Embryo–maternal interactions at the implantation site: a delicate equilibrium

European Journal of Obstetrics & Gynecology and Reproductive Biology 83 (1999) 85–100

Original Article

Embryo–maternal interactions at the implantation site: a delicate equilibrium P. Duc-Goiran*, T.M. Mignot, C. Bourgeois, F. Ferre´ INSERM U. 361, Universite´ Rene´ Descartes, Pavillon Baudelocque, 123 Bvd de Port-Royal, 75014 Paris, France Received 10 July 1998; accepted 17 November 1998

Abstract Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal environment. During preimplantation, maternal / embryo communication is mediated by the trophectoderm. In the late luteal phase, physiological changes occur in the endometrium to allow blastocyst implantation. The ‘‘window of implantation’’ represents the period of maximum uterine receptivity for implantation. In response to signals from the embryo, pregnancy-specific proteins are released in maternal serum and a series of morphological, biochemical and immunological changes occur in the uterine environment. These systemic and local modifications can be considered to constitute ‘‘the maternal recognition of pregnancy’’. The human hemochorial placenta arises primarily through proliferation, migration and invasion of the endometrium and its vasculature by the embryonic trophoblast. The complex invasive processes accompanying implantation of the embryo are controlled at the embryo–maternal interface by factors from decidualized endometrium and the trophoblast itself. An inflammatory reaction and a proper maternal immune response allow survival and development of the feto–placental unit. In this review, we focus on interactions between trophoblast and uterine tissues and on cellular mechanisms and molecular signals involved in the closely regulated process of implantation.  1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Implantation; Blastocyst; Endometrium; Trophoblast; Growth factors; Cytokines; Integrins; Proteinases; Interferons

1. Introduction Implantation, initial placentation and trophoblast development occur in early pregnancy. The human blastocyst forms and penetrates the endometrium which has become receptive to implantation. Then, the blastocyst invades the stroma and underlying maternal capillary spaces resulting in the formation of a hemochorial placenta. Two processes unfold: formation of the blastocyst and endometrial modifications. These modifications create a uterine environment which is favorable to the development of the embryo and immunologically tolerant of the semi-allogenic graft (i.e.,

*Corresponding author. Tel.: 133-1-4326-2826, Fax: 133-1-43264408, E-mail: [email protected]

the embryo). This acceptance of the embryo represents an immunological paradox which is not discussed in this article (for a review see Ref. [1]). Synchronization between the development of the blastocyst and modifications in the endometrium is necessary for successful pregnancy. The pregnancy loss rate in spontaneous pregnancies is about 15–19% of all pregnancies diagnosed [2], but the true figure is probably much higher because of the high incidence of pregnancy loss before the clinical detection of pregnancy [3]. Implantational failures after in vitro fertilization (IVF) / embryo transfer (ET) are high. Approximately 80 to 90% of transferred embryos do not implant into the uterus. In the study of Ezra and Schenker [2], pregnancy results in 18.56% of cycles treated. Miscarriage rates are much higher (20–30%) after assisted reproduction than in

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spontaneous pregnancies, and vary with the treatment program [2]. During the peri-implantation period, many molecules such as cytokines, growth factors and hormones play key roles in the communication which occurs between the embryo and the maternal endometrium. Given the broad array of these signals, special attention was given here to substances which are released at the implantation site, and particularly, on growth factors and cytokines which are likely to play an important role in regulating trophoblast differentiation and invasion.

2. Early stages of embryo development Trophectoderm differentiation occurs after entry of the human embryo [morula consisting of 12 to 16 blastomeres (Fig. 1a)] into the uterus on the third to fourth day after fertilization [4,5]. In mice, compaction of the morula is accompanied by restriction of the expression of certain genes such as those of colony-stimulating factor-1 (CSF-1) receptor, the Na 1 / K 1 ATPase enzyme and a cell–cell adhesion molecule, E-cadherin, to trophoblastic mural cells (reviewed in ref. [6]). Homozygous mouse embryos that were rendered genetically deficient in the E-cadherin gene fail to form a trophectoderm epithelium or a blastocyst cavity [7]. In the course of normal gestation, when the blastocyst (Fig. 1b) arrives at its implantation site, it is probably oriented so that its embryonic pole is in contact with the uterine epithelium, as seen in a higher primate, the macaque (reported in Ref. [8]). The disappearance of the zona pellucida (hatching) seems to occur when the human trophoblast contacts the uterine epithelium, around the fifth day after fertilization [5]. Incorrect orientation of the blastocyst may explain some early implantation failures.

