In the baboon model, the first detectable molecular response to CG in uterine stromal cells is an increase in the expression of α-SMA in the subepithelial region in early gestation after implantation and after CG infusion, indicating that remodeling of the stromal cell cytoskeleton is necessary for decidualization of endometrial stromal cells (Fazleabas et al. 1999a; Jones and Fazleabas 2001; Strakova et al. 2005). This stimulation has been attributed to binding of ECM proteins to integrin heterodimers on stromal cells (Fazleabas et al. 1997a). The remodeling of the cytoskeleton of stromal cells is essential for their differentiation. In vivo, in the absence of α-SMA, endometrial stromal cells in baboons are not able to predecidualize until induced by CG signaling from the implanting blastocyst or infusion of CG into the uterine lumen (Enders 1991; Kim et al. 1999a; Ramsey et al. 1976). However, these stromal cells can be induced to decidualize by cAMP and steroid hormones since they induce expression of α-SMA during in vitro culture (Kim et al. 1998). Furthermore, disruption of actin filaments by cytochalasin D sensitizes the cultured baboon endometrial stromal cells response to inducers of decidualization characterized by expression of IGFBP1 within 24 h after treatment (Kim et al. 1999b) compared to 6 days under standard conditions (Kim et al. 1998). Stromal cells isolated from the endometrium primed by embryonic or infused CG in vivo exhibit the decidualization response in vitro as rapidly as cytochalasin D-sensitized stromal cells (Kim et al. 1999b), indicating the importance of CG to initiate decidualization of endometrial stromal cells. Expression of the decidualization marker IGFBP1 is also regulated by the conceptus and CG (Fazleabas et al. 1997b). In early gestation (day 28), LHCGR is expressed by stromal cells around spiral arteries where decidualization is initiated, indicating that those cells are a direct target of CG to induce initiation of decidualization (Cameo et al. 2006). Responses of endometrial stromal cells to CG during decidualization are summarized in Fig. 10.1b.

Full differentiation of endometrial stromal cells requires a decrease in abundance of α-SMA to allow for an increase in the expression of IGFBP1. In vivo, α-SMA disappears between days 32 and 40 of pregnancy, which is the time when expression of IGFBP1 is detectable (Christensen et al. 1995; Strakova et al. 2005; Tarantino et al. 1992). In vitro, an increase in IGFBP1 is associated with the decrease in expression of α-SMA (Kim et al. 1998, 1999b). This decrease in α-SMA is also associated with a decrease in expression of LHCGR in later stages of gestation (days 40–50) and at the completion of in vitro decidualization (Cameo et al. 2006). Collectively, the embryonic signal CG first induces expression of α-SMA to promote remodeling of the cytoskeleton of stromal cells and differentiation of endometrial stromal cells around spiral arteries via its membrane receptor. Subsequently the decrease in CG signaling appears to be necessary for the completion of decidualization. Our recent studies demonstrated that NOTCH1, a membrane receptor of Notch signaling, may mediate CG-regulated decidualization (Afshar et al. 2012; Su et al. 2015).

CG is believed to rescue the endometrium from its apoptosis cascade, which usually occurs at the end of each menstrual cycle, and direct it toward a decidualization response. CG inhibits this apoptotic fate of endometrial cells (Lovely et al. 2005) and, with ovarian hormones, differentiates them into the decidualized phenotype (Jasinska et al. 2006). CG prevents apoptosis by inducing anti-apoptosis genes like BCL-2 (Jasinska et al. 2006).

Notch signaling is a highly conserved pathway across most multicellular organisms. It plays an important role in cell-cell communication and mediates cell fates such as proliferation, differentiation, and apoptosis (Rizzo et al. 2008). Notch signaling is associated with four transmembrane receptors (Notch 1–4) and five transmembrane ligands of the jagged/delta-like families (Afshar et al. 2012). Activation of Notch signaling is generally initiated by interactions between adjacent cells expressing receptor and ligand. This results in a series of receptor-mediated cleavage events and the release of the Notch intracellular domain (NICD) which translocates to the nucleus where it binds and activates the Notch family transcription factor, recombination signal binding protein Jκ (RBP-Jκ). RBP-Jκ then initiates the expression of Notch target genes, such as the “hairy enhancer of split” (Hes) and Hes-related (Hey) transcription factor families (Su et al. 2015).

Notch signaling mediates cellular processes that are essential for successful decidualization. Expression of NOTCH1 and its target α-SMA are both induced by CG in baboon endometrial stromal cells in vivo. Silencing of NOTCH1 in human uterine fibroblast (HuF) cells cultured in vitro leads to the impairment of decidualization, suggesting that NOTCH1-α-SMA mediates CG function in rescuing endometrial stromal cells from apoptosis and differentiating them to decidual cells (Afshar et al. 2012). In vivo CG infusion upregulates expression of both NOTCH1 and α-SMA in human endometrium, which further supports our findings from studies of the baboon model (M.R. Strug and A.T. Fazleabas, unpublished data). Furthermore, activation of Notch signaling is regulated by progesterone which indicates a significant interaction between the CG-Notch pathway and progesterone signaling during decidualization (Afshar et al. 2012). Additional studies have shown that silencing NOTCH1 in HuF cells inhibits decidualization only during the initiation of the differentiation process. To inhibit decidualization of HuF cells in vitro, NOTCH1 must be silenced before the induction of decidualization, whereas silencing NOTCH1 three days after induction of decidualization does not inhibit the expression of decidualization markers (Su et al. 2015). Furthermore, comparative microarray analysis indicated that Forkhead box protein O1 (FOXO1) is a downstream target of NOTCH1 during in vitro decidualization, since FOXO1 and its specific target genes are downregulated when NOTCH1 is silenced during in vitro decidualization (Su et al. 2015). FOXO1 is one of the earliest genes induced during decidualization (Brar et al. 2001; Christian et al. 2002b). A number of in vitro experiments conclusively demonstrated the importance of FOXO1 for the induction of decidualization (Buzzio et al. 2006; Grinius et al. 2006; Labied et al. 2006). Overexpression of FOXO1 in human endometrial stromal cells can induce expression of IGFBP1 and PRL independent of cAMP and hormones induced in vitro decidualization (Buzzio et al. 2006; Christian et al. 2002b; Kim et al. 2005; Takano et al. 2007). A recent study demonstrated that FOXO1 is functionally required for the binding of PGR to genomic targets during decidualization (Vasquez et al. 2015). These results demonstrate that NOTCH1 acts downstream of CG and plays a critical role during the decidualization response of endometrial stromal cells by regulating the expression of its targets, α-SMA and FOXO1 (Fig. 10.1b).

