Molecular Signaling Regulating Endometrium–Blastocyst Crosstalk

Implantation of the embryo into the uterine endometrium is one of the most finely-regulated processes that leads to the establishment of a successful pregnancy. A plethora of factors are released in a time-specific fashion to synchronize the differentiation program of both the embryo and the endometrium. Indeed, blastocyst implantation in the uterus occurs in a limited time frame called the “window of implantation” (WOI), during which the maternal endometrium undergoes dramatic changes, collectively called “decidualization”. Decidualization is guided not just by maternal factors (e.g., estrogen, progesterone, thyroid hormone), but also by molecules secreted by the embryo, such as chorionic gonadotropin (CG) and interleukin-1β (IL-1 β), just to cite few. Once reached the uterine cavity, the embryo orients correctly toward the uterine epithelium, interacts with specialized structures, called pinopodes, and begins the process of adhesion and invasion. All these events are guided by factors secreted by both the endometrium and the embryo, such as leukemia inhibitory factor (LIF), integrins and their ligands, adhesion molecules, Notch family members, and metalloproteinases and their inhibitors. The aim of this review is to give an overview of the factors and mechanisms regulating implantation, with a focus on those involved in the complex crosstalk between the blastocyst and the endometrium.


Introduction
Implantation requires a complex crosstalk between the endometrium and the blastocyst and is highly regulated by a variety of factors, such as soluble growth factors, hormones, prostaglandins, adhesion molecules, and the extracellular matrix (ECM) [1][2][3][4][5]. These factors, produced by the receptive endometrium in response to the presence of the blastocyst and vice versa, are able to synchronize the development of the embryo to the blastocyst stage and the differentiation of the uterus to the receptive state [6,7]. This complex network of signaling accounts for implantation being one of the major limiting steps in mammalian reproduction. Indeed, the implantation rate in humans is about 30% per cycle [8,9]. Alterations of these signaling pathways may result in pathological conditions leading to infertility.
The WHO has designated infertility as "a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse" [10,11]. cultures or endometrial derived stromal cells with progesterone induces expression of PRL [109,110], but with higher efficiency if steroid hormones are used in combination with cAMP [111,112]. cAMP alone can induce decidualization of human endometrial stromal cells (HESCs) but for few days only [113][114][115], since for the stabilization of the process it is necessary the presence of both cAMP and progesterone [112].
In addition to the steroid hormones produced by the ovary, other hormones are involved in the establishment of pregnancy, among which one of the most studied is the chorionic gonadotropin (CG). CG is produced by the trophectoderm of the blastocyst and is one of the main players involved in endometrium-embryo crosstalk at the time of implantation. The ovaries respond to CG, which acts as an agonist of LH, by maintaining the corpus luteum, thus producing the progesterone necessary for the establishment and progression of pregnancy [116]. The responses of the endometrium are multiple, including the inhibition of apoptosis, which usually occurs at the end of the menstrual cycle, by activating anti-apoptotic genes as B-cell lymphoma 2 (BCL-2) [117,118], and the induction of the decidualization process [118][119][120]. Both epithelial and stromal cells possess the LH/CG receptor (LHCGR), a seven transmembrane G protein-coupled receptor, which shows the highest expression during the secretory phase of the menstrual cycle [119,121,122]. Endometrial epithelial cells respond to CG by expressing cyclooxygenase-2 (COX2) and prostaglandin E synthase (PGES), through the activation of extracellular signal-regulated protein kinases 1/2 (Erk1/2) signaling pathway. The increased production of prostaglandin E2 (PGE2) [122][123][124] induces cAMP in endometrial stromal cells and promotes their decidualization [124,125]. COX-derived PGE2 plays an important role in the increase of endometrial vascular permeability, which characterizes the inflammatory reaction typical of implantation [126,127]. In endometrial stromal cells CG activates Erk1/2 signaling pathway, thus increasing the expression of the PR and regulating the expression of genes controlling endometrial receptivity [121]. Moreover, in primates, endometrial stromal cells respond to CG and progesterone by activating Notch receptor 1 (NOTCH1) pathway, as discussed later. NOTCH1 induces the expression of α-smooth muscle actin (α-SMA), which positively regulates remodeling of cytoskeleton and the initial changes typical of the decidualization process [128]. Subsequently, a decrease in CG and NOTCH1 levels is necessary for the completion of decidualization, which is accompanied by an increase in the expression of insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL), markers of decidualization [129][130][131], and a downregulation of LHCGR [120,[132][133][134].