3. Signals during the preimplantation period A large number of hormonal and non-hormonal agents are produced by both the preimplantation embryo and the maternal endometrium. They include hormones such as hCG, signalling factors such as PGE 2 and PAF, growth factors, cytokines and receptors of the last two (Fig. 2).

3.1. Embryo signals and receptors Embryo signals include several factors: (1) Early pregnancy factor (EPF). It appears that the egg signals its presence to the maternal organism very early. As in mice, the so-called EPF is detected in human maternal serum by the rosette inhibition assay within hours of fertilization [9]. Recently, EPF was reported to be chaperonin 10 (cpn 10), a member of the molecular chaperones, most of which, including cpn 10, are heatshock or stress proteins [10]. (2) Preimplantation factor (PIF). Using the lymphocyte / platelet binding assay, the PIF can be detected in maternal serum four days after fertilization and before implantation of a viable embryo [11]. It is a low-molecular-mass protein which has also been found in culture media from viable preimplantation embryos, thus suggesting embryonic origin [12]. (3) Growth factors. Between the 8-cell and early blastocyst stage, epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) have been shown to be co-expressed with EGF receptors (EGF-R), suggesting autocrine stimulation in human development [13]. Transcripts of platelet-derived growth factor (PDGF)-A and the two receptor subunits, a and b, were also detected in human embryos from the 8-cell stage onwards [14], while the morula and blastocyst have been found to secrete insulin-like growth factor-II (IGF-II) (reported in Ref.

Fig. 1. Differentiation until the blastocyst stage can be obtained by cultivating human embryos after IVF, as shown in photographs of a compacted morula (a) and an expanded blastocyst before hatching (b) after four and six days, respectively, of co-culture with Vero cells. In (b), zona pellucida surrounding the blastocyst, polar trophectoderm overlying the inner cell mass and mural trophectoderm lining the blastocyst cavity are clearly visible. Magnification (a) ˆ 1503; (b) 2003. (C. Poirot, J.P. Wolf, Laboratoire de Biologie de la Reproduction, Hopital Cochin, Paris, France).

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Fig. 2. Embryo–endometrial interactions before implantation, schema. A variety of growth factors and receptors are expressed by the human embryo. Factors detected in 2-8 cell embryo are shown by an asterisk *. EGF-receptor ligands and LIF are expressed by the human endometrium in the secretory phase: uterine epithelial cells express TGF-a and LIF, while stromal cells express predominantly EGF and EGF receptors.

[15]). A large review of peptide growth factors and their role in preimplantation embryos is summarized by Kane et al. [16]. (4) Cytokines. In the 2- to 8-cell human embryo, CSF-1 has been detected by bioassay (reported in Ref. [15]). Interleukin (IL)-1, IL-6 and TGF-b are detected by bioassay during the first 48 h in human embryo culture fluids after IVF. The exact contribution of the embryo to cytokine production is unknown, since granulosa, cumulus and sperm cells are also possible sources [17]. IL-1a has also been detected by enzyme-linked immunosorbent assay (ELISA) in media from cultured human oocytes and early embryos. High concentrations of IL-1a have been correlated with the success of implantation after IVF. [18]. Transcripts of IL-6 and its receptor have been detected by RT-PCR in human blastocysts [15]. Some authors have found another cytokine, tumor necrosis factor (TNF)-a before the morula stage (reported in Ref. [15]). (5) Prostaglandin E 2 and PAF. Two cell signalling molecules, a prostanoid, prostaglandin E 2 (PGE 2 ), and a phospholipid, the platelet-activating factor (PAF), are also released by preimplantation human conceptuses [19,20]. The secretion of PAF by human embryos has been reported to be correlated with embryo viability and predictive of successful implantation following embryo transfer. It may be responsible for transient thrombocytopenia in the mother. However, other authors failed to confirm embryo production of PAF or its correlation with embryo viability (reported in Ref. [21]).

