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Biology of Reproduction 66, 516-523 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Effect of Iron Deficiency on Placental Cytokine Expression and Fetal Growth in the Pregnant Rat1

Lorraine Gamblinga, Zehane Charaniaa, Lisa Hannaha, Christos Antipatisa, Richard G. Leaa, and Harry J. McArdle2,a

a The Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Iron deficiency anemia is the most common nutritional disorder in the world. Anemia is especially serious during pregnancy, with deleterious consequences for both the mother and her developing fetus. We have developed a model to investigate the mechanisms whereby fetal growth and development are affected by maternal anemia. Weanling rats were fed a control or iron-deficient diet before and throughout pregnancy and were killed at Day 21. Dams on the deficient diet had lower hematocrits, serum iron concentrations, and liver iron levels. Similar results were recorded in the fetus, except that the degree of deficiency was markedly less, indicating compensation by the placenta. No effect was observed on maternal weight or the number and viability of fetuses. The fetuses from iron-deficient dams, however, were smaller than controls, with higher placental:fetal ratios and relatively smaller livers. Iron deficiency increased levels of tumor necrosis factor {alpha} (TNF{alpha}) only in the trophoblast giant cells of the placenta. In contrast, levels of type 1 TNF{alpha} receptor increased significantly in giant cells, labyrinth, cytotrophoblast, and fetal vessels. Leptin levels increased significantly in labyrinth and marginally (P = 0.054) in trophoblast giant cells. No change was observed in leptin receptor levels in any region of the placentas from iron-deficient dams. The data show that iron deficiency not only has direct effects on iron levels and metabolism but also on other regulators of growth and development, such as placental cytokines, and that these changes may, in part at least, explain the deleterious consequences of maternal iron deficiency during pregnancy.

cytokines, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most species, maternal blood volume increases and hematocrit and hemoglobin concentration fall during pregnancy; this is known as the anemia of pregnancy. However, in a high percentage of women, the fall in hemoglobin levels is greater than that which is regarded as both physiological and safe [1]. A significant proportion of these anemias arise as a result of iron (Fe) deficiency [2]. The consequences are serious, both for the mother and her developing fetus, and many studies have shown that anemia during pregnancy results in an increased risk of mortality and morbidity (reviewed in [3, 4]).

One of the consequences of Fe deficiency during pregnancy is smaller size at birth, and Godfrey et al. [5] have shown that maternal Fe status may be a risk factor for adult disease. Several studies have shown that Fe deficiency during pregnancy, both in humans and in animal models, results in long-term problems for the offspring, such as increases in blood pressure [6], diminished brain function [7–12], and compromised immune system development [13–16]. The mechanisms underlying these effects have not been clarified, but clearly, growth and development in utero may play an important part.

The placenta is the pathway for delivering the majority of nutrients to the developing fetus. Consequently, any stress that alters placental development 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, which are produced both locally and distally. One cytokine that has attracted a lot of interest is tumor necrosis factor {alpha} (TNF{alpha}). Indeed, a pivotal role for this cytokine during pregnancy has been suggested [17]. Elevated levels of TNF{alpha} at the maternal-fetal interface are associated with early and midpregnancy failure in rodents and with premature labor in humans [18–20]. However, TNF{alpha} is also produced at low levels in placental and decidual immune cells during normal, healthy pregnancies and, therefore, is thought to be beneficial for pregnancy. It is reported to induce apoptosis of placental cells and, therefore, may be important in trophoblast turnover and remodeling [21]. Data also suggest that TNF{alpha} may regulate placental steroid production by the placenta and down-regulate amino acid transfer [22]. Because the suggested beneficial and detrimental roles of TNF{alpha} are concentration dependent, the regulation of TNF{alpha} expression at the maternal-fetal interface must be crucial for successful placental development and function.

The relationship between Fe status and cytokines has been the subject of many studies, most of which have concentrated on the effect of cytokines on Fe uptake or metabolism (see, for example, [23] for a recent report). Recently, however, several groups have examined the effect of Fe status on TNF{alpha} production. Scaccabarozzi et al. [24] have shown that Fe supplementation increased and that desferrioxamine (DFO), an Fe chelator, decreased the production of TNF{alpha} by monocytic cells. This same effect has been obtained in the leukemic cell line THP-1 [25] and in mice treated with DFO [26]. Similar data were also obtained in Kupffer cells, in which loading with Fe reduced the sensitivity to lipopolysaccharide [27]. To our knowledge, how this relationship operates in the placenta has not been examined, but given the association between elevated concentrations of TNF{alpha} and problems with pregnancy, this question is clearly of considerable importance. In this paper, therefore, we examine the effect of maternal Fe deficiency on TNF{alpha} and TNF{alpha} receptor levels in the placenta.

