Endometrial responses to embryonic signals in the primate

Abstract

The delicate interaction between an embryo and the uterus to initiate implantation and maintain pregnancy is one of the most elegant and fascinating interactions in human biology. Understanding the molecular events of embryo-maternal interaction is of interest to reproductive biologists, clinicians and couples affected by infertility. We have established the baboon as the non-human primate model for studying embryo implantation. Infusion of chorionic gonadotropin (CG), the major embryonic signal of primates, into the uterine cavity of normal cycling baboons during the window of receptivity induces a myriad of morphological, biochemical and molecular changes in the estrogen and progesterone primed endometrium. The luminal epithelium responds by forming plaques, the overall secretory function of the glandular epithelium increases and the stromal response is characterized by induction of α-smooth muscle actin (αSMA). Cross talk between ovarian and embryonic hormones is evidenced by the fact that these responses are inhibited upon treatment with a progesterone receptor antagonist. CG signals principally through the seven transmembrane LH/CG G-protein coupled receptor, and activates a mitogen activated protein kinase pathway in the endometrial epithelium that is unique and independent of all the classical signaling pathways. In the stromal compartment, CG both rescues stromal fibroblasts from their apoptotic demise and also differentiates them into the decidualized phenotype. We propose that stromal cell survival and differentiation is mediated by a critical modulator of cell fate, Notch-1. Thus, CG is an important embryonic signal which modulates communication between the embryo and the endometrium and induces changes that are critical to successful implantation.

Implantation

One of the most elegant and fascinating interactions in human physiology is the one that takes place between an embryo and the uterus to initiate and maintain the process of implantation. However, the relative inefficiency of this process in human reproduction remains to a great degree, unexplained. Fecundity (the probability of conception during one menstrual cycle) is recorded at about 30 percent and over 75 percent of the failed pregnancies are attributed to implantation defects (Wilcox et al., 1999). Thus failed implantation is a major cause of failure of both normal pregnancies as well as those resulting from assisted reproductive techniques (Margalioth et al., 2006). As a result, understanding the molecular events of embryo-maternal interaction has long been of interest to reproductive biologists, clinicians and couples affected by infertility. However, it is not possible, for both practical and ethical reasons, to study the physiological process of implantation in women. Studies in other animal models e.g. mice and rats have distinct advantages (genetic modification, cost, maintenance) but they remain essentially limited in their ability to elucidate the physiological mechanisms of human implantation. In contrast, non-human primate models have menstrual cycles that are similar to humans and comparable hormonal profiles, making them the best possible model for comparative studies of human implantation.

Across a multitude of species ranging from the rodent to the primate, the period of time for the initiation of implantation is very limited. This short window of time is aptly termed the “window of uterine receptivity”. Limitations to this time period are set by the degree of maturation of the embryo, the development of the endometrial lining and a mutual signal that confirms the initiation of implantation. Broadly, the process of embryo implantation involves a coordination of two simultaneous processes: development of the newly formed embryo and the maturation of the uterine endometrium. The embryo, entering the uterine cavity a few days after fertilization, begins an intimate dialog with the endometrium in order to ensure its own survival and development. On the maternal end, upon recognizing a cycle involving conception, the endometrium, one of the most dynamic entities in the body, alters its normal course of sloughing and regeneration, and prepares itself to receive this newly formed embryo. These two processes are inseparable and successful implantation requires a precise coordination brought about by an eloquent interplay between the embryo, ovary and the endometrium.

Embryonic signals

The initial identification of a gonad stimulating substance produced by an embryo dates back to the pioneering work by Aschheim and Zondek in the 1920s (Aschheim and Zondek, 1928). These discoveries led to the establishment of the first pregnancy test for women. Subsequently, this substance was identified, purified and characterized as chorionic gonadotropin (Bahl, 1969).

Fertilization in primates, as in most species occurs in the oviduct. Around the sixth day following fertilization, the developing embryo (called a blastocyst at this stage) hatches from its shell, the zona pellucida, prior to its attachment to the uterine epithelium. Trophoblast and syncytiotrophoblast cells of the blastocyst produce various hormones and cytokines e.g. chorionic gonadotropin (CG), interleukin -1 and insulin- like growth factor II, displaying profound effects on the endometrium (Cameo et al., 2004; Fazleabas et al., 2004; Strakova et al., 2005). In primates, the first indication of the presence of a viable embryo is the detection of CG in the peripheral circulation and maternal urine; this event occurs even when the blasocyst is still free-floating in the uterine cavity in its pre-implantation stage. Subunits of CG are reportedly transcribed by 8-cell embryos (Bonduelle et al., 1988), and the fully functional bio –active hormone is detectable in serum and urine as early as day 9 following the lutenizing hormone (LH) surge (Lohstroh et al., 2005).