Role of Pinopodes
One of the major structural changes of the endometrium during the luteal phase is the formation of apical protrusions on the epithelial cells called pinopodes (also known as uterodomes). These dome-like structures are formed in response to progesterone, but regress upon estrogen stimulation [135][136][137][138]. The function of pinopodes is not clear. Some authors suggest that pinopodes are responsible of pinocytosis and endocytosis of uterine fluid and macromolecules, which facilitates adhesion of the blastocyst to the endometrium [139,140]; others have suggested that they might be directly involved in blastocyst-endometrial interaction through the expression of adhesion molecules, such as integrins [141][142][143][144], or of LIF [145], although co-localization of these molecules and pinopodes has been questioned [146,147].
Pinopodes formation has been initially demonstrated to coincide with the WOI [137], hence their role as potential markers of endometrial receptivity was proposed [148,149]. However, this role is still currently a topic of great debate. Several studies demonstrated that pinopode are present beyond the WOI [150], questioning their utility to identify endometrial receptivity. Moreover, no major differences in the coverage and morphology of pinopodes was observed in endometrial samples from fertile women compared to those of women with recurrent pregnancy loss, suggesting no direct correlation between pinopode density/morphology and pregnancy success [151]. However, recent studies re-evaluated pinopode utility to identify endometrial receptivity, by demonstrating a strong correlation between pinopode quality and pregnancy rate [152][153][154]. These contrasting results may be explained, at least in part, by sampling variability, and lack of standardization for morphological identification and staging of the pinopodes. As recently reported, computer-assisted analysis of endometrial tissue images could be used to overcome operator subjectivity [140]. It should also be considered that absence or presence of pinopodes might not be the solely parameter to consider for endometrial receptivity, as quality and molecular content of pinopodes could also be of relevance.

Growth Factor of the EGF Family
Uterine receptivity is also regulated by members of the epidermal growth factor (EGF) family, whose expression pattern in the peri-implantation uterus has been widely investigated in murine models [155][156][157][158][159][160][161][162]. Among the EGF family members, amphiregulin (AREG) has been identified in the luminal epithelium exclusively at the site of blastocyst apposition and its expression appears to correlate initially with the increase of progesterone levels and then with the attachment reaction [157]. Similarly, the expression of heparin binding-EGF (HB-EGF), which is under the control of both estrogen and progesterone [160], requires the presence of a competent blastocyst and it occurs in the luminal epithelium when pinopodes are fully formed at the site of blastocyst apposition [155,161], while epiregulin (EREG) is expressed in both the luminal epithelium and stroma during blastocyst attachment [158]. This unique expression pattern suggests a role for AREG, HB-EGF, and EREG in uterine receptivity and subsequent embryo adhesion. The role of HB-EGF in blastocyst adhesion to the uterus has been further demonstrated in vitro in a co-culture of a mouse cell line synthesizing transmembrane human HB-EGF (TM HB-EGF) and mouse blastocysts. Cells synthesizing TM HB-EGF adhered to mouse blastocysts more than parental cells or cells synthesizing a constitutively secreted form of HB-EGF [163]. These results are confirmed by a more recent study using HB-EGF mutant mice, which demonstrates that maternal deficiency of HB-EGF limits pregnancy success [162].

NOTCH Signaling Pathway
NOTCH signaling pathway is involved in the regulation of various cellular processes such as cell proliferation, invasion, adhesion, survival, apoptosis and differentiation [164][165][166][167]. All four NOTCH receptors, the ligands Jagged1 (JAG1) and Delta-like (DLL) 4 and the target genes hairy enhancer of split (HES) and Hes-related 1 (HEY1) are known to be expressed by the endometrium [168][169][170][171]. Several ligands and receptors of the NOTCH signaling pathway are expressed in both the inner cell mass (ICM) and trophectoderm of the human blastocyst [172][173][174]. NOTCH1 plays an important role in the process of decidualization, by inducing pro-survival signals in the endometrium, thus avoiding apoptosis normally occurring at the end of the menstrual cycle. Hess et al. showed that blastocyst-conditioned medium induces an increase in the expression of NOTCH family members in decidual cells, suggesting a role for this pathway in decidualization [175]. Moreover, it has recently been shown that NOTCH signaling pathway is dysregulated in the endometrium of women with unexplained recurrent pregnancy loss [176]. Activation of NOTCH1 pathway in the endometrium is stimulated by CG and progesterone and leads to increased expression of α-SMA and Forkhead box protein O1 (FOXO1) [1,128,177]. FOXO1, in turns, induces expression of PRL and IGFBP1 and it is essential for the decidualization process [178][179][180][181][182]. NOTCH1 is involved in the inhibition of cAMP/protein kinase A (PKA) signaling pathway [183], so that NOTCH1 has to be downregulated to allow cAMP response of stromal cells. Similar to what described for α-SMA and LHCGR expression, a downregulation of NOTCH1 is necessary for the induction of IGFBP1 and the completion of decidualization [111,120,128].

