Dose- and sex-dependent effects of phlebotomy-induced anemia on the neonatal mouse hippocampal transcriptome

Background: Phlebotomy-induced anemia (PIA) is universal and variable in degree among preterm infants and may contribute to neurodevelopmental risk. In mice, PIA causes brain tissue hypoxia, iron deficiency, and long-term sex-dependent neurobehavioral abnormalities. The neuroregulatory molecular pathways disrupted by PIA underlying these effects are unknown. Methods: Male and female pups were phlebotomized daily from postnatal day (P)3–P14 via facial venipuncture to target hematocrits of 25% (moderate, mPIA) and 18% (severe, sPIA). P14 hippocampal RNA from non-bled control and PIA mice was sequenced by next-generation sequencing to identify differentially expressed genes (DEGs) that were analyzed using Ingenuity Pathway Analysis. Results: mPIA females showed the least DEGs (0.5% of >22,000 genes) whereas sPIA females had the most (8.6%), indicating a dose-dependent effect. mPIA and sPIA males showed similar changes in gene expression (5.3% and 4.7%, respectively), indicating a threshold effect at mPIA. The pattern of altered genes induced by PIA indicates sex-specific and anemia-dose-dependent effects with increased pro-inflammation in females and decreased neurodevelopment in males. Conclusion: These gene-expression changes may underlie the reduced recognition memory function in male and abnormal social-cognitive behavior in female adult mice following neonatal PIA. These results parallel clinical studies demonstrating sex-specific behavioral outcomes as a function of neonatal anemia. Impact: Phlebotomy-induced anemia (PIA) in neonatal mice results in an altered hippocampal transcriptome and the severity of changes are dependent upon degree of anemia and sex of neonatal mice.The reported findings provide context to the sex-specific outcomes that have been reported in transfusion threshold clinical trials of preterm infants and therefore may inform treatment strategies that may be based on sex.These data advance the field by showing that consequences of PIA may be based in sex-specific transcriptomic alterations. Such changes may also result from other causes of neonatal anemia that also affect term infants.