al. 2002). It is believed that
the effects of Fe deficiency in early development lead
to later problems in cognitive and behavioural functions
(Lozoff, 2000). Poor fetal Fe status is associated with
diminished language ability, fine motor skills and tracta-
bility, as assessed at 5 years of age (Tamura et al. 2002).
These findings in the human population are supported by
extensive studies in animal models. Offspring born to rats
fed an Fe-deficient diet either in early or late gestation
show reduced activity and homing ability (Felt & Lozoff,
1996). Investigations carried out in a murine model
provide evidence that maternal Fe deficiency leads to
persistent alterations in behaviour and cognitive function
that cannot be reversed by postnatal Fe supplementation
(Kwik-Uribe et al. 2000).
Possible mechanisms of action
Direct effects
The investigations into the mechanisms by which maternal
Fe deficiency exerts its effect on fetal growth and devel-
opment are only in the early stages compared with those
for Cu. Again possible mechanisms can be subdivided into
direct and indirect effects. As with Cu, Fe deficiency may
exert effects directly by reducing the activity of the
enzymes that use Fe as a cofactor. Rodent models have
indicated that enzymes involved in neurotransmitter syn-
thesis and neuronal energy may be perturbed in maternal
Fe deficiency. For example, Taneja et al. (1990) have
demonstrated that maternal Fe deficiency leads to a
reduction in g-aminobutyric acid metabolism that is not
reversible by postnatal Fe supplementation. In addition,
areas of the brain that are involved in higher cognitive
functions have lower cytochrome c oxidase activity in
Micronutrient interactions and public health 557

neonatal rats born to mothers who were Fe deficient during
pregnancy (Deungria et al. 2000).
Indirect effects
Most of the abnormalities and developmental problems
associated with maternal Cu deficiency can be directly
related to the perturbation of the activity of a particular
enzyme. With the exception of brain development and
function there does not appear to be a similar relationship
with maternal Fe deficiency. In fact, data from recent
studies (Gambling et al. 2002; L Gambling, A Czopek, HS
Andersen, R Wojak, Z Krejpcio and HJ McArdle, unpub-
lished results) indicate that birth weight is dependent on
the mother’s Fe status and not that of the neonate. Thus,
maternal Fe deficiency may also affect fetal development
by more indirect mechanisms. Fe-deficiency-induced
changes in maternal metabolism may have downstream
effects on placental structure, endocrine and transport
functions, nutrient interactions and fetal organ develop-
ment.
Placental function. The placenta is the pathway for
delivery of the majority of nutrients to the developing
fetus. Consequently, any stress that alters placental devel-
opment or function is likely to have consequences for the
developing fetus. Placental function is regulated, at least
in part, by a wide spectrum of cytokines produced both
locally and distally. TNFa has been suggested to play an
important role in pregnancy (Hunt et al. 1996). Elevated
levels of TNFa are associated with early to mid-pregnancy
failure and premature labour in man (Silen et al. 1989;
Chaouat et al. 1990; Tangri & Raghupathy, 1993). How-
ever, TNFa is also produced at low levels in placental and
decidual immune cells in normal healthy pregnancies, and
is therefore thought to be beneficial for pregnancy. It may
also be important in trophoblast turnover and re-modelling
(Yui et al. 1996). Leptin has also been suggested to be
important in the maintenance of pregnancy (for review, see
Ashworth et al. 2000) and is thought to be a growth factor
for the fetus. Studies on TNFa and leptin expression in
placentas from control and Fe-deficient litters have been
carried out (Gambling et al. 2002). Maternal Fe deficiency
increases levels of TNFa in the trophoblast giant cells of
the placenta. Levels of the type 1 TNFa receptor are
increased in giant cells, cytotrophoblasts, the labyrinth and
fetal vessels. Leptin is also increased in the labyrinth and
marginally in trophoblast giant cells. There is no change in
leptin receptor levels in any region of the placentas from
Fe-deficient litters. Growth and development are clearly
dependent not on a single cytokine but on the presence of
an appropriate profile of factors, so that here the increased
leptin may be acting to counter some of the worst effects
of the increased TNFa levels.
In human pregnancy the placental structure is also
altered in maternal anaemia (Mayet, 1985). Maternal
anaemia has been shown to be associated with increased
placental weight and fetal weight:placental weight
(Beischer et al. 1970; Godfrey et al. 1991). This increase
in placental weight has been interpreted as compensatory
placental hypertrophy. The effect of maternal Fe deficiency
on placental structure has been investigated in both rodent
and human pregnancies. The Wistar rat model has been
used to investigate the effect of maternal Fe deficiency on
placental structure. This study has shown that there is
decreased capillary length and surface area in the placentals
from Fe-deficient litters (Lewis et al. 2001a). Low ferritin
concentrations in early human pregnancy are associated
with increased placental vascularisation at term. The
surface area of capillaries involved in gas exchange is
strongly and inversely related to serum ferritin concen-
trations (Hindmarsh et al. 2000). The relationship between
maternal Fe deficiency and placental size and birth weight
exists across the normal range for these measures and
is not just restricted to severely-anaemic mothers. Altera-
tion in placental structure would clearly have an impact
on its ability to transport nutrients to the fetus. Maternal
Fe deficiency has been shown to cause fetal plasma amino
acid and cholesterol and triacylglycerol levels to be
decreased, clearly suggesting decreased placental trans-
port of amino acid and NEFA to the fetus (Lewis et al.