CG is a member of the glycoprotein family of hormones, which includes the pituitary hormones lutenizing hormone, follicle stimulating hormone and thyroid stimulating hormone. These hormones are heterodimers comprising a common α- subunit and a species specific β- subunit. The CGβ subunit shares extensive homology with the LHβ subunit (Bo and Boime, 1992; Pierce and Parsons, 1981). Synthesis of CG occurs in distinct stages: the cytotrophoblasts produce the CGα subunit, and following multiplication and differentiation of the cytotrophoblasts to form the syncytiotrophoblasts, the latter produce the CGβ subunit (Muyan and Boime, 1997).

Other embryonic signals include growth factors such as the epidermal growth factor, which plays a role in trophoblast differentiation and invasion (Bass et al., 1994; Dakour et al., 1999) and cytokines such as interleukin -1, which along with CG is considered to be one of the earliest signals from the embryo and modulates communication between the embryo and the maternal endometrium (Simon et al., 1997; Strakova et al., 2000; Strakova et al., 2005; Vigano et al., 2003). However, in this review we shall focus on the effect of CG as the primary embryonic signal and discuss the responses of the primate endometrium to CG.

The endometrium is a dynamic entity which, under the control of ovarian hormones – estrogen and progesterone, alters itself in a cyclic pattern between phases of proliferation, secretion, attrition and regeneration. The endometrial lining is composed of a transient superficial functional layer and a germinal or basal layer. The luminal epithelium is the first maternal surface that is encountered by the implanting embryo. In a cycle involving conception, subsequent to ovulation, a critical function of the ovary is the role played by the corpus luteum in producing progesterone which can then maintain the endometrial lining and prevent it from undergoing apoptosis. Thus, one of the vital requirements for establishing pregnancy is the rescue of the corpus luteum from its impending demise and to extend the luteal production of progesterone. For a very long time the proposed role of embryonic signals such as CG was limited to this. However, in the last decade many studies have clearly demonstrated that CG exerts both autocrine effects on the trophoblast, promoting cytotrophoblast differentiation (Shi et al., 1993) and migration of extravillous trophoblasts (Zygmunt et al., 2005), and paracrine effects on the maternal ovary and the endometrium (Filicori et al., 2005).

A valuable primate model for investigating the modulation of the endometrium during pregnancy is the baboon. Uterine receptivity in these animals can be demarcated into three distinct phases. Phase I, regulated by the ovarian hormones – estrogen and progesterone, is distinguished by the presence of columnar epithelium with microvilli and an increase in stromal cell proliferation. Biochemical changes such as an increase in smooth muscle myosin and a decrease in levels of Muc1 are characteristics of this phase (Hild-Petito et al., 1996). Phase II is characterized as the ‘blastocyst response phase’ and features changes induced by the embryonic signals viz. CG, superimposed on the hormonally primed endometrium. The final phase, Phase III encompasses the responses following attachment and implantation, which includes decidualization of endometrial stromal cells.

In our initial studies in establishing the baboon as a non-human primate model for studying human implantation (Fazleabas et al., 1999), we extensively investigated the changes in Phase II of uterine receptivity. Upon infusion of CG into the oviduct of normal cycling animals in a manner that mimics blastocyst transit between days 6 and 10 postovulation, we observed dramatic morphological and biochemical changes in all of the different cellular components of the uterine endometrium. The luminal epithelium responded by forming plaques (a distinctive early maternal response to pregnancy), characterized by hypertrophy of the epithelium and rounding up of the cells to form acinar clusters (Tarara et al., 1987). The overall secretory function of the glandular epithelium was increased, with glycodelin being the major secretory product and the stromal response was characterized by induction of α - smooth muscle actin (αSMA). Evidence of cross talk between ovarian and embryonic hormones was established by the fact that the responses were inhibited upon long term treatment with a progesterone receptor antagonist (Banaszak et al., 2000).

