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* Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB UK and Biomathematics and Statistics Scotland, Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB UK
2 To whom correspondence should be addressed. E-mail: H.McArdle{at}rowett.ac.uk.
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ABSTRACT |
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KEY WORDS: • fetal programming • developmental origins • embryo culture • micronutrients
Iron (Fe) deficiency during pregnancy can have serious consequences for both the mother and her baby. In addition to an increased risk of low birth weight, and increased morbidity and mortality, there are also longer-term sequelae. For example, babies that are anemic at birth have an increased risk of stroke, even up to 18 mo later (1). There are also several studies that show behavioral defects in both humans (1) and mice (2,3), possibly as a consequence of inappropriate myelination (4). We developed a rat model of Fe deficiency during pregnancy (5,6). Pups born to Fe-deficient dams are smaller than controls, and have enlarged hearts. They also develop increased blood pressure, which persists into adulthood (7). These data provide support for the "fetal origins" hypothesis, which states that imbalances in the nutritional environment in utero can have long-term effects such as increased risk of heart disease, hypertension, obesity, and Type II diabetes (8).
Identification of the critical periods during development in which the fetus or embryo is most susceptible to disturbances in its milieu is an essential part of understanding the mechanisms underlying these observations. It seems logical that nutritional deprivation would have its greatest effect on those tissues and systems that are developing during that period. There are some data support this. Women who were pregnant during the Dutch "hunger winter" of 1944 gave birth to offspring with different adult outcomes. For example, Stein et al. (9) identified changes in birth parameters such as size occurring primarily in those who were in the last trimester of pregnancy, whereas Brown et al. (10) identified increased affective disorders in males whose mothers were in their second trimester during the famine. In contrast, Stanner et al. (11) did not find changes in children born during the siege of Leningrad, which lasted for a much longer period than the Hunger Winter. They suggested that this demonstrates the importance of postnatal catch-up growth, which is more likely to have happened in Holland than Leningrad, in the development of disease (11).
There are surprisingly few animal data. Fleming and colleagues proposed that rats exposed to a low-protein diet for the first 4 d of gestation have long-term changes in function (12), implicating the embryonic period as an important critical window [although there are some concerns about the interpretation (13)]. More work has been carried out in large animals such as pigs, where feeding strategies are used to maximize embryo survival and animal productivity [For a review see (14).] Currently, we are testing the hypothesis that different systems in the developing embryo are particularly vulnerable to nutritional stress during their periods of maximum growth and differentiation. For the developing cardiovascular system in rats, this is most apparent about halfway through gestation, between approximately embryonic d 9 (E9)3 and E13. Although this may not be the only such critical period, we hypothesize that this may be a sensitive period in which nutritional imbalance can result in cardiovascular abnormality.
Rodents offer several advantages for studying the relation between nutrients at different stages and adult outcome. The gestation period is comparatively short, the developmental profile is well established, and the periods in which different organs develop are known. Additionally, it is possible to culture embryos taken from different stages in vitro in a culture medium that is at least partly defined. This is especially true in the postimplantation whole embryo culture system. Embryos are taken from dams at E10.5 and can be maintained in culture for up to 48 h. During this period, they grow and develop to the same extent as in vivo, with the advantage that the medium can be manipulated. The system is used extensively for pharmacologic testing but much less so for nutritional manipulation except for the work of Keen et al. [e.g., (15,16) and some early studies of our own (17)]. In this paper, using postimplantation embryos, we identified a critical window during which Fe deficiency might result in the occurrence of increased blood pressure in the offspring of Fe-deficient rats.
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MATERIAL AND METHODS |
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Serum production. Male Rowett hooded Lister rats weighing 430–560 g were fed a commercial standard, starch-based diet (Special Diets Services, Fe, 0.13 g Fe/kg) or a Fe-deficient [0.0075 g Fe/kg (FeSO4) other components as described above] diet for 8 wk before blood was drawn for serum production. The rats were anaesthetized with isoflurane and blood was withdrawn from the deep abdominal aorta using serum gel S-Monovette syringes and metal free needles (Sarstedt). The rats were killed by cervical dislocation and livers were collected for further analysis. Serum was collected by centrifugation (2000 x g 15 min, twice) and heat inactivated for 30 min at 56°C. Serum from rats within each dietary group was pooled. Penicillin G sodium/streptomycin sulfate was added to a final concentration of 50 kU/L and 50 mg/L, respectively (Gibco, Invitrogen). Serum was stored in 10-mL aliquots at –80°C until used. The Fe in serum would all be bound to transferrin.