Since its identification in placenta [28], leptin has been the subject of considerable investigation. Results of several studies suggest it is important in the maintenance of pregnancy [29–31] (reviewed in [32]). For example, diabetes in pregnancy is clearly associated with increased leptin levels, whereas intrauterine growth retardation is related to decreased leptin. In contrast, leptin in pre-eclamptic placentas is actually increased [33]. Generally, leptin is thought to be a growth factor for the fetus, and several pieces of evidence support this hypothesis. For example, several groups have shown that leptin concentrations in cord blood are positively correlated with birth weight, whereas no correlation is seen with maternal leptin [34]. Both placental and cord blood leptin are reduced in intrauterine growth retardation and increased in maternal diabetes [30]. Additionally, leptin concentrations are higher in venous umbilical blood than in arterial blood, which is indicative of placental leptin targeting the fetus [35]. The developing murine fetus also expresses leptin receptors and, therefore, is a potential target of placental leptin [36]. As far as we are aware, no studies to date have addressed the relationship between Fe status and leptin production, either in the placenta or in other tissues. In this paper, therefore, we examine whether altered leptin levels in the placenta can be associated with changes in the fetal growth of Fe-deficient rats.

Our data show a clear correlation between maternal Fe status, placental cytokine levels, and fetal development. Although we cannot demonstrate causality, the data suggest a relationship that may explain at least some of the effects of maternal Fe deficiency on her offspring.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Diets

The experimental diets used were based on a dried egg albumin diet [37] and conformed to American Institute of Nutrition guidelines for laboratory animals [38]. FeSO4 was added to achieve dietary levels of added Fe of 50 (control diet), 37.5, 12.5, and 7.5 mg kg-1. Dietary ingredients were purchased from Mayjex, Ltd. (Chalfont-St Peter, U.K.); BDH Chemicals (Poole, U.K.); or Sigma (Poole, U.K.).

Experimental Animals

Experiments were performed using weanling female rats of the Rowett hooded lister strain. They were group-housed in cages under constant temperature and humidity. Controlled illumination with a 12L:12D photoperiod was maintained to ensure regular estrous cycles. Forty female weanling rats were fed control diet for 2 wk before being randomized into four groups. The first group of rats (n = 16) remained on the control diet (50 mg kg-1), whereas the remaining three groups (n = 8 each) were placed on experimental diets of reduced Fe content (37.5, 12.5, and 7.5 mg kg-1). All diets were freely available, and body weights were recorded three times per week throughout the experiment. All groups were fed these diets for 4 wk before mating. The rats were mated with males of the same strain. Mating was confirmed by detection of a vaginal plug, and this day was denoted as Day 0. The female rats were maintained on the same diet throughout pregnancy and were killed at Day 21 of gestation. All experimental procedures were approved and conducted in accordance with the U.K. Animals (Scientific Procedures) Act of 1986.

Tissue Samples

On Day 21 of gestation, the dams were killed by stunning and cervical dislocation. The numbers of fetuses and placentas were counted, and the number of resorption sites observed in the uterus was recorded. Placentas associated with healthy fetuses were weighed and either fixed in neutral buffered formalin (10% [v/v]) overnight (4°C), followed by storage in 70% (v/v) ethanol, or frozen in liquid nitrogen before being stored at -70°C. Livers, hearts, kidneys, lungs, and brains from eight fetuses, chosen from each mother at random, were rapidly dissected, weighed, and frozen in liquid nitrogen. Livers were dissected from all dams, removed, and immediately frozen in liquid nitrogen before being stored at -70°C.

Hematological Measurements

Maternal and fetal hematocrit were measured by drawing blood into heparinized capillary tubes, which were then centrifuged in a high-speed hematocrit centrifuge (Universal 32R, Hettich; Scientific Laboratory Supplies, Coatbridge, U.K.) and read in a microhematocrit reader. Five hundred microliters of maternal blood were collected into heparinized tubes for hemoglobin measurement and centrifuged at 12 000 rpm at 25°C for 7 min. The remaining maternal blood was collected in nonheparinized tubes and centrifuged at 1000 x g at 4°C for 10 min. The resulting serum was stored in metal-free eppendorfs at -70°C. Maternal hemoglobin was measured colorimetrically using the cynanmethemoglobin method (Diagnostic Kit no. 525; Sigma). Serum Fe- and total Fe-binding capacity (TIBC) were measured by the ferrozine-based colorimetric assay (Diagnostic Kit no. 565; Sigma). Transferrin saturation was calculated by expressing the ratio of serum Fe:total Fe and TIBC.

Atomic Absorption Spectrophotometric Analyses

For estimation of serum Fe levels, samples were treated with 20% (w/v) trichloroacetic acid (TCA) and heated to 95°C for 15 min before centrifugation (12 000 x g at 25°C for 5 min) and collection of the supernatant. For the estimation of tissue Fe, samples were baked at 100°C before being treated with nitric acid (Ultrapure; Merck, Poole, U.K.). To differentiate between heme and non-heme Fe in the placenta, homogenized samples were treated with 20% TCA as described for serum. The Fe contents of these samples were determined by graphite-furnace atomic spectrophotometry (Model 3100; Perkin-Elmer, Norwalk, CT). A standard curve for Fe was prepared from commercially available standards (Spectrosol; BDH). Appropriate quality controls were included as necessary.