Molecular biology of the LHCGR

CG signals principally through the same seven transmembrane G-protein coupled receptor as LH – the LH-CG receptor (LHCGR) (Mcfarland et al., 1989). In fact, CG binds with a greater affinity to the LHCGR as compared to LH. The LHCGR is an 880kb gene comprising 10 introns and 11 exons. The greater part of the receptor including the entire carboxyl terminal consisting of the seven transmembrane helices, the three interconnecting extracellular and intracellular loops and the cytoplasmic tail is encoded by the 11^th exon. The first ten exons only comprise the extracellular N-terminal exodomain consisting of numerous leucine-rich repeats involved in protein-protein interactions.

The LHCGR is shown to be present and to play diverse roles in various tissues of reproductive (gonadal and extra-gonadal) and non-reproductive origin (Apaja et al., 2005; Pakarainen et al., 2007; Segaloff and Ascoli, 1993). Studies both in vivo (Fazleabas et al., 1999) and in vitro (Zhou et al., 1999) have also confirmed that the LHCGR receptor is present in the baboon and human endometrium. These studies have also demonstrated that this receptor is functional and regulates biological processes related to implantation. Although much work has been done to understand the regulation and expression of the LHCGR in the endometrium, a large number of questions still remain unanswered. For example, in the human, the full-length LHCGR is expressed in the endometrium during both the proliferative and secretory phases of the cycle (Reshef et al., 1990). However, during early pregnancy, the decidua expresses only a truncated form of the receptor (Licht et al., 2003). In the baboon, we have shown that the LHCGR was absent in the endometrium during the proliferative phase, while in the secretory phase, both the luminal and the glandular epithelial cells expressed the receptor. With the onset of pregnancy, the expression of the LHCGR was limited to the stromal cells surrounding the spiral arteries. This expression persisted until day 25 of pregnancy and subsequently decreased between days 40–60 as pregnancy progressed. This decrease, together with the truncated form of the receptor, was comparable to that reported in the humans (Licht et al., 2003). We confirmed these results in vitro by demonstrating that decidualization of endometrial stromal cells was associated with a decrease in LHCGR expression (Cameo et al., 2006). The downregulation of receptor levels during pregnancy and in decidual cells coincided with the period of decreased levels of CG in the peripheral serum of pregnant baboons (Fortman et al., 1993). Additionally, we showed that CG was capable of regulating the levels of its own receptor and treatment of decidualized cells with CG restored LHCGR expression (Cameo et al., 2006). Receptor levels in the endometrium however showed a decrease upon treatment with cAMP, as previously shown in gonadal cells in vitro (Menon et al., 2004). Furthermore, rescue of the LHCGR by CG was not possible in the presence of cAMP, indicating a cAMP independent regulation of the LHCGR. Incidentally, CG has also been shown to induce a cAMP- independent pathway in endometrial cells (Srisuparp et al., 2003). Recent studies indicate the regulation of LHCGR expression in the rat ovary by a novel protein, the LH-receptor binding protein, which utilizes a cAMP-dependent pathway to inhibit LHCGR expression (Peegel et al., 2005). Hence, decidualization, which is a cAMP dependent mechanism, may also possibly mediate the regulation of the receptor in this manner. Thus, details of the molecular mechanisms and potential cross-talk between the various regulatory components are far from clear and are open to investigation.

Endometrial responses

Using the in vivo baboon model (Fazleabas et al., 1999) we clearly elucidated the paracrine effects of CG on the endometrium, which mimicked the early maternal response to pregnancy. In order to elucidate changes in endometrial gene expression with CG, we carried out microarray analysis on whole endometrial samples taken from biopsies of CG- treated and control baboons on day 10 post- ovulation. We identified a set of novel genes that are differentially regulated in animals infused with CG as compared to controls, whose expression levels were verified using real time PCR and the presence of the protein product was confirmed using immunohistochemistry (Sherwin et al., 2006).

Among the genes significantly influenced by CG are the leukemia inhibitory factor (LIF), an interleukin 6 class cytokine, the complement component C3, the radical scavenging enzyme superoxide dismutase 2 (SOD2), the matrix metalloproteinase 7 (MMP7) and the immunomodulatory compound glycodelin, all of which were up-regulated in response to CG and the regulator of the Wnt-signaling pathway, soluble frizzled receptor protein 4 which was down-regulated in response to CG treatment.

The importance of LIF and gp130 (the signaling element of the LIF receptor) has been shown in the murine model (Stewart et al., 1992) and in other primates (Yue et al., 2000) as well as in humans (Licht et al., 2001). LIF has been recently shown to regulate extravillous trophoblast adhesion to the extracellular matrix (ECM) suggesting an important role in trophoblast invasion and early placental development (Tapia et al., 2008).