(6) hCG and IFN-t. Some signals of a hormonal character are specific to the trophoblast such as chorionic gonadotrophin (hCG) in humans and interferon (IFN)-t in ruminants. These two signals are responsible for the maintenance of the corpus luteum and the production of progesterone by the persistent corpus luteum. They act either directly, as in the case of the luteotrophic hormone hCG, which stimulates progesterone production via the luteal luteinizing hormone (LH)-receptor, or indirectly, as in the case of IFN-t, which interferes with the pathway of the pulsatile uterine release of prostaglandin F 2a (PGF 2a ), a luteolytic prostaglandin (reported in Ref. [22]). Production of hCG is crucial for both the establishment and maintenance of pregnancy during the first seven to nine weeks, from which time the placenta becomes the primary source of progesterone. Anti-hCG vaccines have been developed to control fertility. One of them, targeted against the carboxyl terminal part of the b-subunit, undergoes trials under the aegis of the World Health Organization (WHO). These vaccines act before implantation of the fertilized egg occurs (reported in Ref. [23]) and thus are considered contragestive. Receptors of cytokines and growth factors are expressed in the embryo: (1) c-fms. In mice, CSF-1 receptor transcripts, encoded by the c-fms proto-oncogene, are expressed by preimplantation embryos from the 2-cell stage onwards and become restricted to the trophoblastic lineage after the blastocyst stage (reported in Refs. [6,24]). In preimplanta-

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tion human embryos, c-fms expression parallels the murine pattern [15]. (2) EGF and LIF receptors. As soon as it is formed, the murine trophectoderm expresses an epithelial marker (cytokeratin) (reported in Ref. [6]), receptors for a growth factor (EGF) and receptors for a cytokine leukemia inhibitory factor (LIF) (reported in Ref. [25]). In humans, LIF receptors (LIF-R) are detectable in first trimester chorionic tissue and in BeWo choriocarcinoma cells [26], suggesting their expression in human trophoblast. Transcripts for receptors of both EGF and LIF were also detected in human preimplantation embryos [13,15,27,28]. Hence, it may be extrapolated that the human embryo is the target of these growth factors and cytokines.

3.2. Endometrial factors Preparation of the endometrium for implantation is assured by a precise mechanism involving cyclic sequential secretions of 17b-estradiol and progesterone. In humans, secretory activity in glandular epithelium is maximal on days 5–7 post-ovulation, while stromal cell differentiation, known as pre-decidual change, is spontaneous and begins later, on days 9 to 10 [1]. During the preand peri-implantation periods, estrogens and / or progesterone regulate expression of several growth factors and cytokines in the uterus. These endometrial factors (EGFreceptor ligands, LIF, etc.) may serve as mediators of steroid actions (reported in Ref. [29]) and may act on the blastocyst through paracrine action (Fig. 2): (1) EGF-receptor ligands. It has been shown in the mouse that ovarian steroids induce the expression of growth factors, in particular those of the EGF family: TGF-a, heparin binding EGF-like growth factor (HBEGF) and amphiregulin. The latter two factors are expressed in murine uterine epithelium exclusively at the attachment site of the blastocyst [30]. HB-EGF is induced by the blastocyst and interacts with the EGF receptor expressed by the trophoblast [31], while amphiregulin expression is correlated first with rising progesterone levels and then with the attachment reaction. It is related to epithelial cell differentiation and could be a sign of uterine receptivity [30]. In human endometrium during the secretory phase, TGF-a has been immunohistochemically localized to the glandular and surface epithelium [32], while EGF immunostaining was predominant in stromal cells [33]. HBEGF mRNA was found by RT-PCR in the human glandular and stromal endometrium throughout the menstrual cycle and persists in pregnancy [34]. The intense immunoreactivity for EGF receptors in stromal cells suggests an autocrine / paracrine role in differentiation of the stroma [35]. (2) LIF production. The endometrium also secretes cytokines, one of which, LIF, seems to play an essential

