**10. Molecular Regulation of Decidualization**

Proper decidualization is a critical determinant of pregnancy success. The endometrium must be optimally primed prior to and shortly after the arrival of the blastocyst (Figure 2). In addition to the aforementioned maternal hormonal, metabolic, and immunological factors, a number of other local factors are involved (discussed below). Some of these local factors are of embryonic origin, such as lactate, relaxin, CRH, and hCG [57–59,80–83], although a detailed discussion of these embryo-derived factors in perpetuating the process of decidualization is beyond the scope of this review.

During decidualization, differentiating ESCs carry a molecular signature of mesenchymal–epithelial transition (MET) as they are reprogrammed to become DSCs with widespread changes in gene expression, including the induction of such genes as HOXA10, HOXA11, FOXO1, WNT4, IGFBP1, and prolactin (PRL) [84–87]. Many of these are known upstream regulators of genes critical for implantation and placental development [17,18]. The signal transduction pathways involved in the genetic reprogramming and terminal differentiation of ESCs into DSCs (summarized in Figure 3) can be classified into several categories:

• Genomic progesterone signaling pathways mediated by the nuclear progesterone receptor (nPGR). nPGR is the dominant member of the 3-ketosteroid nuclear receptor family that responds to progesterone and cyclic AMP/protein kinase A (cAMP/PKA) signaling during decidualization [88,89]. A recent study that employed both RNA-sequencing and PGR chromatin-immunoprecipitation (ChIP)-sequencing of endometrium during the window of implantation showed that the PGR signaling network is made up of multiple different classical signaling pathways and involves numerous downstream regulators [90], including Indian hedgehog (IHH) [91], heart and neural crest derivatives-expressed (HAND2) [92], transcription factors Forkhead Box O1 (FOXO1) [93], SPR-related HMG-box gene 17 (SOX17) [94] and signal transducers and activators of transcription (STAT) transcription factor members (STAT1, STAT3, STAT5) [95], Notch signaling [96], insulin receptor substrate 2 (IRS2) [97], BMP2 and WNT signaling [72], HOXA10 [98], CCAAT/enhancer-binding protein β (CEBPB) [99], EGFR [100], mammalian target of rapamycin complex 1 (MTORC1) [101], and the tumor necrosis factor alpha-nuclear factor kappa-light-chain-enhancer of activated B cells' (TNFα/NFκβ) pathway [102]. These pathways play an important role in the embryo–uterine, epithelial–stromal, and stromal–immune cell crosstalk that occurs in the peri-implantation period and is responsible for such functions as EMT, insulin resistance, focal adhesion, trophoblast invasion, regulation of the complement and coagulation cascade, cytokine-cytokine receptor interactions, xenobiotics metabolism, inflammatory response, ECM receptor interaction, angiogenesis and vasculature development, apoptosis, cytoskeleton remodeling, and the secretion of glycogen and other decidualization markers, such as PRL and insulin-like binding factor (IGFBP1). In a proteome and secretome screening study of in vitro decidualized ESCs, Garrido-Gomez et al. [103] reported that, in addition to PRL and IGFBP1, a number of other secreted decidualization markers might be involved in the attendant angiogenesis, including platelet/endothelial cell adhesion molecule-1 (PECAM-1) and myeloid progenitor inhibitory factor-1 (MPIF-1). In another study of 23 secreted factors derived from primary ESCs prior to ART, coordinated and synchronized changes in the secretome were associated with successful implantation, whereas cultures from the failed implantation group typically demonstrated a disordered secretome profile [104].

**Figure 3.** Signal transduction pathways involved in the genetic reprogramming and terminal differentiation of endometrial stromal fibroblast cells (ESCs) to decidual stromal fibroblast cells (DSCs). **Figure 3.** Signal transduction pathways involved in the genetic reprogramming and terminal differentiation of endometrial stromal fibroblast cells (ESCs) to decidual stromal fibroblast cells (DSCs).


decidualization of ESCs [63,64]. COX2 in turn activates uterine peroxisome

proliferator-activated receptor-delta (PPAR-δ) and retinoid X receptor (RXR), which are critical regulators of decidualization and implantation [116]. Omega-3 polyunsaturated fatty acids have been shown in numerous animal and clinical studies to be beneficial for pregnancy outcome [117]. The receptor GPR120, a member of the rhodopsin family of G protein-coupled receptors, mediates potent anti-inflammatory and insulin- sensitizing effects [118]. Huang et al. showed that GPR120 could promote decidualization by upregulating FOXO1 and glucose transporter-1 (GLUT1) expression, glucose uptake, and pentose-phosphate pathway activation in ESCs [119].

