Genes Regulated: number of genes regulated.

In female SPTB placentas, the important upstream regulators identified by IPA are listed in Table 6. Insulin (INS) was predicted as an inhibited upstream regulator in female SPTB placentas with a z-score = −1.09. Phosphatase and tensin homolog (PTEN), an inhibitor of PI3K signaling, was also predicted to be inhibited with a z-score of −1, suggesting that PI3K signaling was also activated in female SPTB placentas. Vascular endothelial growth factors (VEGFs) were predicted as inhibited upstream regulators with a z-score = −1.09. They are important for angiogenesis and vascular remodeling, a process critical for placental function and healthy pregnancies.

The IPA analysis can expand predictions and identify the potential novel master regulators responsible for the changes in the gene expressions. LGALS1, NOX1, DNAJA3, PPP2R2A, PPP2CA, and BRCA1 were a few examples of master regulators identified in male SPTB placentas (Table 7). Galectin 1 (LGALS1) is a β-galactoside-binding protein and regulates maternal–fetal immune tolerance and maintaining a normal pregnancy [68–70]. The dysregulation of LGALS1 expression is associated with preeclampsia [71], and interestingly, maternal serum LGALS1 levels are significantly higher in pregnancies with premature ruptures of the membranes [72]. LGALS1 was predicted as an activated master regulator in male SPTB placenta and interacts with 15 upstream regulators to regulate the gene expression. NADPH oxidase 1 (NOX1), a member of the NADPH oxidase family, was predicted as an activated master regulator in male SPTB placenta. NADPH oxidase is the major source of superoxide in placentas and plays a role in early placental devel-

opment [73,74]. However, the overexpression of NOX1 increases oxidative stress and is associated with preeclampsia [75]. DnaJ heat shock protein family member A3 (DNAJA3) is a mitochondrial protein regulating protein folding, degradation, and complex assembly. It plays a critical role in maintaining the mitochondrial membrane potential and mitochondrial DNA integrity; however, its role in the placenta is unknown [76]. DNAJA3 was predicted to be inhibited in SPTB placentas (z-score = −3.16) and interact with 11 upstream regulators to regulate the gene expression. Both PPP2R2A, a regulatory subunit of protein phosphatase 2 (PP2A), and PPP2CA, the catalytic subunit of PP2A, were predicted as inhibited master regulators in male SPTB placentas with a z-score of −3.03 and −1.51, respectively. PP2A acts as a negative regulator of cell growth and division and controls energy metabolism and redox homeostasis via modulating AMP kinase (AMPK) and PI3K-AKT-mTOR signaling [77]. AMPK is a master metabolic regulator controlling glucose sensing and uptake, lipid metabolism, glycogen, cholesterol, and protein synthesis, and the induction of mitochondrial biogenesis [78,79]. mTOR regulates energy-sensing pathways and functions as an important placental growth signaling the sensor to regulate trophoblast proliferation [80–83]. Furthermore, PP2A can control genome integrity by coupling the metabolic processes with DNA damage responses [84]. The breast cancer type 1 susceptibility protein (BRCA1), a tumor suppressor, regulates the cell cycle and DNA damage repair in the placenta [85]. It also functions as a regulator of glucose and lipid metabolism, as well as mitochondrial respiration [86–88]. BRCA1 was predicted as an inhibited master regulator in the male SPTB placentas (z-score = −2.08) and interacts with 27 upstream regulators to modulate the gene expression.

**Table 6.** Top upstream regulators altered in female SPTB placentas.