role in the beginning of implantation. In the mouse, LIF expression is under maternal control and is not dependent on the presence of the blastocyst. The high level of expression of this glycoprotein by the endometrial glands, specifically on day 4 of pregnancy, precedes implantation [36] and is essential for it to occur. Hence, Stewart et al. [37] have demonstrated that normal murine embryos cannot implant in transgenic mice homozygous for an endogenous gene mutation rendering the LIF gene nonfunctional. If exogenous LIF is supplied by a peritoneal micropump, a decidual response which may allow embryo implantation is re-established [37]. Furthermore, also in mice, Harvey et al. [25] suggested that LIF, like EGF, upregulates the activity of proteases produced by the blastocyst and may act in a paracrine manner, given that LIF receptors, like those of EGF, are expressed by the trophectoderm. These authors also showed that LIF later downregulates protease activity in the blastocyst after nine to ten days in culture. Likewise, LIF is expressed in human endometrium, where it is produced preferentially by the uterine glandular epithelial cells which, in culture, produce more LIF than do the stromal cells. The timing of LIF expression by these glandular epithelial cells corresponds to the predicted moment of implantation, in the middle of the luteal phase [27,38]. (3) CSF-1, predominantly expressed in the uterine epithelium, increases from the proliferative to the secretory phase of the cycle. It is also expressed by first trimester cytotrophoblasts (reported in Refs. [24,39]). (4) Placenta protein 14, a progesterone-dependent glycoprotein, is the main secretory product of glandular epithelium, starting from the mid-luteal phase [39]. (5) Decidual prolactin (PRL), a hormone identical to primate pituitary PRL, is expressed in stromal cells prior to implantation and expression continues throughout pregnancy (reported in Refs. [40,41]).

4. Developmental changes in the endometrium: ‘‘the window of implantation’’ During the luteal phase of the menstrual cycle, a series of changes occurs in the different endometrial components, namely in glandular and surface epithelium, stromal cells, stromal vessels and the extra-cellular matrix [39]. These changes converge to make the endometrium receptive to embryo implantation. Whereas some of these changes persist into early pregnancy, some have a limited lifespan. The moment of maximal uterine receptivity is defined by morphological and biochemical studies as well as by clinical investigations during IVF or embryo transfer trials:

4.1. Structural marker During the peri-implantation period, the presence of

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pinopodes at the apical pole of uterine epithelial cells has been described as a structural marker of receptivity. The presence of fully developed pinopodes, which does not exceed a period of 48 h, varies from patient to patient and is affected by hormonal treatment [42].

4.2. Glycocalyx layer and MUC-1 protein on the surface epithelium The non-adhesive nature of the uterine epithelium may be partially attributed to the glycocalyx layer coating the surface epithelial cells. During the receptive period, a decrease in the thickness of the endometrial glycocalyx coating the apical plasma membrane of surface epithelial cells has been described in the mouse [43]. In contrast, the glycocalyx becomes thicker in cynomolgus monkeys, where it may promote trophoblast migration (reported in Ref. [44]). A mucin-type glycoprotein designated MUC-1 is expressed on the surface epithelium in both murine and human species. Its expression peaks at the implantation stage in the human, while it falls to minimum levels in the mouse at this stage. Its exact role in implantation remains to be defined. Changes in protein distribution and binding cell junctions have been reported on the surface epithelium of many diverse animals (reported in Ref. [5]).

4.3. Integrins Studies by Lessey and co-workers [45,46] have shown the involvement of adhesion molecules called integrins in the definition of the implantation window. Integrins form a family of heterodimeric cell surface receptors consisting of two subunits (a-chain and b-chain). They are subdivided into eight subfamilies based on their b-subunit. Three integrin subunits, a1, a4 and b3, are specifically expressed in glandular epithelium during a four-day interval, from cycle days 20 to 24 corresponding to the putative window of implantation. The vitronectin receptor, avb3, appears abruptly on cycle day 20, ‘‘opening’’ the window, while a4b1, a fibronectin receptor, is present from ovulation to cycle day 24 when its disappearance ‘‘closes’’ the window. Both a4b1 and avb3 may participate in the endometrialtrophoblast interactions that take place during the implantation process [46]. Aberrant expression of integrins has been observed in the endometrium of women with reproductive failure [39]. Absence of the a4b1 integrin which recognizes fibronectin present on the fetal trophoblast may result in incomplete maternal recognition of the embryo. Lack of avb3 integrin was noted in women with retarded endometrial development or endometriosis [39].

4.4. Blastocyst attachment Implantation consists of two processes: apposition, common to all viviparous mammals, and invasion of the

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endometrium in rodents and primates (including humans). In these latter two groups, implantation is precocious, starting after the blastocyst hatches from the zona pellucida (reported in Ref. [22]). The apparent adhesion of the trophoblastic and uterine epithelia represents a biological paradox. At the implantation site, two genetically different epithelia, the blastocyst epithelium and the non-adhesive apical epithelial surface of the endometrium, seem to secondarily adhere by their apical poles [47]. Early interactions may involve binding of extracellular matrix protein (oncofetal fibronectin) expressed at the outer surface of the trophoblast by integrins located at the apico–lateral borders of epithelial uterine cells [46]. This association may contribute to the loss of epithelial polarity and the loosening of lateral borders [47].

