Impact of Maternal Iron Restriction on Offspring Figure 5. The experimental dietary regimens established iron deficiency anemia (IDA) and caused changes in body weight and core body temperature in the offspring. The hematological status, weight, and core temperature were evaluated in each animal after the ABR test completion on postnatal day 45 in the various dietary groups (depicted in Figure 4). A. The table summarizes the results of the hematological tests. All groups of experimental iron deficient animals (ID -2wks, ID E0, ID E7, and ID E14) presented with significantly decreased hematocrit (Ht) values, significantly lower hemoglobin (Hb) levels, profoundly decreased red blood cells (RBC) counts and markedly reduced mean corpuscular volume (MVC) compared to controls, demonstrating the presence of anemia in dietary iron restricted rats. Serum iron concentrations in iron-deficient diet treated animals were significantly lower compared to control animals, confirming the anemic condition of experimental animals. (Ht measurement: n = 11 CTL rats, n = 9 ID -2wks rats, n = 11 ID E0 rats, n = 9 ID E7 rats, and n = 9 ID E14 rats; Hb, RBC, and MCV determination: n = 6 CTL rats, n = 4 ID - 2wks rats, n = 4 ID E0 rats, n = 4 ID E7 rats, and n = 6 ID E14 rats; Mean 6 SEM, * p,0.0001; serum iron analysis: n = 5 rats for all groups, Mean 6 SEM, * p,0.01; each CTL and experimental group included rats from 3 separate litters). B. Experimental iron deficient animals exhibited marked growth retardation compared with controls. (n = 35 CTL rats, n = 15 ID -2wks rats, n = 14 ID E0 rats, n = 27 ID E7 rats, and n = 29 ID E14 rats; all CTL and experimental groups contained rat pups from at least 3 separate litters; Mean 6 SEM, * p,0.0001). C. Experimental group ID -2wks (n = 18 rats from 3 separate litters) offspring exhibit significantly reduced core body temperature in comparison to CTL animals (n = 17 rats from 3 different litters; * p,0.05). These changes are not seen in the other three experimental groups (n = 13 ID E0 rats, n = 13 ID E7 rats, and n = 14 ID E14 rats). All groups are composed of animals from 3 separate litters. doi:10.1371/journal.pone.0017483.g005 still disrupt fetal iron homeostasis as early as E15 and result in severe IDA in the offspring. We provide evidence showing that maternal iron restriction that occurs prior to the onset of pregnancy and during the first trimester has the most severe impact on the neural development of the offspring. Maternal ID during the second trimester still negatively affects the neural conduction velocity (measured by ABR) of the offspring but to a lesser extent. Our data further demonstrate that the development of neural impairments in the offspring is not dictated by the presence of IDA, low serum iron concentration or low birth weight, but is determined by the time window during which the dam received the iron-deficient diet. Discussion The purpose of this study was to determine the impact of dietary maternal iron deficiency (ID) on fetal development, with the main focus on the identification of critical periods of gestation when the developing CNS is most vulnerable to maternal ID. The animal model we established was intended to mimic the extremely prevalent human condition of marginal maternal ID that occurs in an estimated 30 to 50 percent of pregnancies worldwide. After careful titration of the iron concentration in the diet, we were able to establish a model where the pregnant dam was rendered iron deficient but did not develop severe anemia during pregnancy. The analysis of the most prolonged form of diet restriction, in which iron levels were already reduced prior to the onset of pregnancy led to some unexpected findings. For example, we found that despite the fact that the dams received the iron- deficient diet not only prior to conception but during the entire pregnancy, the dams did not develop severe anemia that had an impact on hematocrit levels, which remained in the normal range. However, maternal serum iron concentrations decreased contin- PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17483