Experimental animals. All experimental procedures were approved by the Home Office and the Ethics Committee at the Rowett Research Institute and conducted in accordance with the UK animals (Scientific Procedures) Act, 1986. Experiments were performed using weanling female rats of the Rowett Hooded Lister strain, bred at the Rowett Research Institute. They were grouped housed in cages, under a 12-h light:dark cycle with constant temperature and humidity. All rats consumed food and distilled water ad libitum.
Female weanling rats were fed the control diet for 4 wk, before being randomly assigned to control or Fe-deficient diet for 4 wk before mating. The females were synchronized with progesterone and pregnant mare serum according to standard animal husbandry procedures before mating. Mating was confirmed by detection of a vaginal plug (E0.5). The females were fed the same diet until gestation E10.5.
Whole embryo culture. The technique of whole rat embryo culture is described in detail elsewhere (19). On E10.5 of gestation, the dams were anesthetized with isoflurane, and maternal blood samples collected from the heart. The uterine horns were removed by caesarean section and placed in Tyrode's solution (Sigma) until dissected for whole embryo culturing. The dams were then killed by cervical dislocation and livers were collected for further analysis. The number of embryos was counted, and the number of resorption sites observed in the uterus was recorded.
The embryos were dissected free of the uterine wall, deciduas, and Reichert's membrane. Only those embryos of 6–8 somites and with an intact yolk sac were used. A maximum of 2 embryos were placed in each 30 mL-glass cylindrical tube containing 3.8 mL serum and 0.2 mL Tyrode's solution. The cultures were rolled at 
30 rpm in an incubator (BTC Engineering) maintained at 37°C. Twice daily, the culture flasks were gassed with the appropriate O2, CO2, and N2 gas mixture (20). Partial pressures of the different gasses were not measured directly.
Experiment 1: Effect of Fe deficiency on rat embryonic development. Dams (n = 12) initially fed the control diet were randomly assigned to 1 of the 2 diet treatments, control or iron deficient (as described above) with 6 rats/group. At E10.5, they were killed and their embryos cultured for 48 h in serum collected from males that had been fed either a control or a Fe-deficient diet. The medium contained final Fe concentrations of 26.8 ± 0.004 µmol Fe/L (control) or 14.32 ± 0.003 µmol Fe/L (Fe deficient). In this experiment, embryos from control dams were cultured in control serum (+/+) and those from deficient dams in deficient medium (–/–).
Experiment 2: Can the effect of Fe deficiency on cardiovascular development be reversed by culturing in control serum? To determine whether the effects were reversible by providing iron during the 48 h culture period, we repeated Expt. 1, but cultured deficient embryos in both deficient and control medium and vice versa. For this study, 20 dams were randomly assigned to 1 of the 2 diet groups (as described above) with 10/group, and embryos collected at E10.5. The embryos were randomly assigned to medium containing serum collected from either control or Fe-deficient males. This resulted in 4 groups; control embryos cultured in control (+/+) or Fe-deficient serum (+/–), and Fe-deficient embryos cultured in Fe-deficient (–/–) or control (–/+) serum.
Embryo analysis. After 48 h, the morphology of the embryos and their yolk sacs was examined for developmental abnormalities under a stereomicroscope; the development was scored using an adaptation of the system of Brown and Fabro (21). Structures examined included yolk sac, optic system and otic placode, neural tube, branchial arches, hind-, mid-, and forebrain, fore- and hindlimbs, body curvature, and heart. For example, yolk sac circulation was given a score of 0 if there were no blood vessels, 1 for blood islands but no movement of blood, 2 for vessels and slow movement, 3 for full vessels and rapid movement of blood, and 4 for split vitelline vein and arteries. Heart development was scored using criteria for the presence of a tube and whether it was looped or had 2 or 4 chambers. A similar approach was used for the otic and optic vesicles (21). The scoring was carried out by 1 trained observer who was unaware of the treatment. The variation in scoring for any 1 embryo was <1 score unit. Any defects for any organs were noted and tested for significance as described below.