4.5. Clinical data: hCG as a marker of implantation Experience with ovum donation and transfer has demonstrated that the optimal period of transfer for two-day-old embryos occurs on normalized cycle days 15 through 19–20. In these studies, the detection of the first embryonic signal (hCG) in maternal serum occurs between cycle days 20 and 24 and is considered as indicative of and temporally related to embryo implantation. Within this window, implantation is dependent on embryonic age: the first embryonic signal detected is found around the embryonic age of 7.1 days, irrespective of endometrial maturation [48].

5. Factors involved in trophoblast invasion and its control

5.1. The invasion process and trophoblast differentiation Penetration of the endometrium by the trophoblast may be considered as similar to the invasion by tumor cells [49]. However, while tumors tend to invade as individual migrating cells, the implanting blastocyst invades en masse. In primates, guinea pigs and carnivores, this aggressive migration into the endometrium has been referred to as ‘‘intrusive implantation’’ and may also be regarded as ‘‘inter-epithelial implantation’’, since there is no damage to the uterine epithelium [44]. From observations conducted on primates [50,8] and in vitro models, it has been deduced that syncytial transformation may be induced in the trophoblast by contact with the surface epithelium [1] and that penetration of the human blastocyst into the endometrium occurs by protrusion of cytoplasmic processes from syncytiotrophoblast. Intrusion of these extensions between epithelial cells reaches the basal membrane. Then, the trophoblast invades the endometrium by proteolysis of the underlying basement membrane and the extracellular matrix (ECM) of the stroma. While cytotrophoblasts, the original unicellular layer, act as stem

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proliferating cells leading to continual expansion of the trophoblast, the primitive syncytiotrophoblast, formed by cellular fusion, constitutes a large continuous acellular outer layer facing maternal tissue [51]. Around the 9th day, the syncytiotrophoblast hollows out gaps, the trophoblastic lacunae, which will fuse with newly formed maternal lacunae and uterine capillary spaces around the 12th day [50]. Trophoblast lineage: two differentiation pathways. Placentation results from trophoblast proliferation, migration and invasion into the endometrium and its vasculature. Distinct trophoblast populations arise through two main differentiation pathways: villous and extravillous. Cytotrophoblasts (CTBs) proliferate between the radially distributed lacunae and also into side branches which protude into lacunae, leading to the formation of primary trophoblastic villi. In these villi, CTBs fuse to form hormonally active villous syncytiotrophoblast which represents the endstage of human intravillous trophoblast differentiation and possesses important endocrine and transport properties which are beyond the scope of this article. At the sites where villi contact the uterine wall, anchoring villi develop (2nd and 3rd week [51]). Near the tips of these anchoring villi, some CTBs proliferate towards the basal plate. These trophoblast cells, which are extravillous trophoblast (EVT), form multilayered columns of nonpolarized proliferative CTBs which connect anchoring villi

to the placental bed [51,52]. Arising from these columns, some EVT cells acquire a transiently invasive phenotype; hence, they no longer proliferate and they start to migrate deeply through the decidua and the inner third of the myometrium, where they infiltrate the walls and lumen of spiral arteries. This invasive activity peaks during the twelfth week of pregnancy and declines rapidly thereafter [53]. Furthermore, intervillous blood flow establishment, determined by color Doppler imaging, occurs at a gestational age $11.7 weeks and is associated with the peak and subsequent decline in circulating hCG concentrations [54]. The complex processes of invasion and their control are reported in greater detail in several reviews [5,55,56].

