In this study, the expression of all three major molecules involved in ATP catabolism and hypoxia including CD39, CD73, HIF-1a, and their regulatory miRNAs in the decidua of PE patients was investigated. These molecules can influence placenta environment by altering ATP, adenosine and cellular oxygen status which is observed in pregnancy related complications like recurrent pregnancy loss (RPL) and PE.
The PE molecular mechanisms are not fully revealed and different biological processes are involved in the pathogenesis of PE. Improper trophoblasts invasion and proliferation are two critical factors PE development and spreading. Therefore, it is necessary to examine the basis of these defects to identify reputable and effective targets for the treatment of PE . High levels of ATP are one of the danger signals, which are now found in PE. Disrupted balance of adenosine and ATP with a low ratio of adenosine/ATP is found in PE, which may be resulting in the activation of endothelial cells, systemic inflammation, and hypertension .
CD39 is an ectonucleotidase cell surface enzyme with a high variety of cell distribution, which is most dominantly expressed in human trophoblasts and vessels and regulates the function of trophoblasts in an ATP-dependent manner . CD73 is a glycosyl-phosphatidylinositol (GPI)-linked cellular surface enzyme with 70 kDa moleculare weight found in various tissues and act as an ecto-5′-nucleotidase. CD73s first function was described as a lymphocyte differentiating antigen by participating in T lymphocytes signaling as ligand which also can acts as adhesion molecule via binding to endothelium. A long variety of biological processes like precondition of ischemia, platelet function, vascular leac, tissue injury, hypoxia, and transportation of ion and fluid in epithelial are done by CD73 involvement. CD73 functions are outcome of the regulated hydrolytic activity on extracellular nucleotides . Conversion of ATP and ADP to AMP and consequence changing of AMP to adenosine via CD39 and CD73 activities notify critical role of these enzymes in calibrating duration, magnitude, and particularity of purinergic signals. It is necessary to note that CD39 acts as rate-controling enzyme in ATP/CD39/CD73/adenosine cascade .
There are several studies on CD39 and CD73 expression and their role in pregnancy disorders especially in PE based on CD39-CD73 roles in different inflammatory and hypoxic condition and their expression pattern. Our results showed decreased and increased expression of CD39 and CD73 in decidua tissue of PE patients compared to healthy pregnant women at both mRNA and protein level. In line with our results, Zhu et al.  showed that decreased expression of ZDHHC14 and CD39 by hypomethylation is related to late-occurred PE cases via the these genes regulatory effects on trophoblast cell lines. They concluded that ZDHHC14 and CD39 may consider as potential targets and markers in PE clinical diagnosis and treatment . In another study was done on mouse model of PE by McRae et al. , they concluded that inducing gestational hypertension in mice could be done by transferring TH1-polarized lymphocytes which are isolated from maternal blood and CD39 overexpression has protective effects in this model. In supporting our outcomes for CD73 expression in PE cases, elevated expression of CD73 as key regulatory enzyme which cause increase of adenosine in placenta was observed by Iriyama and coworkers in their study . Huang et al.  observed reduction of adenosine in placenta and attenuated preeclampsia features and hypomethylation of placental DNA in preeclampsia mouse model which is induced by autoantibody.
Hypoxia-inductive Factor-1α (HIF-1α) is a critical transcription factor that alters response of the cell to hypoxia in pathological situations . HIF-1 has a basic and heterodimeric helix-loop-helix structure and is composed of two alpha and beta subunits. It found that the determination of HIF-1 activity is measured by HIF-1a. There is a close correlation between CD73 and HIF-1a activity, which is confirmed by Lu et al.  in a study was done on human gastric carcinoma. Adenosine receptors comprise ADORA2A in promoter section of placenta tissue, which is respond to hypoxia. HIF-1α and HIF-2α controls ADORA2A, and ADORA2B, respectively. It is expected that the chronic hypoxic state of PE patient's placenta is result of elevated expression of placental ADORA2A and ADORA2B . Also, increased transcription of soluble endoglin (sEng), soluble fms-like tyrosine kinase-1 (sFlt-1), and endothelin-1 (ET-1), which plays key role in PE pathogenesis are related to elevated expression of HIF-1α in the PE patient's placenta .