Immunohistochemical Analyses

Fixed placentas from the control and 7.5 mg kg-1 diet groups were processed into wax blocks. Sections (thickness, 5 µm) were cut for immunohistochemistry. All epitopes were exposed by microwaving at high power in 10 mM sodium-citrate buffer (pH 6.0). Following a further wash with PBS or Tris-buffered saline (TBS) buffer, nonspecific endogenous peroxidase activity was blocked by treatment with 3% (v/v) hydrogen peroxide (Sigma) in distilled water for 5 min at room temperature. All tissue sections were exposed to a nonimmune block with normal horse or rabbit serum (150 µl in 10 ml of buffer; Vector Laboratories, Ltd., Bretton, Peterborough, U.K.) and incubated with the appropriate primary antibodies as follows: Tissue sections were incubated with goat polyclonal anti-rat TNF{alpha} (final concentration, 4 µg/ml in TBS and 1% (w/v) BSA at 4°C overnight; R&D Systems, Ltd., Abingdon, Oxfordshire, U.K.) followed by biotinylated rabbit anti-goat antibody (50 µl in 10 ml of TBS for 30 min at room temperature; Vector Laboratories). The type 1 TNF{alpha} receptor (TNF{alpha}R1) was detected with mouse polyclonal rabbit anti-human TNF{alpha}R1 (p55; final concentration, 3.3 µg/ml in PBS and 1% BSA at 4°C overnight; Santa Cruz Biotechnology, Ltd., Santa Cruz, CA) followed by biotinylated goat anti-rabbit antibody as described above. Leptin was immunolocalized with an in-house-produced polyclonal rabbit anti-human leptin antibody (42 µg/ml in PBS and 1% BSA at 4°C overnight) raised against recombinant human leptin (Peprotech EC, Ltd., London, U.K.) [30] followed by incubation with biotinylated goat anti-rabbit antibody. Leptin receptor was examined using polyclonal goat anti-human leptin receptor antibody (8 µg/ml; Insight Biotech, Middlesex, U.K.), which is reactive against all splice variants. Subsequent treatment was as described above.

Negative controls were performed by replacing the primary antibody with mouse immunoglobulin (Vector Laboratories) at the same concentration as the primary antibody. Sections were labeled with the avidin-biotin-peroxidase detection system (Vector Laboratories). Thereafter, sections were counterstained with hematoxylin, dehydrated, cleared in xylene (20 min), and mounted in Pertex (Cellpath Plc, Newton Powys, U.K.).

Statistical Analyses

For each dam, the litters were averaged and the data recorded as a single point (rather than treating each fetus as a single point). This is the statistically more accurate option, although it may obscure trends that might be identified with high numbers of animals. Relative organ weights were calculated as the percentage of total body weight (g 100g-1). All results are presented as mean ± SEM. A minimum of five measurements for each diet group was used for all analyses. Linear regression tests were used to determine statistical significance between continuous variables, whereas ANOVA was used to determine statistical significance between diet groups.

For immunohistochemical analyses, slides were scored for staining intensity on a four-point scale (0, no staining; 1, weak staining; 2, medium staining; 3, intense staining throughout region). The placenta was divided into discrete regions: trophoblast giant cells, spongiotrophoblast, labyrinth, and cytotrophoblasts. The thin, adherent rim of decidua on the maternal side of each placenta was also assessed. The data were collected from one placenta from each dam (minimum of seven in each group), and each section was scored independently by three observers. The data were analyzed by the Mann-Whitney test. Significance was assumed at P <= 0.05, and all analyses were carried out using Excel 6.0 (Microsoft, Seattle, WA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduced maternal dietary Fe content had no effect on fertility and growth of the dams or on the viability and number of fetuses (Table 1). A diet-induced decrease in maternal hematocrit and hemoglobin concentration, however, was observed (Fig. 1). Maternal serum Fe and transferrin saturation were markedly decreased, but TIBC did not increase significantly. The Fe content of the liver is generally considered to be the most accurate measure of Fe status. In the pregnant dams, liver Fe levels were reduced as a result of decreasing Fe content of the diet (Fig. 2).


