**1. Introduction**

Preeclampsia (PE) is a pregnancy related disorder that remains a major cause of maternal and fetal mortality by affecting 3% to 8% of pregnancies worldwide [1]. Although it is diagnosed after 20 weeks of gestation, based on the maternal symptoms, such as high blood pressure and proteinuria [2], the events leading to PE may be triggered during trophoblast invasion and embryonic implantation [3]. Insufficient trophoblast invasion into the decidua and the spiral arteries leads to dysfunctional placentation, altered placental blood flow, subsequent hypoxia, and cell senescence [4–8]. Changes in cellular metabolism, increased placental oxidative stress, and placental barrier damage lead to leakage of placental and fetal factors into the maternal circulation [5,9]. One of these factors is free fetal hemoglobin (HbF), which is found to be elevated in the maternal circulation as early as the first trimester in women who later develop PE [10]. Free hemoglobin triggers inflammation, vasoconstriction, and tissue damage [11], and particularly impairs the placenta barrier [12].

The negative consequences of a PE pregnancy affect the mother as well as the developing fetus [13–16]. Our group has previously demonstrated higher levels of total hemoglobin in the arterial and venous UCB [17], suggesting possible accelerated fetal erythropoiesis. Higher erythroblast count in the UCB is a common observation in PE pregnancies [18–21] that has been linked to elevated erythropoiesis induced by hypoxia and enhanced fetal erythropoietin (EPO) levels [22–25]. However, previous studies investigating in vitro colony formation of UCB HSPCs have not demonstrated any significant differences in the erythroid differentiation capacity of the cells obtained from PE vs. normotensive pregnancies [26,27]. This suggests that a mechanism other than hypoxia-induced EPO-dependent enhanced fetal erythropoiesis may affect erythroid maturation, underlying the higher erythroblast count documented in the UCB of PE pregnancies. For instance, levels of glucose, glucose/insulin ratio [28,29], lactic acid [30], and inflammatory cytokines, such as tumor necrosis factorα (TNFα) [31], are reported to be altered in the fetuses from PE pregnancies. All these factors are significant in regulating erythropoiesis and erythroid maturation [32–36]. However, the effects of these changes on the molecular pathways that control HSPC differentiation and late erythroid maturation in the fetus have not been investigated in detail.

Another important factor in regulating fetal hemoglobin levels is fetal sex [37,38], which also determines the onset of the disease and severity of maternal symptoms in PE [39,40] as well as maternal adaptation to pregnancy [41]. Sex-specific fetal responses in PE have been assessed in both an animal model [42] and in humans [43–46]. However, these studies are limited to comparing clinical parameters, such as blood pressure, height, and weight, indicating the importance of further analysis to investigate the molecular changes.

The focus of this study was to evaluate any changes in the migration/homing or differentiation capacity of fetal HSPCs by analyzing the expression of surface adhesion molecules (SAMs), the transcriptome, and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs) in fetuses from PE pregnancies. Moreover, the proteomic profile of in vitro differentiated erythroid cells was studied to explore possible differences in erythroid maturation. Finally, using an established flow cytometry analysis [47], the profile of terminally differentiating erythroblasts in the UCB was compared between PE and normotensive pregnancies to explore any alterations in fetal erythroid populations. In addition, transcriptome analysis on isolated erythroid cells was performed, with particular attention to sex-specific differences, using SE50bp RNA sequencing.
