ID for US children under 2 years of age is estimated to be 25% [23,50,51,52], which might well be an underestimation due to the difficulty in diagnosing ID in the absence of anemia. Despite the recognition that ID during pregnancy can have multiple adverse effects on the developing fetus, it remains elusive to what degree and during which gestational time window maternal ID has to occur to affect fetal development to a degree that leads to functionally relevant long-term impairments. We addressed this question using a highly controlled animal model system that allowed us to stage the initiation of ID during pregnancy using a defined feeding regimen and to analyze the fetal development as well as the neural function in the young adult offspring. Our prior studies on the effects of IDA during pregnancy suggested that IDA affects a very early arising precursor cell pool. The impairment of this cellular population is likely to contribute to an ultimate disruption of proper CNS development during postnatal development [53]. Based on those studies, we now address the fundamental questions of whether (i) the generation of neural impairment in the offspring is limited to cases of maternal IDA as opposed to ID without anemia and whether (ii) the time window during which the iron depletion occurs in the dam determines the degree of functional impair- ments in the offspring. Results Maternal iron restriction that does not cause severe maternal anemia renders the embryo iron depleted with postnatal onset of anemia One of the major challenges in understanding the impact of iron on development is the difficulty of measuring iron concentrations in the developing embryo. Animal models allow us to gain insight into the relationship between maternal iron intake and fetal iron levels. We established a feeding protocol in which rat dams were provided a customized iron-deficient diet that led to maternal iron deficiency (ID) but not to severe iron deficiency anemia (IDA). As shown in Figure 1A, this diet did not generate a discernable Impact of Maternal Iron Restriction on Offspring difference in hematocrit values in the pregnant dams compared to control dams, although hemoglobin (Hb) and red blood cell counts (RBC) were slightly below the control range by 21 days of gestation. Even though severe anemia was absent in the dams, we found progressively reduced serum iron levels, ranging from a .40% reduction at 15 days of gestation up to an 82% decrease at 21 days of gestation, relative to controls (CTLs; Figure 1B). To determine the impact of the decrease in serum iron concentration in the dam on the iron status in the developing embryo, we measured the iron concentration of whole embryos using atomic absorption spectroscopy (AAS; Figure 2A). While we did not see a significant reduction in tissue iron concentrations at E13, at E15 embryos from iron deficient dams had only 44% of the levels of iron seen in control embryos. Despite a developmental increase in total iron concentration in the iron deficient embryos from an average of 8 ng Fe/mg tissue at E15 to approximately 14 ng Fe/mg at E19, these values were far below the normal Fe concentrations that increased from 19 ng Fe/mg at E15 up to 63 ng Fe/ml at E19. This lack of iron accumulation in the embryos was correlated with the respective total body weights. At E13, when the embryos did not yet show a significant difference in Fe concentration compared to controls, there also was not a difference in the weight of the embryos. At E15, E17, and E19, however, there was a persistent lack of weight gain in the iron deficient embryos compared to controls, rendering ID fetuses nearly 40% lighter than control fetuses at E15. By E19, the weight increased although ID embryos never reached the same weight as controls (Figure 2B). In order to determine whether a continuation of the maternal feeding protocol postpartum has a persistent impact on the developing pups, we maintained the dams on the diet they received throughout gestation and tested the iron status of the pups one week after birth. As expected, the iron status of the pups continued to deteriorate and after one week serum iron concentrations were reduced by 63% and hematocrit levels showed a 40% decrease (Figure 2C, D). While the reduction of iron concentrations in whole embryos and in the serum of Figure 1. Iron restriction two weeks before conception induces maternal iron deficiency without severe anemia. A. Hematocrit levels of pregnant rats exposed to an iron-deficient diet two weeks prior to conception (ID -2wks) (red bars) were evaluated at gestational day E15, E17, E19, and E21 and compared to time matched pregnant rats that received a control diet (black bars). CTL pregnant dams: n = 6 for each gestational age; ID - 2wks pregnant dams: n = 6 for each gestational age; expressed values are mean 6 SEM. B. The serum iron concentrations in iron-deficient diet treated animals (ID -2wks) and control animals (CTL) were determined via atomic absorption spectroscopy (AAS) at E15, E17, E19, and E21 (CTL pregnant rats: n = 3 for each time point; ID -2wks pregnant rats: n = 3 for each time point; Mean 6 SEM, * p,0.001). While there were no significant changes in the hematocrit values between the two groups, iron restricted dams showed significantly lower serum iron concentrations compared to control dams, confirming their iron deficiency (ID) status. doi:10.1371/journal.pone.0017483.g001 PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17483