HIF-1α known as one of main molecules involved in hypoxia condition which is observed in preeclamtic placenta. We observed significant increase in expression of HIF-α mRNA and protein in PE patients. In supporting our data, up-regulation of HIF-1α in placenta of PE patients was confirmed in several studies [35, 37–39]. Persistent enhancement of placental adenosine and bilateral up-regulation of HIF-1α promotes PE pathogenesis which is observed by Iriyama et al.  in a mouse model study. Zhao confirmed a significantly increased expression of HIF-1a and TLR4 in placental tissues from severe PE cases (late-onset and early-onset) compared to healthy control. In addition, he suggested that HIF-1α could promote human placental micro-vascular endothelial cells (hPMECs) apoptosis by upregulation of TLR4 expression during PE pathogenesis .
MicroRNAs are small molecules with a large impact on the cellular and physiological function of the body and play an undeniable role in the pathogenesis of disorders like PE. In biogenesis of miRNA, pri-miRNA converts to single-hairpin precursor by a nuclear protein complex which has a binding site for double-strand RNA (dsRBP). After that, pri-miRNA delivers to the cytoplasm by exportin-5. Dicer is an RNase-III family cytoplasmic enzyme and modifies long dsRNA to 19–25 nucleotides named double-strand miRNA (ds-miRNA). The function of the human Dicer is consonant with dsRBP. At the end of miRNA biogenesis, produced ds-miRNA is hand-over to the Argonaute in an RNA-induced silencing complex process, and finally, one strand discard and the other was chosen to become mature miRNA .
We evaluated expression level of miR-30a, miR-206, and miR-18a in PE cases decidua in the current study. Our results showed decreased expression level of miR-18a in PE cases. MiR-18a regulates HIF-1α and SMAD-2 in positive and negative manner, respectively [25, 40]. In confirmation of our results, decreased levels of miR-18a in PE cases have been reported in several studies [40–42]. As result of miR-18a immune system related positive target, our group observed decreased levels of SMAD-2 and TGF-β in PE patients. SMAD-2 is a signal transduction molecule which is involved in TGF-β signaling pathway . In a study was don Xu and coworkers , they found a reduction of miR-18a contributes to PE by down-regulating Smad-2 and reducing TGF-b Signaling. Zhu was found that promotion of apoptosis in human trophoblast cells and inhibited invasion of trophoblast occurs by targeting the ESRα gene via miR-18a suppression . We reported increased expression of miR-30a and miR-206 in the decidua of preeclamptic cases. Also, our study showed decreased levels of serum IGF-1 as a common target of miR-30a and miR-206 in blood samples of PE patients. Several studies revealed decreased increased expression of miR-30a and miR-206 in PE and their regulatory effect on IGF-1 levels in these patients [43–45]. In a study which is done by Akehurst et al. , up and down-regulation of miR-206 and IGF-1 was seen in PE cases maternal circulations and placental tissue. They reported that mir-206 can be introduced a novel PE factor which is up-regulated during pregnancy of these patients.
One of the weaknesses of our study is the small sample population, which returns to the difficulty of sampling decidua tissue. Another weakness of this study was the lack of blood sampling at different times of the pregnancy process in PE patients, which could identify the changes in the blood variables we studied during pregnancy.
In conclusion, PE is a multifactorial pregnancy disorder with physiological, environmental, immunological, genetic, and anatomical aspects. It's difficult to know the exact etiology of PE because of such a variety of signs. We examined molecules that are involved in ATP catabolism and hypoxia as the main elements of hypertension which is the most dominant sign of PE. Our results showed that differential expressions of miR-18a, miR-30, miR-206, and their target genes could be considered as PE factors. These molecules are a few from a large scale of targets which is need to survey in future studies to find PE exact etiology.