5.2. The integrin switch in cytotrophoblasts Depending on their location, CTBs are surrounded by different ECM components which specifically regulate the collagenolytic activity and cellular behavior probably via integrins, which mediate adhesion to the extracellular matrix. During trophoblast migration from the villous zone into the decidua, CTBs modulate their integrin repertoire (the so-called ‘‘integrin switch’’) showing a decrease in a6b4, a laminin receptor, and an increase in a5b1 and a1b1 heterodimers, respectively a fibronectin and a collagen / laminin receptor (Fig. 3). The depolarization of a6b4 integrins in CTBs situated in the proximal trophob-

Fig. 3. Diagram showing a floating villus (FV) bathing in maternal intervillous blood and an anchoring villus (AV) attached to the maternal decidua basalis and the uterine wall by EVT cell columns (CCs). The special organization of the junctional zone recapitulates the differentiation of EVTs and integrin expression changes along the invasive pathway. Invasion is controlled by tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2 and TIMP-3) and factors such as TGF-b, mainly secreted by decidual cells.

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lastic columns marks the acquisition of invasiveness by EVT cells [1,52,56,57]. Concomitantly, these cells express histocompatibility-linked antigen-G (HLA-G) protein considered a non-classic major histocompatibility complex class I antigen, which may play an important role in maternal tolerance of the fetus. HLA-G positive cells also secrete significantly more gelatinases and less fibronectin than HLA-G negative cells [52]. In distal columns, EVT cells express the a5b1 heterodimer, the major fibronectin receptor and also produce fibronectin [1,52]. Once EVT cells leave the cell columns and invade the placental bed, they continue to express a5b1 fibronectin receptor and upregulate the expression of the a1b1 collagen / laminin receptor [58], which is particularly strong in EVTs that have invaded maternal blood vessels [59]. Immunocytochemical data and antibody perturbation experiments suggest that upregulation of the a1b1 integrin is critical for acquisition of an invasive phenotype by cytotrophoblasts [58]. Adhesion molecule switching by invasive cytotrophoblasts is abnormal in preeclampsia. In the placental bed from preeclamptic patients, EVT cells display the a5b1 integrin but fail to express the a1b1 integrin complex. In this pregnancy disorder, cytotrophoblast invasion is limited to the superficial decidua and does not progress to the maternal blood vessels [59].

5.3. Mediators of invasion Invasion through the epithelium and stroma of the

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endometrium is mediated by proteinases present at the trophoblast cell surface or in the extracellular environment. These proteinases include members of the matrix metalloproteinase family, plasminogen activators belonging to the serine proteinase family, and plasmin. (1) Matrix metalloproteinases (MMPs). MMPs are a multigene family of zinc-dependent proteinases which are able to degrade components of the ECM. Most are secreted as inactive proenzymes and are activated in the ECM. They are grouped into three subfamilies (collagenases, stromelysins and gelatinases) according to their substrate specificity [60]. The human preimplantation embryo produces MMPs capable of degrading collagen IV of the basal endometrial membranes, and this proteinase activity increases with time in culture [61]. Invasive first trimester human CTBs synthesize a number of MMPs (Fig. 4) showing ECM degradative activity in vitro. Only some of these MMPs are found in later gestation in non-invasive cells [53]. Among the type IV collagenases, the 72 000 gelatinase (gelatinase A or MMP-2) is produced by noninvasive fibroblasts, while the 92 000 gelatinase (gelatinase B or MMP-9) is unique to CTBs and is critical for invasion [53,62,63]. Other data (reported in Ref. [56]) indicate that MMP-2 and MMP-9, whose mRNAs are both expressed by EVT cells in situ, are important mediators of trophoblast invasion. The activation of proMMP-2 occurs at the surface of the trophoblast and requires the participation of an active membrane-type (MT)-MMP, MT1-MMP. MT1-MMP and an interstitial collagenase (MMP-1) are

Fig. 4. Proteinase cascade involved in trophoblast invasion. Trophoblast uPA activates plasmin which directly activates MMP-1, MMP-9 and MT1-MMP. The latter MT1-MMP in turn activates MMP-2 (adapted from Lala and Hamilton, 1996) [56]. (*) Stromelysin-3 and uPA are also expressed in syncytiotrophoblast.