Crown-rump length, head length, and yolk sac diameter were measured with an eyepiece graticule 5 mm:100 divisions (Leica Microsystems). Image J analysis (program version 1.32j) was used to measure the heart area from digital pictures (photographed using a Nikon camera 5 M pixels) of the embryos. Briefly, the heart and the pericardial sac can be identified separately under the microscope. The heart area was delineated and the pixel count collected. Because there were no gross changes in heart morphology, we considered this a reasonable, indeed the only practical, way of estimating heart size. At the end of the experiment, the embryos were killed by snap freezing in liquid nitrogen.
Atomic absorption spectrophotometric analysis. Fe and Cu levels in serum and in tissue from donor males and dams were measured using graphite furnace atomic absorption spectroscopy as previously described (7). Quality controls were included in all assays. Repeat measurements gave variances <15%. Standards were obtained from BDH Laboratory Supplies. The laboratories are certified to ISO 9001.
Statistical analysis. Maternal data and data from Expt. 1 were analyzed with a 2-sample t test, except for hematocrit and the number of resorption sites (maternal data), and the circulation score, which were analyzed with Mann-Whitney nonparametric t test. All values are expressed as means ± SEM.
Data from Expt. 2, except for abnormality data and morphology scores, were analyzed with 1-way ANOVA to look for overall treatment effect. In addition, data were also analyzed with 2-way ANOVA, with dam (control or Fe-deficient), serum (control or Fe-deficient), and their interaction as treatment effects. When any of these effects was significant, post hoc t tests were applied to test for significant differences between treatment means. For the morphology scores, Kruskal-Wallis nonparametric, one-way ANOVA was used to test for an overall treatment effect. When the treatment effect was significant, Mann-Whitney's nonparametric test was used to test for significant differences between treatment means. Abnormality data were treated as binomial outcomes, with a score of 0 when a defect was absent and a score of 1 when a defect was present. A binomial model with logistic link (22) was fitted to these data, with dam, serum, and their interaction as explanatory variables. In addition, Fisher's exact test was used to investigate a possible relation between blood pooling and thin yolk sac vasculature, in which embryos were combined across all treatments.
Initially, variation between dams and between culture tubes was allowed for by fitting residual maximum likelihood (REML) models to the data, except for abnormality data to which initially a generalized linear mixed model (GLMM) was fitted. Both the REML and GLMM models included random effects for dam and embryos from the same dam, but these were estimated to be 0 and were omitted from subsequent analyses, resulting in the simpler models described above. P-values for treatment effects were not substantially affected by this simplification.
All statistical analyses were performed in Genstat 8th ed., Release 8.1 (VSN International). P-values 
5% were considered significant.
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RESULTS |
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The importance of iron during pregnancy is well established. Early studies, in both humans and animals, showed that iron deficiency leads to problems for both the mother and her developing fetus (7,23–25). However, it is only more recently that the sequelae were shown to extend into the adult period (23,24). Inducing a mild iron deficiency, in which the maternal hematocrit does not drop significantly until late in pregnancy, and weight gain and general well-being are not affected at all (5,7), results in increased blood pressure in the offspring, despite their having normal iron status. Further, we showed that the effect is determined prenatally, most likely early in pregnancy (23). The current results confirm these data, and cast light on the possible mechanisms underlying the in vivo observations.