Interleukin-1b in Blastocyst-Endometrium Dialogue
Interleukin (IL)-1β is another important factor supporting blastocyst-endometrium dialogue, playing a fundamental role in decidualization of stromal cells and in successful blastocyst implantation. IL-1β is secreted by cytotrophoblast cells isolated from first trimester placenta, while its expression is lower in cultures from second and third trimester placenta [184]. In endometrial stromal cells IL-1β induces the expression of COX2 and PGE2, known to increase the levels of cAMP, which are necessary for decidualization, as above described [185,186]. Moreover, in vivo infusion of IL-1β and CG promotes the expression of IGFBP1 in apical surface stromal cells [133]. It has been demonstrated that inhibition of COX2 in human and baboon endometrial stromal cells is able to block the decidualization induced by IL-1β in the presence of steroid hormones, suggesting that IL-1β acts upstream of COX2 [185]. On the contrary, inhibition of COX2 does not affect decidualization induced by cAMP and steroid hormones, suggesting that cAMP acts downstream of COX2 and PGE2 [185]. Interestingly, cAMP is able to block decidualization induced by IL-1β indicating a negative feedback between IL-1β and cAMP [185,187]. In baboon, IL-1β positively regulates the expression of matrix metalloproteinase (MMP) 3 in endometrial stroma, thus inducing degradation of the ECM. Considering that disruption of the ECM might reflect in cellular cytoskeleton remodeling, IL-1β may play an important role in the decidualization also by promoting cytoskeleton changes typical of this process [188,189]. All these data clearly indicate that IL-1β plays a relevant role in blastocyst-endometrium crosstalk.

Thyroid Hormone in Endometrial Receptivity
Endometrial receptivity is regulated also by thyroid hormone (TH). Both thyroid hormone and thyroid-stimulating hormone receptors (TR and TSHR, respectively) are expressed in the endometrium with variations during the menstrual cycle [190]. Two of the isoforms of TR, TRα1, and TRβ1, are expressed during the mid-luteal phase in glandular and luminal epithelium, showing an increase during the secretory phase, followed by a drastic decrease. Interestingly, the expression of TRα1 and TRβ1, and also of TRα2 and TSHR, in endometrial cells is concomitant to the appearance of the pinopodes and the establishment of endometrial receptivity. The expression of TRα1, TRβ1, TRα2 and also of type 2 deiodinase (DIO2) is regulated by progesterone. In fact, the administration of mifepristrone, an anti-progestinic drug that makes the endometrium unreceptive and induces menstrual bleeding, reduces the expression of TRα1 and TRα2, while it up-regulates TRβ1 and DIO2 expression, suggesting a role for progesterone in regulating molecules involved in TH synthesis and metabolism [191]. The role of TH pathway in endometrial function is also demonstrated by the observation that hypothyroidism is able to reduce uterine endometrial thickness, and also interferes with estrogenic response of the endometrium [192]. TH regulates endometrial receptivity also by acting on LIF pathway, since TSH induces increased expression of LIF and LIF receptor (LIFR) in endometrial stromal cells obtained from human endometrial samples, suggesting a major role for TSH in the implantation process [190].

Immune Cells in Implantation
A role for the immune system in embryo implantation has been widely investigated for obvious reasons. The decidua plays a fundamental role in ensuring immune tolerance toward the semi-allogenic conceptus, protecting it from the mother's immune system. Regulatory T cells (Tregs) are CD4+ CD25+ T cells, having the role to suppress the immune response [193]. During early pregnancy, in the decidua there is an increase in Tregs, which produce immunosuppressive cytokines, such as IL-10, for inducing immune tolerance [194][195][196][197]. Other cells involved in maternal immune tolerance are the uNK, a particular type of NK cells, which lose their cytotoxic functions during pregnancy andplay a supportive role by enhancing angiogenesis. uNK cells induce immune tolerance by reducing inflammation through interferon-γ (IFN-γ) [198] and by inhibiting the function of T cells through the expression of immunomodulatory molecules such as galectin-1 and glycodelin A [199].

Endometrial Receptivity Array
Recently, a customized endometrial receptivity array (ERA), containing 238 genes related to endometrial receptivity, was created [200]. These genes, differentially expressed in the receptive phase, encode for factors involved in several biological processes, such as processes related to the immune system, circulation, response to external stimulus, behavior, cell cycle, cell adhesion, anatomical structure development, cell-cell signaling, and mitotic cell cycle [200]. Beside the many above mentioned genes suggested to regulate endometrial receptivity, additional genes have been identified by ERA, highlighting the great complexity of factors regulating implantation. ERA has been suggested as a more accurate and reproducible approach to assess endometrial receptivity compared to histological analysis [201] and its use has been proposed for RIF patients [202]. Considering how critical the molecular signature of the endometrium is for embryo implantation, a test which unequivocally assess if the embryo and the uterus are in synchrony may be of great value to avoid ineffective embryo transfers. However, the utility of ERA in the clinical practice is still debated [203]. More recently, a smaller set of genes has been proposed to assess the receptivity status of the endometrium in biopsies obtained in the secretory phase [204]. It is reasonable to foresee new additional advances in this area, that is of potential great clinical utility in the management of infertile women undergoing IVF, as well as in women with RIF and unexplained RPL.