Decidualization results in vascular remodeling with fluctuations in oxygen tension and the generation of reactive oxygen species (ROS). DSCs are programmed to resist a range of cellular stress signals to maintain the integrity of the feto–maternal interface and survival of the conceptus. Several molecular mechanisms have been implicated, including the inhibition of stress pathways such as c-Jun N-terminal kinase [120], attenuated inositol trisphosphate signaling [121], resistance to microRNA-mediated gene silencing [122], and the upregulation of free radical scavengers [123]. Another implicated pathway involves O-GlcNAcylation, a post-translational modification that links glucose sensing to cellular stress resistance. Muter et al. reported that the upregulation of the glycosyltransferase enzyme, EGF domain-specific O-linked N-acetylglucosamine transferase (EOGT), in decidualizing ESCs is responsible for the N-acetyl-glucosamine modification of a number of secreted and membrane-associated proteins involved in glucose and fatty acid metabolism [124]. Finally, in a uterine-specific *p53*-ablation PTB mouse model, decreased mitochondrial β-oxidation and ATP-production led to changes in lipid signaling and premature senescence of the decidua with subsequent PTB and/or stillbirth [125,126]. The inhibition of mTORC1 activity using rapamycin in this *p53*−/<sup>−</sup> murine model attenuated the premature decidual senescence and rescued the PTB phenotype [127].

• MicroRNA (miRNA) and epigenetic regulation. Using the miRNA profiling of ESC primary cultures before and after in vitro decidualization, Estella et al. reported an upregulation of 26 miRNAs and the downregulation of miR-96, miR-135b, miR-181 and miR-183 [128]. The addition of miR-96 and miR-135b in decidualizing ESCs decreased the expression of FOXO1 and HOXA10 as well as IGFBP-1 secretion [128]. In another study, Jimenez et al. reported that the upregulation of the miR-200 family during in vitro decidualization of ESCs correlated with the downregulation of IHH signaling and expression of the EMT regulator, ZEB1 [129]. Similar studies have demonstrated the functional importance also of miR-181a [130], miR-542-3p [131], and miR-194-3p [132] in decidualization. While individual miRNAs can regulate a range of target genes, there is growing evidence that endometrial cells undergo genome-wide chromatin remodeling for the access of transcription factors or epigenetic modifiers during decidualization [133,134]. In particular, the expression of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) appears to be reduced in endometrium beginning in the mid-secretory phase of the menstrual cycle and specifically in decidualizing ESCs [135]. The knockdown of *Ezh2* in decidualizing human ESCs resulted in reduced levels of trimethylated lysine 27 of histone 3 (H3K27me3), a repressive histone mark for silenced genes, in the proximal promoter regions of the *PRL* and *IGFBP1* genes, with a reciprocal enhancement of histone acetylation and concomitant higher expression of these two gene products [136]. A recent combined H3K27me3 ChIP-Seq and RNA-Seq analysis of mouse decidual cells harvested at different gestation stages confirmed the H3K27me3-induced transcriptional silencing of target genes that specifically suppress inflammation and contractile function in early gestation. In late gestation, genome-wide H3K27me3 demethylation was observed, thereby allowing de-repression and target gene upregulation to lead to the onset of labor [136]. Moreover, the pharmacological inhibition of H23K27 demethylation was able to inhibit labor and delivery while maintaining pup viability in a PTB murine model [136], thereby demonstrating the functional importance of this molecular mechanism. These data are consistent with the hypothesis that parturition in humans is nothing more than a delayed menstruation [11]. Although intriguing,

it should be noted that the function of EZH2 and genome-wide chromatin remodeling in the process of human decidualization and implantation remains unclear. Additional studies are needed to further investigate these epigenetic regulatory mechanisms within the various uterine compartments and their association with pregnancy outcome.