The regulation of C3 and SOD2 indicates that CG plays a role in regulating the immune system and protection against oxidative damage during pregnancy. In response to CG treatment, C3 was primarily expressed in the stromal compartment and SOD2 in the glandular epithelial compartment of the baboon endometrium in comparison to controls. C3 plays an integral role in triggering the complement pathway to promote phagocytosis, local inflammatory responses and activating the humoral response (Sahu and Lambris, 2001). The up-regulation of SOD2 by CG indicates the role played by CG in regulating oxidative stress during pregnancy.

In addition, CG up-regulated the expression of glycodelin, a protein demonstrated to have immunosuppressive functions (Bolton et al., 1987; Karande et al., 2005). Glycodelin, a progesterone regulated gene is one of the most abundant secretory glycoproteins in the primate endometrium during implantation and early pregnancy (Seppala et al., 2002) and is proposed to play a significant role in protecting the embryo from maternal immune responses. It has been shown inhibit proliferation and induce apoptosis in T cells (Mukhopadhyay et al., 2001). Apart from this, the additional regulation of the endometrial stromal protein, SCP, a potent regulator of T-cells (Tagoh et al., 1996) by CG (Lobo et al., 2001) emphasizes the immune-modulatory role of this embryonic signal.

Metalloproteinases play an important role in remodeling the ECM of the endometrium during normal cycle and also during implantation (Goffin et al., 2003). Invasion of the trophoblast into the endometrium requires a delicate balance between tissue degradation and maintenance, regulated by a stromal-epithelial interaction and a balance between ovarian and embryonic hormones. MMP-7, a metalloproteinase shown to be present in the glandular epithelium and known to degrade ECM proteins like proteoglycans, fibronectin, elastin, and casein, has been shown to be suppressed by progesterone in humans thus promoting preservation and cellular differentiation of the endometrium (Osteen et al., 2003). On the other hand, CG up-regulates the expression of MMP-7 in both baboons and humans (Rodgers et al., 1994; Sherwin et al., 2007; Yanaihara et al., 2004), thus being able to overcome the inhibitory effect of progesterone that is seen during the menstrual cycle.

Finally, CG also regulates the members of the Wnt signaling pathway, a vital regulator of embryo implantation, which controls processes including embryogenesis, cell proliferation and differentiation (Xie et al., 2008). CG up-regulated the expression of the Wnt pathway inhibitor Dickopff -1 and down-regulated levels of soluble frizzled receptor protein 4, indicating its role in maintaining a balance between the members of the Wnt pathway during implantation.

Overall, these studies revealed a direct effect of CG on the uterine endometrium and specifically the ability of CG to modulate genes related to and important for embryo attachment and endometrial remodeling for implantation.

Epithelial responses

To examine the molecular mechanisms of CG signaling in the endometrial epithelium, we initiated an in vitro study using primary baboon endometrial epithelial cells and a human endometrial epithelial cell line, HES (Srisuparp et al., 2003). Mechanisms of LHCGR action include stimulation of multiple signal transduction effector systems, including adenylyl cyclase and the cAMP pathway, one of the classical activator systems of the steroidogenic pathway in gonadal cells (Hirakawa et al., 2002; Parakh et al., 2006). Alternatively LHCGR can work via the induction of inositol phospholipid pathway involving phospholipase-C and diacylglycerol. Both these conduits lead to activation of the mitogen activated protein kinase (MAPK) pathway in various cell models (Ryu et al., 1998; Steele and Leung, 1992). Our in vitro studies indicated that CG induced a MAPK pathway in endometrial epithelial cells leading to prostaglandin E₂ (PGE₂) production. Prostaglandins have been shown to regulate the initial attachment of the embryo to the epithelium by playing a role in endometrial proliferation, differentiation and vascular permeability (Dey, 2005; Kennedy et al., 2007; Wang and Dey, 2005). The expression of cyclooxygenase – 2 (COX-2), the rate limiting step in PGE₂ synthesis, has been well characterized in the human (Marions and Danielsson, 1999) and the mouse (Chakraborty et al., 1996; Ni et al., 2002; Ni et al., 2003) uterus during pregnancy. In addition, COX-2 induction by CG has been demonstrated in human endometrial glandular epithelial cells (Zhou et al., 1999) and adenocarcinoma cells (Munir et al., 1999; Tsai et al., 2008). COX-2 is also expressed in the epithelium and the decidualizing stroma in the baboon (Kim et al., 1999) and the endometrium of the Rhesus monkey from day 20–25 of the menstrual cycle (Milne et al., 2001; Sun et al., 2004). Murine models have demonstrated that the absence of prostaglandins lead to multiple reproductive failures particularly due to implantation defects (Kennedy et al., 2007; Lim et al., 1997).