In animals with a hemochorial placenta, such as rodents or humans, transfer of iron from the mother to her fetus increases as pregnancy develops (26). This is as a consequence not only of increased size and surface area of the placenta, but also of increased density of transferrin receptors in the apical side of the placenta. This iron is stored in the fetal liver, possibly to provide a reserve during the early neonatal and weaning periods. Earlier in pregnancy, the role of iron is more equivocal. Early data from in vivo studies in mice suggested that Fe could inhibit the growth of embryos past the 2-cell stage (27), although there was a more recent suggestion that chelators act in another fashion that is not correlated with their ability to remove divalent ions (28). Fe metabolism in the postimplantation embryo has also been studied. We showed that transferrin was taken up by yolk sac, and the iron was utilized by the embryo. Interestingly, both transferrin and albumin could be broken down by the yolk sac, and the amino acids produced were transferred to the embryo (17,29). These data were supported by other studies that showed that methionine and iron were growth factors for the postimplantation embryo (30). Fe deficiency certainly results in inhibition of the cell cycle in totipotent mouse cells; in cancerous cells, it was shown to block cells at the G1/S phase (31).
Our current data also show that Fe deficiency early in pregnancy has marked effects on development. The fact that growth is inhibited, and that providing Fe during the E10.5–12.5 period does not reverse the inhibition, points to a critical window that occurs earlier than E10.5. However, because growth is further inhibited by Fe deficiency during culture, it would appear that the sensitivity goes beyond the early embryonic period.
These results contrast with the cardiovascular variables. The culture period seems to be absolutely critical, and providing Fe during this period, even to embryos that were deficient, results in normal development of both the heart and the circulatory system. In rats, cardiovascular differentiation is similar to that described in other rodents (32). A period of elongation is followed by looping, septation, and enlargement of the heart itself. Concurrently, vascularization develops so that the circulation is complete by E12.5. This process is inhibited in Fe deficiency. At present, we do not know whether the cardiovascular system that is finally formed is normal or whether structural disorders remain that will persist into adulthood. This latter option is one possible explanation for the increased blood pressure in the adult offspring of iron-deficient mothers (7), but there are also others. For example, it is possible that there is a decrease in the number of cells in the developed system as a consequence of delay, with a concurrent loss of efficiency. Alternatively, there could be ancillary damage as a consequence of reduced oxygen supply, in turn arising as a consequence of poor vascularization.
The increase in heart size in these embryos is intriguing. We showed previously, using this model, that the heart is larger in pups born to deficient dams at birth (7). These rats will go on to develop hypertension. We assumed, therefore, that hypertension is a problem for the pups at birth and hence there is an increase in heart size (hypertrophy, perhaps). This is an appealing hypothesis for the embryos. If there is increased vascular resistance because of decreased angiogenesis, it is quite feasible that there would be an increase in heart size.
It is important to remember that Fe deficiency per se may not be the major mediator of the changes. For example, altering Fe in the diet of the animals supplying the serum may change lipid (33,34) or zinc metabolism (35) and may also interfere with copper metabolism (36). In support of this latter possibility, there are changes in the levels of Cu in the yolk sac (data not shown). We can exclude maternal endocrine influences because the offspring were cultured in male serum. Further, we can exclude earlier effects on maternal reproductive competence because there was no difference in viability, fertility, or survival to birth between control and deficient fetuses (7).
Whichever is the case, the current results strongly support the data that we presented previously in which we showed that maternal Fe deficiency results in increased blood pressure in the offspring as adults. These data now indicate that the fetus is sensitive to deficiency, as far as cardiovascular development is concerned, during the period in which differentiation and growth occur. This does, of course, make teleological sense. The data also show that growth, per se, is not the cause of the "fetal origins" symptoms, but rather occurs as a parallel effect of nutritional imbalance. Although this has generally been accepted, it has been difficult to prove directly.
This period of cardiac development corresponds to wk 3–5 in humans. Although the 2 situations cannot be compared directly, the data do indicate that to produce the best outcome possible, it is important to ensure that a pregnant women's iron status is optimal early in pregnancy (25).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: E10.5, embryonic day 10.5; Fe, Iron; (+/+), embryos of control dams cultured in control serum; (+/–), embryos of control dams cultured in Fe-deficient serum; (–/–), embryos of Fe-deficient dams cultured in Fe-deficient serum; (–/+),embryos of Fe-deficient dams cultured in control serum.
Manuscript received 24 November 2005. Initial review completed 15 December 2005. Revision accepted 8 February 2006.
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