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  		TABLE 1. Effect of maternal Fe deficiency on maternal growth and fertility.a



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  		FIG. 1. Effect of maternal Fe deficiency on maternal blood and serum hematological parameters. Hematological parameters were measured in maternal blood and serum at Day 21 of gestation. A significant (P < 0.02) diet-dependent decrease was observed in hematocrit (A), hemoglobin (B), serum Fe (C), and transferrin saturation (D). No significant change was observed in TIBC (E). Data are presented as mean ± SEM (n = 34). Statistical analysis was carried out by one-way ANOVA



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  		FIG. 2. Maternal liver Fe content in Fe deficiency. Maternal Fe deficiency leads to a significant (P < 0.0001) diet-dependent decrease in maternal liver Fe content. Liver samples were taken at Day 21 of gestation; the samples were baked and treated as described in Materials and Methods. The Fe content of the samples was then measured by atomic absorption spectroscopy. Data are presented as mean ± SEM (n = 37). Statistical analysis was carried out by one-way ANOVA

Overall, changes in maternal Fe status were reflected by alterations in placental and fetal parameters. The placental non-heme Fe level decreased (Fig. 3), as did the fetal hematocrit and fetal liver Fe (Fig. 4). Importantly, the proportional drops in Fe content in the placenta (50%) and the fetal liver (46%) were much less than that seen in the mother (69%; P < 0.001).



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  		FIG. 3. Maternal Fe deficiency decreases placental non-heme Fe content. Maternal Fe deficiency leads to a significant (P < 0.0001) diet-dependent decrease in placental non-heme Fe content. Placental samples were taken at Day 21 of gestation and were homogenized and treated as described in Materials and Methods. The Fe content was then measured by atomic absorption spectroscopy. Data are presented as mean ± SEM (n = 30). Statistical analysis was carried out by one-way ANOVA



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  		FIG. 4. Effect of maternal Fe deficiency on fetal Fe status. Maternal Fe deficiency leads to a significant (P < 0.0001) diet-dependent decrease in fetal hematocrit levels (A). Fetal liver Fe levels are significantly (P < 0.0001) decreased in maternal Fe deficiency (B). Samples were taken at Day 21 of gestation and were baked and treated as described in Materials and Methods. The Fe content was then measured by atomic absorption spectroscopy. Data are presented as mean ± SEM (n = 37). Statistical analysis was carried out by one-way ANOVA

Data regarding fetal development can be presented in two ways: related to maternal dietary intake, or related to maternal liver Fe levels. The latter measurement is clearly a better indicator of status, taking into account individual variations in absorption, for example. Therefore, it has been used wherever appropriate as the independent variable.

Placental weight was not significantly related to maternal Fe status (data not shown), but a clear decrease was observed in fetal weight (P = 0.01) (Fig. 5A). Between the two extremes of dietary Fe, 50 mg kg-1 (control) and 7.5 mg kg-1 (15% of control), a 10% decrease in weight was observed. This decrease led to significant changes in the placental:fetal ratio (P = 0.02) (Fig. 5B). In addition to changes in total fetal weight, the fetal liver weights, expressed as a fraction of fetal size, were decreased by 30% (P = 0.03) (Fig. 5C), indicating disproportionate fetal growth. No other tissues showed a significant alteration in relative size (data not shown).



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  		FIG. 5. Effect of maternal Fe deficiency on fetal development. Fetal weight (A), placental:fetal ratio (B), and fetal liver relative organ weight (ROW) (C) were all significantly (P < 0.03) related to Fe levels in the maternal liver. Each point represents the mean value for each litter. Statistical analysis was carried out by linear regression

Placentas from Fe-deficient dams did not show gross morphological changes. In both control and Fe-deficient placentas, TNF{alpha} was localized to placental trophoblast lineages (i.e., trophoblast giant cells, spongiotrophoblast, and labyrinth) (Fig. 6, A and B). In the placentas from Fe-deficient dams, TNF{alpha} was significantly increased in the trophoblast-giant-cell region of the placenta (P < 0.05) (Figs. 6B and 7A). No significant change in the labyrinth, spongiotrophoblast, cytotrophoblasts, or adherent maternal deciduas was observed.



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  		FIG. 6. Immunolocalization of TNF{alpha}, TNF{alpha}R1, and leptin in late-gestation placentas (Day 21) from control and Fe-deficient rats. In control placentas, TNF{alpha} (A), TNF{alpha}R1 (C), and leptin (E) were detected in all trophoblast lineages. Relative to controls, placentas from Fe-deficient rats exhibited increased TNF{alpha} (B), TNF{alpha}R1 (D), and leptin (F). The inset shows the immunoglobulin G-negative control. Significance was achieved in specific trophoblast lineages: TNF{alpha} (TGC), TNF{alpha}R1 (TGC, lab, cytotrophoblasts: data not shown), and leptin (lab). Lab, Labyrinthine placenta; Sp, spongiotrophoblast; TGC, trophoblast giant cells. Bars = 100 µm

The distribution of TNF{alpha}R1 in control placentas was similar to that of TNF{alpha}, with staining in most regions of the placenta. However, Fe deficiency induced a significant increase in levels in all regions except the spongiotrophoblast (P = 0.054). No changes were found in the maternal decidua (Figs. 6, C and D, and 7B).