Implantation of the Competent Blastocyst
In order for a healthy pregnancy to proceed, the embryo needs to synchronize its developmental program with endometrial receptivity and to acquire the ability to implant, defined as competence. A competent blastocyst is characterized by distinctive morphological and molecular features, which are discussed in this section.

Blastocyst Transport and Orientation
After fertilization, the embryo encased in a non-anchored glycocalix, the so-called zona pellucida, which prevents ectopic implantation, descends the Fallopian tube and reaches the uterine cavity, while undergoing profound morphological changes ending in the formation of the blastocyst [205,206]. For successful implantation into the maternal tissues, a correct orientation of the blastocyst towards the uterine wall is needed. In most eutherian mammals, at the time of first contact of the blastocyst with the endometrial epithelium, the ICM of the various embryos has an almost constantly specific orientation toward the uterus. In humans, the ICM faces the uterine wall. This positioning of the ICM usually correlates with the site of trophectoderm attachment to the endometrium, as well as with subsequent development of the fetal membranes and placental structures [207,208]. In rodents, implantation occurs in anti-mesometrial position with the ICM facing the mesometrium [209]. Why, within most species, the ICM of the blastocyst, or the placenta, should be positioned consistently in the same way with respect to the uterine wall is not completely understood. Moreover, how the blastocyst becomes correctly oriented [210,211] or what directs the process has not been well clarified, for even the most commonly-studied mammals. However, it has been postulated that orientation depends on signals from the endometrium rather than from the embryo, since embryo-mimicking structures (beads, bubbles or cells) end up in the position that the embryo would occupy [212][213][214][215]. For example, in mice endometrial expression of the transcriptional regulator Rbpj is required to instruct embryo orientation, and its conditional deletion determines loss of ventral-dorsal orientation [216]. A role for endometrial glands in embryo orientation has been also proposed. Indeed, recent data indicate that in mice endometrial gland development is confined to the anti-mesometrial side of the uterus [217]. In consideration of the above discussed essential role of uterine glands in implantation, it can be speculated that glands may drive the anti-mesometrial orientation of the implanting mouse embryo, possibly through the expression of specific factors. In this respect, it has been shown that Wnt signaling activity in the mouse uterus is limited to the anti-mesometrial region and a role for Wnt proteins in anti-mesometrial localization of the implanting embryo has been proposed [217].