The MAPK pathway in the endometrial epithelial cells is activated independent of the classical cAMP/protein kinase A induction since CG failed to induce production of intracellular cAMP in these cells and phosphorylation of the extracellular regulatory kinase occurs independent of inhibitors of protein kinase A (Srisuparp et al., 2003). Preliminary work from our laboratory indicated that this MAPK pathway is activated in a manner distinct from the classical signaling paradigms including activation of the G_i subunit or transactivation of the epidermal growth factor receptor and instead was mediated via the activation of the phospho-inositol -3- kinase pathway (Banerjee et al., 2008). Receptor desensitization in the presence of high ligand concentrations involving the termination of the G_s mediated classical signaling has been described in gonadal cells (Hunzicker-Dunn and Maizels, 2006). In the presence of the embryo, the uterine endometrium is subjected to high levels of CG. It is probable that the endometrium maintains its function and responsiveness to the embryonic signal by silencing the canonical G_s and G_i pathways and maintaining signaling through the inositol phosphate dependent MAPK pathway. Downstream, this leads to the induction of the microsomal enzyme prostanoid synthase – prostaglandin E synthase, known to enzymatically convert the COX-2 product PGH₂ to PGE₂ (Park et al., 2006). Expression of prostaglandin E synthase has been well characterized in the human endometrium (Milne et al., 2001). Together with our previous data of CG – induced PGE₂ production in HES cells (Srisuparp et al., 2003) these studies indicate that the activation of a single continuous pathway by CG leads to prostaglandin synthesis in the endometrial epithelium.

Stromal responses

The primary effect of CG on stromal fibroblasts is the induction of αSMA (Fazleabas et al., 1999). This stimulation has been attributed to a consequence of stromal integrin binding to the secreted ECM proteins (Fazleabas et al., 1997). We reason that the induction of αSMA by CG is essential to terminate the proliferative process of the endometrial stromal cells and initiate the differentiation process, which is a key to decidualization (Kim et al., 1999). The differentiation of a stromal fibroblast to a decidualized cell with a secretory phenotype is a pre-requisite for the maintenance of pregnancy in the primate. The baboon endometrium undergoes morphological and secretory changes during pregnancy, associated with the development of a functional placenta and a decidualized endometrium (Fazleabas et al., 1993). Decidual cells play an important role by providing nutritional support to the embryo. A very well characterized marker for decidual cells of the primate uterus is the insulin-like growth factor binding protein (IGFBP-1), which is secreted selectively by decidualized stromal cells (Fazleabas et al., 1989). In both the normal and the simulated pregnant model in the baboon, IGFBP-1 expression was clearly shown to be regulated by the conceptus and embryonic signals (Fazleabas et al., 1997; Tarantino et al., 1992). Subsequently, we found that IGFBP-1 expression in vitro in decidualizing fibroblasts requires the presence of ovarian hormones and cAMP (Kim et al., 1998). Differentiation of the stromal fibroblasts to decidual cells is linked intrinsically to reorganization of the cellular cytoskeleton. IGFBP -1 induction was concurrently associated with a decrease in αSMA expression both in vivo (Christensen et al., 1995) and in vitro (Kim et al., 1998). We also showed that alterations in the cytoskeleton of stromal cells were a prerequisite for their differentiation into the decidualized phenotype and subsequent production of IGFBP-1 (Kim et al., 1999). Disruption of actin filaments in vitro in the presence of ovarian hormones and CG both rescues cells from their apoptotic demise and also differentiates them into the decidualized phenotype (Jasinska et al., 2006). Recently we have also shown that interfering with actin-myosin interactions by the phosphorylation of myosin light chain prevented decidualization of endometrial stromal cells in vitro (Ihnatovych et al., 2007). These studies all point to the importance of the CG induced cytoskeletal regulation as a requisite to the initiation of decidualization.