In the control placentas, leptin was immunolocalized to all trophoblast lineages at a uniform intensity (Fig. 6E). In contrast, placentas from Fe-deficient mothers were characterized by significantly increased levels in the labyrinth (P = 0.04) and marginally increased levels in the trophoblast giant cells (P = 0.054) and spongiotrophoblast (P = 0.072) (Figs. 6, E and F, and 7C). Leptin-receptor levels produced little staining in control placentas, and Fe deficiency showed no significant changes in any region of the placenta (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has examined the effects of different degrees of maternal Fe deficiency during pregnancy on Fe metabolism, placental cytokine expression, and fetal growth and development in a rat model. Our data indicate that maternal Fe deficiency causes deficiency in the fetus, but to a lesser extent than in the mother. These data are in keeping with previous studies showing that available dietary Fe is directed toward the formation of new structures and the maintenance of body weight rather than toward Fe stores [39]. In particular, during pregnancy, the fetus has priority for dietary Fe over maternal stores [40], with more than 70% of dietary Fe being delivered to the fetus toward the end of gestation [41]. We have previously identified some of the mechanisms involved in the placental response to Fe deficiency. Transferrin receptor and DMT1, the protein mediating trans-membrane transport of Fe, show increased expression at both protein and mRNA levels, whereas IREG1 (involved in Fe efflux from placenta) expression does not change and the activity of placental copper oxidase increases [42].

The treatment in the present study has no effect on the viability or number of fetuses. This is in contrast to other reports, in which the number of offspring was significantly reduced. Tojyo [43] showed that fetal growth was dependent on the severity of maternal anemia at the beginning of pregnancy, but their treatments were so severe as to lead to a fall in maternal weight, fertility, and fetal viability. Using a marginally less severe maternal dietary restriction, Crowe et al. [6] demonstrated fetal growth restriction as a result of Fe deficiency during pregnancy at Day 20 of rat gestation. However, those authors expressed concern regarding the severity of the maternal dietary regime used, citing it as an explanation for the inconsistent result of a decrease in placental:fetal ratio. A milder dietary restriction used by Sherman and Moran [44] to investigate the effects of Fe deficiency on pregnancy showed no significant effect on fetal number, fetal weight, or placental weight. Our protocol matches more closely the one used in that study, since we also found no change in viability or fertility.

We have demonstrated that maternal Fe deficiency does induce a significant decrease in fetal weight, and that this is associated with a small rise in placental weight, giving a marked increase in the placental:fetal ratio. We can advance some hypotheses regarding possible mechanisms causing these changes. Almost certainly, direct effects of decreased Fe on fetal growth and development are involved. However, in this study, we have also shown, to our knowledge for the first time, that placental cytokine levels are altered during Fe deficiency. The changes are specific to different parts of the placenta, which may give some indication regarding the consequences of the alterations in levels.

Increases in TNF{alpha} are located mostly in the region occupied by the trophoblast giant cells. The function of these cells has been studied extensively. They produce a variety of hormones and endocrine-signaling agents, including prolactin-like proteins [45] and growth hormones [46]. How expression is regulated is not well understood. In other tissues, a clear interaction occurs between TNF{alpha} and, for example, prolactin, which increases TNF{alpha} production and secretion in astrocytes [47], whereas TNF{alpha} can alter steroid regulatory proteins (hence reducing progesterone production) and hormone receptors in corpora lutea [48]. Furthermore, Monoz et al. [49] have shown increased TNF{alpha} production in Fe deficiency anemia by lipopolysaccharide-stimulated mononuclear cells [49]. How these results translate to the placenta are not clear. Studies show that TNF{alpha} induces apoptosis in purified cultures of human syncytiotrophoblast and cytotrophoblast cells [21, 50], and that the pathway is mediated by TNF{alpha}R1 [21]. Furthermore, Rasmussen et al. [51] have also demonstrated, in a complex series of transgenic mouse cell experiments, that TNF{alpha} receptor expression is possibly more important in determining cell fate and function than simple expression of TNF{alpha} [51]. Many pathways, therefore, are possible whereby our observations can be related to altered trophoblast and placental function. At present, we are testing the hypothesis that altered expression of TNF{alpha}R1 is the primary response to altered Fe status.

Leptin was first identified as a placental hormone in 1997 [28], but its function is still unclear. The presence of a placenta-specific upstream enhancer on the gene suggests that it is regulated differently from that of adipose origin [52, 53]. Studies in humans have shown a correlation between cord blood and placental leptin, but not between maternal leptin and birth weight [30, 54] (reviewed in [32]). This suggests that placental leptin may promote fetal growth. Initially, the data in this study appear to contradict the hypothesis. However, growth and development are clearly dependent not on a single cytokine but on the presence of an appropriate profile of factors so that, for example, the increased leptin may be acting to counter some of the worst effects of the increased TNF{alpha} levels. Some evidence supports this idea [55]. Leptin secretion by adipocytes is regulated by TNF{alpha}, acting through TNF{alpha}R1 [56]. Whether this mechanism operates in placenta has not been determined, however, and the interactions between these cytokines are currently under investigation.