Blastocyst Hatching
Embedding of the blastocyst into the maternal endometrium requires hatching from the zona pellucida, which otherwise would prevent adhesion of the embryo to the uterine wall. Blastocyst hatching exposes the trophectoderm and allows the blastocyst to implant in the maternal uterus. The crucial event for blastocyst hatching is the formation of a nick into the zona pellucida, and proteases, such as serine-, cysteine-, and metallo-proteases have been proposed to play a major role in this event depending on the species [218][219][220][221][222][223]. Cathepsins, belonging to the ubiquitous cysteine proteases family [224], have been demonstrated to be involved in blastocyst hatching and zona lysis in mice; the expression of cathepsin L and P (mRNA and protein) and their natural inhibitor, Cystatin C, has been demonstrated in mouse peri-hatching blastocysts [225]; treatment of golden hamster embryos with Cystatin C is able to block blastocyst hatching [221]. The process of murine blastocyst hatching from the zona pellucida is also regulated by two mouse-specific proteinases, Strypsin (ISP1) and Lysin (ISP2). ISP1 and ISP2 are two related S1-family serine proteinases, which are tandemly localized in a cluster of tryptase genes [226,227]. The ISPs are co-expressed in the mouse preimplantation embryos and in the mouse uterine endometrium during the WOI, indicating that they could play a role in the process of blastocyst implantation [226,228]. Expression of ISP genes is positively regulated by progesterone and TH [219,223,226] and ISPs are secreted by the blastocyst and the endometrial glands into uterine fluid just prior to implantation [229]. The use of antibodies against ISP1/ISP2 abrogate murine embryo hatching and outgrowth, ascribing a crucial role for ISPs in this process [228]. This is further supported by our recent observations using mouse blastocysts cultured in the presence of TH, with or without endometrial cells used as the feeder layer. In the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching ( Figure 1) by upregulating the expression of blastocyst produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. On the contrary, in the absence of the endometrial feeder layer, TH does not affect blastocyst hatching, suggesting that TH is one of the players involved in the bidirectional crosstalk between the blastocyst and the endometrium during the WOI [223]. Human homologs of ISPs have not been so far identified, and it is possible that other proteases might be involved in blastocyst hatching in humans.
Embedding of the blastocyst into the maternal endometrium requires hatching from the zona pellucida, which otherwise would prevent adhesion of the embryo to the uterine wall. Blastocyst hatching exposes the trophectoderm and allows the blastocyst to implant in the maternal uterus. The crucial event for blastocyst hatching is the formation of a nick into the zona pellucida, and proteases, such as serine-, cysteine-, and metallo-proteases have been proposed to play a major role in this event depending on the species [218][219][220][221][222][223]. Cathepsins, belonging to the ubiquitous cysteine proteases family [224], have been demonstrated to be involved in blastocyst hatching and zona lysis in mice; the expression of cathepsin L and P (mRNA and protein) and their natural inhibitor, Cystatin C, has been demonstrated in mouse peri-hatching blastocysts [225]; treatment of golden hamster embryos with Cystatin C is able to block blastocyst hatching [221]. The process of murine blastocyst hatching from the zona pellucida is also regulated by two mouse-specific proteinases, Strypsin (ISP1) and Lysin (ISP2). ISP1 and ISP2 are two related S1-family serine proteinases, which are tandemly localized in a cluster of tryptase genes [226,227]. The ISPs are co-expressed in the mouse preimplantation embryos and in the mouse uterine endometrium during the WOI, indicating that they could play a role in the process of blastocyst implantation [226,228]. Expression of ISP genes is positively regulated by progesterone and TH [219,223,226] and ISPs are secreted by the blastocyst and the endometrial glands into uterine fluid just prior to implantation [229]. The use of antibodies against ISP1/ISP2 abrogate murine embryo hatching and outgrowth, ascribing a crucial role for ISPs in this process [228]. This is further supported by our recent observations using mouse blastocysts cultured in the presence of TH, with or without endometrial cells used as the feeder layer. In the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching ( Figure 1) by upregulating the expression of blastocyst produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. On the contrary, in the absence of the endometrial feeder layer, TH does not affect blastocyst hatching, suggesting that TH is one of the players involved in the bidirectional crosstalk between the blastocyst and the endometrium during the WOI [223]. Human homologs of ISPs have not been so far identified, and it is possible that other proteases might be involved in blastocyst hatching in humans. .

Apposition
Histological analysis of uteri of pregnant women allows to recognize three different levels of blastocyst adhesion to the uterine wall, which correspond to the three stages of blastocyst implantation ( Figure 2)

Apposition
Histological analysis of uteri of pregnant women allows to recognize three different levels of blastocyst adhesion to the uterine wall, which correspond to the three stages of blastocyst implantation ( Figure 2) [230,231].

LIF Signaling
Blastocyst apposition is the initial stage representing the first physical contact between the blastocyst and the endometrium, in which the blastocyst finds a site for implantation, guided by the maternal endometrium [232,233]. The site of implantation in the human uterus is usually in the upper and posterior part in the midsagittal plane. During blastocyst apposition, the microvilli placed on the apical surface of trophectoderm interdigitate with the pinopodes localized on the apical surface of the uterine epithelium (Figure 2A). These specialized structures support a stable binding between trophoblast and uterine epithelial cells, so that the plasma membranes of these cells are parallel and separated by a distance of 20 nm [234]. The pinopodes secrete LIF [145]. LIF is a cytokine of the IL-6 family, which in the uterus activates the Janus kinases (JAK)-signal transducer and activator of transcription protein (STAT) pathway, and therefore phosphorylates STAT3, whose activation is required for implantation [235,236]. LIF is indispensable for blastocyst implantation. Mice knockout for LIF are infertile. Although able to develop blastocysts, these mice show implantation failure; ; however successful implantation occurs in surrogate mothers [90]. In Lif-null mice the expression of  B) and invading (C) blastocyst (about 9 to 10 days after conception) and the processes and factors required for uterine receptivity and blastocyst apposition (A), adhesion (B) and invasion (C). hCG denotes human chorionic gonadotropin, LIF leukemia inhibiting factor, IL-1β interleukin-1 beta, EGF-like growth factors epidermal growth factor-like growth factors, AREG amphiregulin, EREG epiregulin, PG progesterone, COX-2 cyclooxygenase-2, PGE2 prostaglandin E2, CSF-1 colony stimulating factor-1, NOTCH1 Notch receptor 1, OPN osteopontin, MUC-1 mucin-1, MMPs metalloproteinases, EGFL7 epidermal growth factor-like domain 7, MAPK mitogen activated protein kinase, AKT protein kinase B, PA plasminogen activator, TGFβ transforming growth factor beta, TIMPs tissue inhibitor of metalloproteinases, PAI-1 plasminogen activator inhibitor-1.