At the end of each menstrual cycle, the endometrium undergoes regression by the process of apoptosis. The level of apoptosis throughout the menstrual cycle has been documented and increases progressively from the proliferative phase and peaks at menses (Vaskivuo et al., 2000). In the event of conception, the embryonic signal is believed to rescue the endometrium from its apoptotic cascade and direct it to a pattern of differentiation. CG has been shown to play a major role in inhibiting this apoptotic fate of endometrial cells (Lovely et al., 2005). The importance of CG in mediating the prevention of apoptosis is emphasized by the fact that CG was capable of rescuing stromal cells from apoptosis upon treatment with a cytoskeletal disruptor. We further elaborated on the role played by CG in the inhibition of apoptosis by demonstrating the induction of anti-apoptotic genes e.g. Bcl-2, by CG. Lastly, the link between differentiation due to decidualization and inhibition of stromal cell apoptosis was re-affirmed as cells undergoing apoptosis were recued upon treatment with decidualization markers IGFBP-1 and prolactin (Jasinska et al., 2006). We currently propose that the regulation of stromal cell survival and differentiation is mediated by a critical modulator of cell fate, Notch-1 (Afshar et al., 2007). Induction of Notch-1 in stromal fibroblasts in response to CG during the window of uterine receptivity upregulates anti-apoptotic genes and induces αSMA, an important mediator of cellular differentiation. We reason therefore, that CG and Notch-1 cooperatively regulate apoptotic rescue and stromal cell differentiation, which are critical to successful pregnancy.

Clinical application and future perspectives

Even eighty years following its discovery, the role of CG in clinical and therapeutic applications continues to be underestimated. The midcycle LH surge is critical for normal oocyte maturation and ovulation and LH and CG have been used interchangeably in stimulating events such as induction of folliculogenesis, oocyte maturation and ovulation in clinical procedures (Griesinger et al., 2006). Studies from oocyte donation programs clearly indicated the possible applications of CG on the uterine response, independent of ovarian functions (Filicori et al., 2005). In women undergoing pituitary desensitization and prior preparation of the endometrium with ovarian hormones, endometrial thickness and implantation rates were significantly higher in groups receiving exogenous CG prior to embryo transfer (Tesarik et al., 2003). Additonally, luteal phase support with CG or progesterone following in vitro fertilization increased pregnancy rates (Daya and Gunby, 2004). Low-dose CG has been used to complete controlled ovarian stimulation and the use of CG is associated with a reduced number of small pre-ovulatory follicles, which could thus reduce the risk of ovarian hyperstimulation syndrome (Filicori et al., 2005). In addition, invasion promoting and angiogenic properties of CG are very well documented (Zygmunt et al., 2002) and these correlate with the fact that CG has been associated with increase in the development of fibroids (Baird et al., 2006). Furthermore, CG is of clinical importance for the diagnosis of pregnancy, monitoring of abnormal and ectopic pregnancies, testing for Down’s syndrome or monitoring therapy of CG-secreting malignancies (Lottersberger et al., 2003). However, administration of antisense chorionic gonadotropin beta gene into choriocarcinoma cells also suppressed the cell proliferation and induced apoptosis (Hamada et al., 2005). Interestingly, administration of a combination of ovarian hormones and CG to virgin animals either before or after carcinogen exposure also confers strong and lasting protection against tumor development (Guzman et al., 1999). Possible long-term protective association for breast cancer risk with elevated levels of circulating CG in the early stages of pregnancy has been reported. Women with high CG levels tended to be at lower risk of breast cancer than those with low levels (Lukanova et al., 2008). A possibility of regulation of myometrial contractivity by CG and hence a mechanism to prevent pre-term labor has also been suggested (Ticconi et al., 2006).

Thus, the early signal from the embryo, CG presents itself as a versatile molecule altering ovarian, endometrial and trophoblastic functions and mediating multiple events related to endometrial receptivity and embryo implantation. Development of molecular techniques and models to study embryonic and endometrial changes during implantation is extremely important to increase implantation rates in assisted reproductive therapies and consequently improve birth rates and fecundity.

Abbreviations used in this paper

αSMA

α-smooth muscle actin

CG

chorionic gonadotropin

COX-2

cyclooxygenase-2

ECM

extracellular matrix

IGFBP-1

insulin-like growth factor binding protein

LHCGR

lutenizing hormone-chorionic gonadotropin receptor

MAPK

mitogen activated protein kinase

PGE₂

prostaglandin E₂