In summary, Fe deficiency during pregnancy exerts a considerable variety of effects, culminating in decreased fetal growth and increased placental:fetal ratio. Fetal growth and development are dependent on placental growth during early pregnancy and on differentiation and function during later pregnancy. These functions are regulated through the activity of local immune and endocrine factors. We cannot, at this stage, be certain, but it seems likely that changing patterns of cytokine production will contribute toward the inhibition of fetal growth. Whether this is the only contributory factor remains to be determined. We consider it to be unlikely, and we believe that other important changes can result in disproportionate fetal growth and postnatal problems in growth and development. This model will clearly help in unraveling the relationship between Fe status and fetal growth, which has significant clinical relevance in both the developed and the developing world. Several reports suggest that inappropriate prenatal growth patterns can be associated with problems later in life [5, 57]. At present, we cannot confirm that the outcome of pregnancy or later development will be compromised by Fe deficiency, but the data suggest that this is likely. We would argue, therefore, that this model has good potential for advancing our understanding of the mechanisms relating in utero nutrition to postnatal development.



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  		FIG. 7. Semiquantitation of TNF{alpha} (A), TNF{alpha}R1 (B), and leptin (C) in placentas from control ({block}) and Fe-deficient ({square}) rats. Immunostaining intensity was assessed on an arbitrary, four-point scale (0, no staining; 3, intense staining). One placenta from each dam (controls, n = 8; deficient, n = 7) was assessed for staining intensity in trophoblast giant cells (TGC), spongiotrophoblast (Sp), labyrinth (Lab), cytotrophoblasts (Cyt), and adherent deciduas (Dec). Values are mean ± SEM. *Significant at the P < 0.05 level (Mann-Whitney test)


   ACKNOWLEDGMENTS
 
We are grateful to the Rowett Research Institute's Bio-Resources and Graphics departments for their technical assistance and to Biomathematics and Statistics Scotland (BioSS) for assistance with the statistical analysis.


   FOOTNOTES
 
First decision: 10 July 2001.

1 Supported by the Scottish Executive Rural Affairs Department, the European Union (QLK1-1999-00337), the Rank Prize Funds, and COST D8. Back

2 Correspondence: Harry J. McArdle, The Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, U.K. FAX: 01224 716622; hjm{at}rri.sari.ac.uk Back

Accepted: October 15, 2001.