2001b).
Nutrient interactions. The initial observation of a link
between Fe and Cu metabolism came with a study that
showed that while Fe supplementation fails to resolve
anaemia in rats, administration of Cu in the form of either
ashed food or acid extracts of the ashes restores Hb levels
(Waddell et al. 1927; Hart et al. 1928). Several subsequent
studies have now confirmed that Fe deficiency has second-
ary effects on Cu metabolism. Generally, Fe deficiency
results in increased Cu levels in the liver and rises in serum
caeruloplasmin concentrations (Sourkes et al. 1968; Evans
& Abraham, 1973; Owen, 1973; Sherman et al. 1977). Few
studies have investigated the effect of Fe deficiency on Cu
metabolism during pregnancy and the interaction of these
metals in the pregnant animal or her offspring (Sherman &
Tissue, 1981; Sherman & Moran, 1984). Thus, in a recent
study the Rowett model has been used to examine the
effect of Fe deficiency on Cu levels in maternal and fetal
tissue (Gambling et al. 2004). Results have highlighted
the fact that maternal Fe deficiency has a differential
effect on Cu metabolism in the mother and fetus. In the
maternal liver Cu levels are inversely correlated with
those of Fe, while in the fetus both Fe and Cu levels are
reduced. A similar differential effect between mother and
fetus is also seen in vitamin A metabolism. Maternal liver
retinol levels are reduced in maternal Fe deficiency, while
in the fetus the opposite is seen, as the level of Fe de-
creases the levels of retinol in the fetal liver increase
(Gambling et al. 2001). Although the reduction in Cu and
vitamin A levels seen in the Fe-deficient fetuses is not as
great as that seen in their respective deficiencies, this
further restriction in nutrient supply may have an impact
on fetal development.
Fetal organ development. It has been proposed that
one possible mechanism for the increased BP in the off-
spring born to Fe-deficient mothers is that maternal Fe
deficiency may interfere with normal kidney development.
Kidney nephron number is an important determinant of
BP. Low nephron number reduces the surface area avail-
able for filtration (Brenner et al. 1988), and therefore limits
the ability of the kidney to excrete Na and maintain normal
extracellular fluid volume and BP (Brenner et al. 1988).
558 L. Gambling and H. J. McArdle

Nephron number is established during kidney develop-
ment, beyond which point the number cannot be increased
(Wintour, 1997). Nephron number is related to birth weight
over the normal range (Merlet-Bencichou et al. 1994), and
intrauterine growth retardation has been associated with
low nephron number (Merlet-Bencichou et al. 1994;
Bassan et al. 2000; Bauer et al. 2002). Fe deficiency
during gestation appears to affect the cellular development
of the kidney, in which DNA concentration has been found
to be lower in 2-d-old neonates (Kochanowski & Sherman,
1985). Expanding on their earlier work with the Wistar Fe-
deficiency model, Hales and colleagues (Lisle et al. 2003)
have recently studied the effect of maternal Fe deficiency
on the renal morphology of the adult offspring. Their
results show a reduction in the number of glomeruli in the
kidney of offspring born to Fe-deficient mothers. Offspring
from both control and Fe-deficient mothers also show an
inverse relationship between glomerular number and BP. It
is suggested that the reduction in nephron number induced
by maternal Fe deficiency may be partly responsible for
the increase in BP seen in these animals.
Conclusions
In conclusion, it is clear that maternal Cu and/or Fe
deficiency during pregnancy has serious consequences
for the offspring. These consequences range from direct
effects of a decreased enzyme activity to indirect results of
changed activities of signalling pathways. Although most
of the data has been obtained in animal models, the ex-
treme examples of Menkes disease and the milder effects
of maternal Fe deficiency on infant cognitive ability pro-
vide strong supportive evidence for similar vulnerability
in man. The animal models discussed in the present re-
view have considerable heuristic value, and will provide
the data underpinning the design of therapeutic strategies
in the treatment of human maternal micronutrient defi-
ciencies.
Acknowledgements
The authors’ work is supported by the Scottish Executive
Environment and Rural Affairs Department, the European
Union Framework V (QLK1–199–00337) and the Inter-
national Copper Association.
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