LIF Signaling
Blastocyst apposition is the initial stage representing the first physical contact between the blastocyst and the endometrium, in which the blastocyst finds a site for implantation, guided by the maternal endometrium [232,233]. The site of implantation in the human uterus is usually in the upper and posterior part in the midsagittal plane. During blastocyst apposition, the microvilli placed on the apical surface of trophectoderm interdigitate with the pinopodes localized on the apical surface of the uterine epithelium (Figure 2A). These specialized structures support a stable binding between trophoblast and uterine epithelial cells, so that the plasma membranes of these cells are parallel and separated by a distance of 20 nm [234]. The pinopodes secrete LIF [145]. LIF is a cytokine of the IL-6 family, which in the uterus activates the Janus kinases (JAK)-signal transducer and activator of transcription protein (STAT) pathway, and therefore phosphorylates STAT3, whose activation is required for implantation [235,236]. LIF is indispensable for blastocyst implantation. Mice knockout for LIF are infertile. Although able to develop blastocysts, these mice show implantation failure; however successful implantation occurs in surrogate mothers [90]. In Lif-null mice the expression of EGF-like growth factors, such as HB-EGF, AREG, and EREG, which, as previously mentioned, are normally expressed by the luminal epithelium adjacent to the blastocyst and are essential for successful pregnancy, is abolished [237]. Since the defects in decidualization caused by the absence of LIF can be rescued by intrauterine administration of EGF ligand [238], it has been hypothesized that LIF favors blastocyst invasion by reducing the expression of cell-cell junction molecules and proliferation of the stromal cells through activation of EGF signaling pathway [239]. In fertile women, LIF expression increases in the endometrium around the time of implantation, while infertile women express low levels of this factor [240,241].

Chorionic Gonadotropin
Once a competent blastocyst makes contact with the maternal endometrium, a dialogue made of signals and responses between them occurs. One of the most important factors secreted by trophoblast cells is CG. CG is expressed very early by the embryo, since its mRNA can be detected starting from the 6-8 cell stage. The protein is secreted by both zona enclosed or hatched blastocysts, and is independent of blastocyst interaction with the endometrium [242]. During pregnancy, CG is firstly detectable in maternal blood during implantation and then rapidly increases [243]. As discussed before, CG plays a fundamental role in inducing the production of progesterone and mediating the decidualization process, thus allowing implantation of the blastocyst.

Adhesion Molecules Mediating Blastocyst Adhesion
Following apposition, stable adhesion of the blastocyst to the endometrium occurs, mediated by the interaction between several receptors and ligands ( Figure 2B). Over the last decades, several of these ligands and receptors have been identified. It has been observed that both the pinopodes of the endometrial epithelium and the trophectoderm of the blastocyst express the integrin αvβ3, together with the endometrial expression of its ligand, the glycoprotein osteopontin (OPN). Their expression during the WOI suggests a role in implantation [160,244,245], and the binding between integrin αvβ3 and its ligand OPN might mediate the stable adhesion between the trophoblast and the endometrium [246]. Using an in vitro model of implantation, Genbacev et al. suggested that trophoblast adhesion to the uterine wall is also mediated by L-selectin expressed on the surface of the trophoblast cells, and uterine epithelial oligosaccharide ligands, such as HECA-452 and MECA-79 [247,248]. More recently it has been also demonstrated that the transmembrane glycoprotein MUC1, abundantly expressed at the apical surface of uterine epithelium under the control of progesterone, acts as a scaffold mediating the binding between L-selectin and their ligands [249].
The adhesion of the blastocyst to the endometrium is also promoted by the expression of adhesion molecules, such as cadherins. The presence of endothelial cadherin (E-cadherin) in both the trophoblasts and endometrial epithelium, regulated by progesterone, indicates that it may play an important role in blastocyst adhesion to the endometrium [250]. As trophoblast cells proliferate, differentiate and invade the stroma, they downregulate E-cadherin and increase osteoblast cadherin (OB-cadherin) [251,252]. This temporal expression of OB-cadherin in the endometrial epithelium suggests that this adhesion molecule later mediates trophoblast-endometrium interactions. Blastocyst adhesion is also favored by the expression of the glycoproteic receptor CD98 on the surface of endometrial cells, which is normally involved not only in amino acids transport but also in cell fusion [253,254]. Using two human endometrial cell lines characterized by low and high receptivity, Dominguez et al. demonstrated that CD98 receptor is significantly associated with the receptive phenotype. In human endometrial samples, CD98 expression was spatially restricted to the apical surface of endometrial cells and temporally restricted to the WOI. Treatment of primary endometrial epithelial cells with hCG, 17-β-estradiol, LIF, or EGF increases expression of CD98, greatly enhancing murine blastocyst adhesion, while its siRNA-mediated depletion reduced blastocyst adhesion rate [255].