Received: June 7, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. World Health Organization. The Prevalence of Anemia in Women: A Tabulation of Available Information. Geneva: World Health Organization; 1992
  2. World Health Organization. Battling Iron Deficiency Anemia. Geneva: World Health Organization; 2001
  3. Allen LH. Anemia and iron deficiency: effects on pregnancy outcome. Am J Clin Nutr 2000; 71:1280-1284
  4. Rush D. Nutrition and maternal mortality in the developing world. Am J Clin Nutr 2000; 72:212-240
  5. Godfrey KM, Redman CW, Barker DJ, Osmond C. The effect of maternal anemia and iron deficiency on the ratio of fetal weight to placental weight [see comments]. Br J Obstet Gynaecol 1991; 98:886-891[Medline]
  6. Crowe C, Dandekar P, Fox M, Dhingra K, Bennet L, Hanson MA. The effects of anemia on heart, placenta and body weight, and blood pressure in fetal and neonatal rats. J Physiol (Lond) 1995; 488:515-519[Medline]
  7. Soewondo S. The effect of iron deficiency and mental stimulation on Indonesian children's cognitive performance and development. Kobe J Med Sci 1995; 41:1-17[Medline]
  8. Walter T. Effect of iron-deficiency anemia on cognitive skills in infancy and childhood. Bailliere's Clin Haematol 1994; 7:815-827[CrossRef][Medline]
  9. Lozoff B, Wolf AW, Jimenez E. Iron-deficiency anemia and infant development: effects of extended oral iron therapy. J Pediatr 1996; 129:382-389[CrossRef][Medline]
  10. Kwik-Uribe CL, Golub MS, Keen CL. Behavior consequences of marginal iron deficiency during development in a murine model. Neurotoxicol Teratol 1999; 21:661-672[CrossRef][Medline]
  11. Kwik-Uribe CL, Golub MS, Keen CL. Chronic marginal iron intakes during early development in mice alter brain iron concentrations and behavior despite postnatal iron supplementation. J Nutr 2000; 130::2040-2048[Abstract/Free Full Text]
  12. Rao R, de Ungria M, Sullivan D, Wu P, Wobken JD, Nelson CA, Georgieff MK. Perinatal brain iron deficiency increases the vulnerability of rat hippocampus to hypoxic ischemic insult. J Nutr 1999; 129:199-206[Abstract/Free Full Text]
  13. Lockwood JF, Sherman AR. Spleen natural killer cells from iron-deficient rat pups manifest an altered ability to be stimulated by interferon. J Nutr 1988; 118:1558-1563
  14. Hallquist NA, McNeil LK, Lockwood JF, Sherman AR. Maternal-iron-deficiency effects on peritoneal macrophage and peritoneal natural-killer-cell cytotoxicity in rat pups. Am J Clin Nutr 1992; 55:741-746[Abstract/Free Full Text]
  15. Krantman HJ, Young SR. Immune function in pure iron deficiency. Am J Dis Child 1982; 136:840-844[Abstract]
  16. Chandra R. Nutrition and the immune system: an introduction. Am J Clin Nutr 1997; 66:460S-463S[Abstract/Free Full Text]
  17. Hunt JS, Chen HL, Miller L. Tumor necrosis factors: pivotal components of pregnancy?. Biol Reprod 1996; 54:554-562[Abstract]
  18. Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal survival in CBA x DBA/2 mice by lymphokine therapy. J Reprod Fertil 1990; 89:447-458[Abstract]
  19. Tangri S, Raghupathy R. Expression of cytokines in placentas of mice undergoing immunologically mediated spontaneous fetal resorption. Biol Reprod 1993; 49:850-856[Abstract]
  20. Silen ML, Firpo A, Morgello S, Lowry SF, Francus T. Interleukin-1 alpha and tumor necrosis factor alpha cause placental injury in the rat. Am J Pathol 1989; 135:239-244[Abstract]
  21. Yui J, Hemmings D, Garcia-Lloret M, Guilbert LJ. Expression of the human p55 and p75 tumor necrosis factor receptors in primary villous trophoblasts and their role in cytotoxic signal transduction. Biol Reprod 1996; 55:400-409[Abstract]
  22. Carbo N, Lopez-Sariano FJ, Argiles JM. Administration of tumor necrosis factor-alpha results in a decreased placental transfer of amino acids in the rat. Endocrinology 1995; 136:3579-3584[Abstract]
  23. Hallmann R, Savigni DL, Morgan EH, Baker E. Characterization of iron uptake from transferrin by murine endothelial cells. Endothelium 2000; 7:135-147[Medline]
  24. Scaccabarozzi A, Arosio P, Weiss G, Valenti L, Dongiovanni P, Fracanzani AL, Mattioli M, Levi S, Fiorelli G, Fargion S. Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells. Br J Haematol 2000; 110:978-984[CrossRef][Medline]
  25. Silver BJ, Hamilton BD, Toossi Z. Suppression of TNF-alpha expression by hemin: implications for the role of iron homeostasis in host inflammatory responses. J Leukocyte Biol 1997; 62:547-552[Abstract]
  26. Vulcano M, Meiss RP, Isturiz MA. Deferoxamine reduces tissue injury and lethality in LPS-treated mice. Int J Immunopharmacol 2000; 22::635-644[CrossRef][Medline]
  27. Olynyk JK, Clarke SL. Iron overload impairs pro-inflammatory cytokine responses by Kupffer cells. J Gastroenterol Hepatol 2001; 16::438-444[CrossRef][Medline]
  28. Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K. Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 1997; 3:1029-1033[CrossRef][Medline]
  29. Linnemann K, Malek A, Schneider H, Fusch C. Physiological and pathological regulation of feto/placento/maternal leptin expression. Biochem Soc Trans 2001; 29:86-90[CrossRef][Medline]
