*2.2. Invasive Pathway*

Numerous daughter villi arise from tertiary villi, some of which extend to the maternal tissue and are called anchoring or stem villi [22–24]. The anchoring sites can be established as early as the second *2.2. Invasive Pathway* 

week of gestation [22]. At proximal ends of anchoring villi, some highly proliferating CTBs break free of the overlying STB layer and invade into maternal endometrium and myometrium [24]. As soon as the detached CTBs make contact with decidual extracellular matrix, they differentiate into interstitial extravillous trophoblast cells (iEVTs) [25]. The iEVTs reach the vascular lumen and differentiate into endovascular extravillous trophoblast cells (enEVTs) [26,27]. The enEVTs remodel the spiral arteries which includes loss of endothelial and smooth muscle cells from arterial walls and their replacement by invasive enEVTs, loss of elasticity, dilation of the arterial lumen, and loss of maternal vasomotor control on the remodeled blood vessels [19,27–29]. second week of gestation [22]. At proximal ends of anchoring villi, some highly proliferating CTBs break free of the overlying STB layer and invade into maternal endometrium and myometrium [24]. As soon as the detached CTBs make contact with decidual extracellular matrix, they differentiate into interstitial extravillous trophoblast cells (iEVTs) [25]. The iEVTs reach the vascular lumen and differentiate into endovascular extravillous trophoblast cells (enEVTs) [26,27]. The enEVTs remodel the spiral arteries which includes loss of endothelial and smooth muscle cells from arterial walls and their replacement by invasive enEVTs, loss of elasticity, dilation of the arterial lumen, and loss of maternal vasomotor control on the remodeled blood vessels [19,27–29]. Spiral artery remodeling is crucial for normal placental development and supplying enough

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 3 of 20

and are called anchoring or stem villi [22–24]. The anchoring sites can be established as early as the

Spiral artery remodeling is crucial for normal placental development and supplying enough nutrients to the fetus (Figure 2). Inadequate remodeling of the spiral arteries is associated with conditions such as preeclampsia (PE), intrauterine growth restriction (IUGR)/fetal growth restriction (FGR), and recurrent miscarriage, that are harmful for both the mother and the fetus. The proliferation and differentiation of trophoblast cells continues throughout gestation. However, unlike cancerous tissues, the proliferation of trophoblast cells is strictly regulated by complex molecular pathways [30,31]. In recent studies, microRNAs (miRNAs) have been shown to play vital roles in trophoblast proliferation and early placental development. nutrients to the fetus (Figure 2). Inadequate remodeling of the spiral arteries is associated with conditions such as preeclampsia (PE), intrauterine growth restriction (IUGR)/fetal growth restriction (FGR), and recurrent miscarriage, that are harmful for both the mother and the fetus. The proliferation and differentiation of trophoblast cells continues throughout gestation. However, unlike cancerous tissues, the proliferation of trophoblast cells is strictly regulated by complex molecular pathways [30,31]. In recent studies, microRNAs (miRNAs) have been shown to play vital roles in trophoblast proliferation and early placental development.

**Figure 2.** Normal vs. abnormal spiral artery remodeling. CTBs from anchoring villi break out of the SCT layer and enter the uterine stroma where they differentiate into extravillous trophoblasts (EVTs). Spiral artery remodeling is accomplished by invasion and migration of EVTs. EVTs replace the vascular endothelial cells, remodel the spiral arteries, and ensure sufficient flow of blood to the placenta. In placenta-associated disorders like preeclampsia, reduced proliferation of CTBs results in less availability of EVTs. This leads to insufficient remodeling of spiral arteries and reduced blood **Figure 2.** Normal vs. abnormal spiral artery remodeling. CTBs from anchoring villi break out of the SCT layer and enter the uterine stroma where they differentiate into extravillous trophoblasts (EVTs). Spiral artery remodeling is accomplished by invasion and migration of EVTs. EVTs replace the vascular endothelial cells, remodel the spiral arteries, and ensure sufficient flow of blood to the placenta. In placenta-associated disorders like preeclampsia, reduced proliferation of CTBs results in less availability of EVTs. This leads to insufficient remodeling of spiral arteries and reduced blood flow to the placenta. Based on different studies listed in Table 1, a different set of miRNAs is upregulated in trophoblast cells during normal vs. preeclamptic pregnancies.

#### flow to the placenta. Based on different studies listed in Table 1, a different set of miRNAs is upregulated in trophoblast cells during normal vs. preeclamptic pregnancies. **3. Functional Analysis of microRNAs in Trophoblast Cells**

**3. Functional Analysis of microRNAs in Trophoblast Cells**  MicroRNAs are 20–25 nucleotide-long single stranded RNAs which bind 3ʹ-untranslated region (3ʹ-UTR) of the target mRNA causing its degradation or translational repression [32–36]. MicroRNAs have been shown to play important roles in deciding the fate of trophoblast cells [37]. Proliferation, MicroRNAs are 20–25 nucleotide-long single stranded RNAs which bind 30 -untranslated region (30 -UTR) of the target mRNA causing its degradation or translational repression [32–36]. MicroRNAs have been shown to play important roles in deciding the fate of trophoblast cells [37]. Proliferation, invasion, and migration of trophoblast cells are critical steps during early human placental development. With increasing evidence for the role of miRNAs in regulation of genes associated with cell proliferation, invasion, and migration, several studies have been conducted to investigate the role of miRNAs in placental development and pathogenesis of placenta-associated disorders.


**Table 1.** Gene regulation by miRNAs in trophoblast cells.


**Table 1.** *Cont.*

miRNAs impose their effect by regulating the expression of different genes and the effect of a specific miRNA on the phenotype of a cell or tissue depends upon the role of genes targeted by that miRNA. Hence, depending upon the function of their target genes in trophoblast cells, some miRNAs support successful placental development by promoting trophoblast cell proliferation, invasion and migration, and inhibiting the apoptosis of trophoblast cells, whereas some miRNAs can lead to abnormal placental development by reducing cell proliferation, invasion and migration, and increasing apoptosis of trophoblast cells. Table 1 describes the genes regulated by miRNAs and their effect on functionality of trophoblast cells as described in some recent studies. All gene symbols used in Table 1 are according to the Human Genome Organization (HUGO) Gene Nomenclature.