NOTCH Signaling in Blastocyst Adhesion to the Endometrium
The expression of NOTCH receptors and ligands in the trophectoderm of the blastocyst and that of NOTCH1, DLL4, and JAG1 in the apical surface of the endometrial epithelium during the mid-secretory phase [170,256] would suggest a role for NOTCH signaling in the adhesion of the blastocyst to the epithelium. Indeed, it has been demonstrated that blastocyst-conditioned medium regulates NOTCH1 and JAG1 expression in the endometrial epithelium [256], suggesting that the blastocyst is able to activate NOTCH signaling in the endometrium, thus possibly regulating its receptivity. This is reinforced by the fact that women with primary infertility show a reduced or absent immunostaining for JAG1 in the luminal epithelium during the mid-secretory phase [256].

Colony-Stimulating Factor-1 in Implantation
A role for colony-stimulating factor-1 (CSF-1) in implantation has been proposed. Indeed, supplementation of CSF-1 in cultures of human trophoblast cells promotes their differentiation in syncytiotrophoblast cells and leads to the production of placental lactogen [257]. In addition, supplementation of CSF-1 to cultures of murine blastocyst induces trophoblast outgrowth [258]. However, using osteopetrotic mutant mice, which lack CSF-1, it has been shown that a maternal source of CSF-1 is not necessary for pregnancy, and possibly the fetus can provide a source of CSF-1 which compensate for the absence of maternally produced CSF-1 [259].

Invasion
Finally, in the third stage, invasion occurs, starting with the penetration of highly invasive trophoblast cells in the uterine epithelium ( Figure 2C), followed by infiltration in the basement membrane and in the stromal compartment, a process known as "interstitial invasion" [233,260,261]. Besides invading the endometrial stroma, trophoblast cells also migrate down the lumen of maternal spiral arteries, replace the vascular endothelial lining and become embedded in the arterial walls. This process of "endovascular invasion" allows to replace small-caliber, high-resistance vessels with large-caliber, low-resistance vessels, ensuring an adequate blood supply to the fetoplacental unit [262,263]. Defects in trophoblast endovascular invasion of maternal spiral arteries can seriously impair placental function, leading to significant complications of advanced gestation, such as intrauterine growth restriction (IUGR) and preeclampsia [264].

Matrix Metalloproteinases in Blastocyst Invasion
The huge invasive ability of the fetal trophoblast is due to a high production of activated gelatinases, in particular MMPs 2 and 9 [265][266][267]. Trophoblastic MMPs are regulated in response to IL-1β, tumor necrosis factor alpha (TNFα) IL-1α, macrophage colony-stimulating factor (MCSF), transforming growth factor β (TGFβ), IGFBP1, leptin, hCG, and EGF [124,[268][269][270][271][272], which are secreted from different cell types at the feto-maternal interface, such as trophoblasts themselves and endometrial cells, promoting trophoblast invasion. As already mentioned above, the expression of MMPs involved in endometrial invasion by trophoblast cells is also under the control of TH, as TH positively regulates MMP expression by endometrial cells [223].

Epidermal Growth Factor-Like Domain 7
Recently, we demonstrated that migration and invasion of trophoblast cells is regulated by the secreted factor Epidermal growth factor-like domain 7 (EGFL7), a novel NOTCH interactor. EGFL7 activates NOTCH1, MAPK and AKT signaling pathways in both trophoblast cell lines and primary cells [273]. Activation of the NOTCH pathway is important in both interstitial and endovascular invasion by trophoblast cells. In vitro functional assays show that invasion of Matrigel by trophoblast cells overexpressing EGFL7 is impaired in the presence of a γ-secretase inhibitor, normally used to inhibit NOTCH activation [264,273]. NOTCH appears to be also involved in trophoblast endovascular invasion, since uNK, involved in the disruption of endometrial spiral arteries integrity, express NOTCH1 and 2 and maternal cells surrounding spiral arteries express DLL1 [264], and NOTCH activation may lead to arterial wall disruption. These results are further confirmed by the fact that NOTCH pathway is dysregulated in placenta of women affected by preeclampsia [264,[274][275][276][277][278][279][280], a common pregnancy disorder characterized by an insufficient trophoblast invasion and an inadequate vascular remodeling. In women affected by preeclampsia, the alteration of NOTCH pathway is accompanied by a concomitant altered expression of EGFL7, in both placenta and maternal circulation [274,281].