On the other hand, NOTCH1 is downregulated at the completion of decidualization (Afshar et al. 2012) similar to α-SMA and LHCGR expression, which is necessary for the induction of IGFBP1 (Cameo et al. 2006; Kim et al. 1998). Results of our most recent studies indicated that constitutively active Notch signaling by overexpressing the NOTCH1 intracellular domain (N1ICD) prevents HuF cells from undergoing decidualization (R. Su and A.T. Fazleabas, unpublished data). The necessity for the decrease in NOTCH1 expression for the completion of decidualization may be because decidualization depends on cAMP stimulation, sustained PKA activity, and CREB activation (Gellersen and Brosens 2014; Kusama et al. 2014), but N1ICD sequesters nuclear CREB and inhibits cAMP/PKA mediated signaling (Hallaq et al. 2015).

In summary, NOTCH1 initially mediates a survival signal in the uterine endometrium in response to CG from the implanting blastocyst together with progesterone, so that menstrual sloughing is averted. Subsequently, NOTCH1 downregulation may be critical for the complete transition of stromal fibroblasts to decidual cells, which is essential for the establishment of a successful pregnancy.

Implantation has been characterized as an inflammatory response, and IL-1β is a key regulator of this response (Fazleabas et al. 2004). Cytotrophoblast cells isolated from first trimester placentae release more IL-1β then those from second and third trimester in culture (Librach et al. 1994), and co-culture of cytotrophoblast cells with HuF cells induces them to decidualize (Jasinska et al. 2004). Numerous studies have provided evidence of the importance of IL-1β as an embryonic signal that affects endometrial responses in primates.

Our laboratory previously reported that IL-1β induces expression of COX2 and PGE2 synthesis in human and baboon endometrial stromal cells (Strakova et al. 2000), which is believed to subsequently increase cAMP in stromal cells and induce decidualization (Fazleabas et al. 2004). Indeed, induced expression of the decidualization marker IGFBP1 by IL-1β in the presence of steroid hormones is blocked by COX-2 inhibitor in human and baboon endometrial stromal cells (Strakova et al. 2000). Induction of IGFBP1 expression by cAMP with steroid hormones is not affected by inhibition of COX-2 which supports the inference that cAMP acts downstream of IL-1β-COX2-PGE2 signaling during decidualization. Interestingly, cAMP prevents decidualization induced by IL-1β which suggests a negative cross-talk between IL-β- and cAMP-induced decidualization responses (Strakova et al. 2000, 2002).

The remodeling of the cytoskeleton of stromal cells is essential to the differentiation of stromal cells (Kim et al. 1998). Cytoskeleton changes can be induced by the disruption of the ECM (Fazleabas et al. 1997a). In baboon endometrial stromal cells, IL-1β induces expression and synthesis of MMP3, which can degrade the ECM (Strakova et al. 2003). This induction of MMP3 is regulated via the MAPK pathway and is critical for decidualization. Inhibition of MMP3, using doxycycline or specific MMP-3 inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl) glycyl hydroxamic acid (NNGE), suppresses the induction of decidualization by IL-1β and hormones (relaxin, estradiol-17β, and medroxyprogesterone acetate) (Strakova et al. 2003). The expression of MMP3 and degradation of ECM may contribute to the decrease in expression of the cytoskeleton protein α-SMA that is induced by IL-1β and hormones during decidualization (Strakova et al. 2000). Additionally, expression of IGFBP1 can be induced in stromal cells close to the apical surface by in vivo infusion of IL-1β in the presence of CG which further supports a role for IL-1β from the blastocyst in regulating decidualization of endometrial stromal cells during implantation (Strakova et al. 2005). The postulated roles of IL-1β during decidualization are summarized in Fig. 10.1.

Successful implantation and decidualization are necessary for providing required maternal support and protection of the developing conceptus. Human reproduction is highly inefficient compared to other primates. In women the average chance of pregnancy is only 15 % per cycle during their reproductive lifespan (Hjollund et al. 2000). In ART, only around 25 % of transferred embryos will successfully implant (Edwards 2006). Understanding the processes and mechanisms required for implantation and the establishment of pregnancy can help improve outcomes of ART. In support of results of studies on the role of CG in modulating the receptive endometrium as discussed in Sect. 10.4, intrauterine injection of CG before embryo transfer significantly improves implantation and pregnancy rates following ART (Mansour et al. 2011). Thus, an understanding of endometrial response to embryonic cells in primates may provide insight into improving pregnancy rates in women who are infertile.