  30. Lea RG, Howe D, Hannah LT, Bonneau O, Hunter L, Hoggard N. Placental leptin in normal, diabetic and fetal growth-retarded pregnancies. Mol Hum Reprod 2000; 6:763-769[Abstract/Free Full Text]
  31. Schulz S, Hackel C, Weise W. Hormonal regulation of neonatal weight: placental leptin and leptin receptors. Br J Obstet Gynaecol 2000; 107:1486-1491
  32. Ashworth CJ, Hoggard N, Thomas L, Mercer JG, Wallace JM, Lea RG. Placental leptin. Rev Reprod 2000; 5:18-24[Abstract]
  33. Mise H, Sagawa N, Matsumoto T. Augmented placental production of leptin in pre-eclampsia: possible involvement of placental hypoxia. J Clin Endocrinol Metab 1998; 83:3225-3229[Abstract/Free Full Text]
  34. Hassink SG, de Lancey E, Sheslow DV, Smith-Kirwin SM, O'Connor DM, Considine RV, Opentanova I, Dostal K, Spear ML, Leef K, Ash M, Spitzer AR, Funanage VL. Placental leptin: an important new growth factor in intrauterine and neonatal development?. Pediatrics 1997; 100:E1-E7
  35. Yura S, Sagawa N, Mise H. A positive umbilical venous-arterial difference of leptin level and its rapid decline after birth. Am J Obstet Gynecol 1998; 178:926-930[CrossRef][Medline]
  36. Hoggard N, Hunter L, Lea RG, Trayhurn P, Mercer JG. Ontogeny of the expression of leptin and its receptor in the murine fetus and placenta. Br J Nutr 2000; 83:317-326[Medline]
  37. Williams RB, Mills CF. The experimental production of zinc deficiency in the rat. Br J Nutr 1970; 24:989-1003[CrossRef][Medline]
  38. American Institute of Nutrition. Second report of the ad hoc committee on standards for nutritional studies. J Nutr 1980; 131:741-744
  39. Rodriguez-Matas MC, Lisbona F, Gomez-Ayala AE, Lopez-Aliga L, Campos MS. Influence of nutritional iron deficiency development on some aspects of iron, copper and zinc metabolism. Lab Anim 1998; 32:298-306[Abstract/Free Full Text]
  40. Murray MJ, Stein N. Contribution of maternal rat iron stores to fetal iron in maternal iron deficiency and overload. J Nutr 1971; 101:1583-1587
  41. Finch CA, Huebers HA, Miller LR, Josephson BM, Shepard TH, Mackler B. Fetal iron balance in the rat. Am J Clin Nutr 1983; 37::910-917[Abstract/Free Full Text]
  42. Gambling L, Danzeisen D, Gair S, Lea R, Charania Z, Solanky N, Joory K, Srai S, McArdle H. Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and in vitro. Biochem J 2001; 356:883-889[CrossRef][Medline]
  43. Tojyo H. Effect of different intensities of iron-deficient anemia in pregnant rats on maternal tissue iron and fetal development. J Nutr Sci Vitaminol (Tokyo) 1983; 29:339-351[Medline]
  44. Sherman AR, Moran PE. Copper metabolism in iron-deficient maternal and neonatal rats. J Nutr 1984; 114:298-306
  45. Hwang I-T, Lee Y-H, Moon B-C, Ahn K-Y, Lee S-W, Chun J-Y. Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cß. Endocrinology 2000; 141:3343-3352[Abstract/Free Full Text]
  46. Lin J, Poole J, Linzer DIH. Two novel members of the prolactin/growth hormone family are expressed in the mouse placenta. Endocrinology 1997; 138:5535-5540[Abstract/Free Full Text]
  47. DeVito WJ, Stone S. Ethanol inhibits prolactin-induced activation of the JAK/STAT pathway in cultured astrocytes. J Cell Biochem 1999;; 74:278-291[CrossRef][Medline]
  48. Chen YJ, Feng Q, Liu YX. Expression of the steroidogenic acute regulatory protein and luteinizing hormone receptor and their regulation by tumor necrosis factor alpha in rat corpora lutea. Biol Reprod 1999; 60:419-427[Abstract/Free Full Text]
  49. Munoz C, Olivares M, Schlesinger L, Lopez M, Letelier A. Increased in vitro tumor necrosis factor-alpha production in iron deficiency anemia. Eur Cytokine Netw 1994; 5:401-404[Medline]
  50. Garcia-Lloret MI, Yui J, Winkler-Lowen B, Guilbert LJ. Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts. J Cell Physiol 1996; 167:324-332[CrossRef][Medline]
  51. Rasmussen CA, Pace JL, Banerjee S, Phillips TA, Hunt JS. Trophoblastic cell lines generated from tumor necrosis factor receptor-deficient mice reveal specific functions for the two tumor necrosis factor receptors. Placenta 1999; 20:213-222[CrossRef][Medline]
  52. Ebihara K, Ogawa Y, Isse N, Mori K, Tamura N, Masuzaki H, Kohno K, Yura S, Hosoda K, Sagawa N, Nakao K. Identification of the human leptin 5'-flanking sequences involved in the trophoblast-specific transcription. Biochem Biophys Res Commun 1997; 241:658-663[CrossRef][Medline]
  53. Bi S, Gavrilova O, Gong DW, Mason MM, Reitman M. Identification of a placental enhancer for the human leptin gene. J Biol Chem 1997;; 272:30583-30588[Abstract/Free Full Text]
  54. Sooranna SR, Ward S, Bajoria R. Fetal leptin influences birth weight in twins with discordant growth. Pediatr Res 2001; 49:667-672[Medline]
  55. Takahashi N, Waelput W, Guisez Y. Leptin is an endogenous protective protein against the toxicity exerted by tumor necrosis factor. J Exp Med 1999; 189:207-212[Abstract/Free Full Text]
  56. Finck BN, Johnson RW. Tumor necrosis factor (TNF)-alpha induces leptin production through the p55 TNF receptor. Am J Physiol 2000;; 278:R537-R543[Abstract/Free Full Text]
  57. Barker DJ, Bull AR, Osmond C, Simmonds SJ. Fetal and placental size and risk of hypertension in adult life [see comments]. BMJ 1990;; 301:259-262



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