Endometrial Control of Blastocyst Invasion
In all the placental species the extent of endometrial decidualization is proportional to the invasiveness of the embryo. The human placenta is the most invasive one known so far, and it has been suggested that the unique invasiveness of the human trophoblast could be due to its high production of hyperglycosylated CG isoform, which is maximal in the first weeks of pregnancy [282,283].
In order to limit the extent of trophoblast invasion, both trophoblast and endometrium balance the expression of growth factors, cytokines, and enzymes. As an example, maternal endometrium increases the production of tissue inhibitors of MMPs (TIMPs), due to a spatial and temporal regulation of cytokines and growth factors, such as IL-10 [284], TGFβ and IL-1α [268]. While IL-1α significantly increases the activity of MMP-9 and MCSF increases MMP-9 immunoreactivity, TGFβ inhibits total gelatinolytic activity, MMP-9 activity and immunoreactivity [268]. TIMP-3, which is up-regulated by progesterone, plays a major role in restricting trophoblast invasion by limiting ECM degradation, and its expression has been detected in the fetal extravillous trophoblast, as well as in the maternal endometrial cells [285,286]. On the contrary, by in situ hybridization in implanting mouse embryos no expression was observed for TIMP-1 or TIMP-2 in the embryo proper, trophoblasts, or in the decidua. Weak signals were demonstrated for TIMP-1 only in the circular layer of myometrial smooth muscle and in some uterine stroma cells distant from the site of embryo implantation. Moreover, the expression of TIMP-1 and TIMP-2 is not dependent on the stage of the menstrual cycle [286]. Trophoblast invasion is promoted by the action of the plasminogen activator (PA) system since it is able to promote trophoblast invasion, by converting plasminogen into the active serine protease plasmin, which in turn, degrades ECM [287]. In endometrial cells, TGFβ regulates trophoblast invasion up-regulating the expression of plasminogen activator inhibitor-1 (PAI-1), which is the main inhibitor of urokinase-type plasminogen activator (uPA) [288][289][290], and decorin, a decidua-derived TGFβ binding proteoglycan, which inhibits proliferation, migration and invasion of trophoblast cells [291]. The blastocyst is completely embedded in the uterine stroma 8 days after fertilization and the site of entry is covered by fibrin, over which the uterine epithelial cells grow [233,292,293].

Blastocyst Competence Profiling in ARTs
Although many of the molecular players involved in the complex process of implantation have been characterized, the selection of competent embryos remains one of the major challenges in ART. A parallel and complementary morphological and molecular profiling analysis of the embryo may represent a successful approach for embryo selection, thus improving IVF outcome. Although morphological characteristics have been significantly associated with euploidy and competence of the embryo [294,295], their evaluation for good quality embryo selection has some limitations, such as operator subjectivity, variability linked to the timing of laboratory observation, culture medium and other culture conditions, hence combined different approaches might be useful [296]. In this respect, metabolomic and proteomic analyses of embryo spent media have been proposed as complementary, non-invasive tools to select embryos with higher implantation ability [297][298][299]. Limitations derive from the variability of commercial culture media, high metabolic plasticity of the embryos which can adapt to different culture conditions and from the fact that embryo development and metabolism vary under different culture conditions [300]. Recently, novel strategies based on gene expression profiling of trophectoderm biopsies have been developed and have linked gene expression patterns with developmental competence [301][302][303]. Although complementary approaches may be used to select the best embryos to be transferred, it should be considered that it has been recently proposed that it is the endometrium itself that selectively discriminates between high-and low-quality embryos in order to guarantee a successful implantation. Based on this, it could be envisioned a test in which the endometrium might be used as a "bio-sensor" to avoid transfer of low-quality embryos, which if implanted would be possibly later rejected resulting in a miscarriage [304,305].

Conclusions
Over the last decades the research aimed to reveal the biomolecular processes and pathways underlying animal and human implantation has greatly progressed for two major reasons. On one hand, the exciting advances in available technologies allowed to define in depth the factors and the pathways involved in proper implantation. On the other hand, the introduction of ARTs and their spectacular development in response to the increasing clinical demands from infertile patients allowed to better understand the determinants of a successful implantation and of several conditions of reproductive failure. Additionally, the large diffusion of ARTs provided new perspectives for studies on implantation, making available biological samples previously unavailable for research; follicular fluid, granulosa cells, oocytes, embryos, culture medium of embryos, and blastocysts are examples of this.
As a general concept, it has become clear that reproduction in humans can be considered a rather inefficient process and in several ways is different from reproduction in other species. An emerging concept is that the proper molecular crosstalk between endometrium and blastocyst is of paramount relevance to ensure a proper implantation. In this context, the studies on animal models, apart for the above differences, may greatly help to increase current knowledge. The specific roles of blastocyst and endometrium are being discovered, although much progress still has to be done in this field. The final objective of this field of research effort is twofold: (1) to improve the understanding of how reproduction and implantation evolved and differentiated among the species; (2) to offer more and more effective treatment options to patients with infertility, RIF and RPL.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.