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also expressed by human trophoblast cells (reported in Ref. [56]). Another member of the metalloproteinase family, stromelysin-3, is expressed in syncytiotrophoblasts of floating villi and in human EVT cells which invade distal regions of decidua and express a1b1 integrins [64]. (2) Plasminogen activators. Cultured human cytotrophoblasts are able to secrete a serine protease, urokinase-type plasminogen activator (uPA) [65], which may directly contribute to the breakdown of stromal ECM or may act as part of a proteinase cascade. Indeed, once activated by uPA, plasmin (derived from cell-bound plasminogen) is a potent proteinase. In addition, plasmin can activate interstitial procollagenase (pro-MMP-1), progelatinase B (pro-MMP-9) and proMT1-MMP (reported in Ref. [56]). Subsequently, MT-MMP induces specific activation of pro-gelatinase A [66] (Fig. 4). Human trophoblast cells in culture also express uPA receptors [67] which, by binding uPA, would focus plasminogen activator activity on the trophoblast cell surface. Polarized expression of uPA-receptors has been localized to the leading edge of migrating EVT cells where the receptors may direct trophoblast migration. Urokinase receptors are also expressed on the apical surface of villous trophoblast where they may facilitate generation of plasmin and limit fibrin deposition in intervillous spaces [68]. Further, immunoreactive uPA and inhibitors of the PA system, plasminogen activator inhibitors (PAI)-1 and -2, are present in early human implantation sites, particularly in invasive EVT cells and syncytiotrophoblast [69]. The uptake and degradation of uPA / PAI-1 complexes may be mediated by a low density lipoprotein (LDL) receptorrelated protein which removes the inactive proteinase complexes from trophoblast surface and allows the trophoblast to continue its invasion [70].

5.4. Factors promoting trophoblast proliferation and invasion Growth factors, cytokines and peptides are likely to play an important role in influencing trophoblast proliferation / differentiation and / or the acquisition of an invasive phenotype, through their receptors and / or binding proteins. Trophoblast proliferation is stimulated by several growth factors: (1) EGF-receptor ligands. EGF, TGF-a and amphiregulin have been found to promote proliferation of EVT propagated in culture without influencing EVT invasiveness in vitro. In fact, they upregulate to similar levels mRNAs for both gelatinase A (MMP-2) and a tissue inhibitor of metalloproteinases, TIMP-1 (reported in Ref. [56]). It should be noted that using freshly isolated first trimester CTBs, a different response to EGF is obtained, with morphological changes and a significant increase in Matrigel invasion [71]. Using term CTBs, Morrish et al. [72] reported the EGF induction of differentiation into

syncytiotrophoblasts with a concomitant increase in hCG and human placental lactogen (hPL). (2) CSF-1. Like EGF and TGF-a, CSF-1 promotes proliferation and stimulates a balanced increase in the production of gelatinase A (MMP-2) and TIMP-1 mRNAs by the EVT cells in culture. In situ expression of CSF-1 receptor (c-fms) has been localized to EVT cell columns (reported in Ref. [56]). (3) VEGF. Recent data indicate that vascular endothelial growth factor (VEGF) synthesized by decidual macrophages promotes proliferation of EVT cells which express VEGF-R ( flt) mRNA and protein (reported in Ref. [56]). (4) IGF-I. Insulin-like growth factor-I (IGF-I) has been reported to have growth promoting effects on the early villous trophoblast (reported in Ref. [56]). Invasion is stimulated by other factors: (1) IGF-II and IGFBP-1. Treatment by IGF-II does not influence the proliferation of EVT cells in culture, but enhances trophoblast invasiveness (reported in Ref. [56]). IGF-II is expressed more abundantly than IGF-I in human placenta and is found only in cells of fetal origin. IGF-II transcript levels are highest at the invading front of the trophoblastic columns [73]. IGF-II binds to specific cellsurface receptors (types 1 and 2) of the trophoblast membranes [74] and also to a family of binding proteins (BPs). IGFBP-1 seems to be the predominant IGFBP synthesized by the maternal decidua [73]. The a5b1 integrins are involved in the action of IGFBP-1 which, along with IGF-II, stimulates first trimester EVT cell migration and invasion [56]. (2) Interleukin-1b. IL-1b also regulates human trophoblast activity and invasion in vitro. It stimulates MMP-9 production in cytotrophoblast cells, while glucocorticoids inhibit it [75]. (3) Endothelin-1. A peptide, ET-1, stimulates the invasion of first trimester trophoblast specifically via the Btype receptor. Its action on proliferation is mediated by both ETA and ET B receptor subtypes [76].

5.5. Factors limiting invasion Invasion is closely regulated within the microenvironment by substances derived from decidual stromal cells, the trophoblast itself and ECM components [56]. Decidual factors. Data indicate that the endometrium controls excessive invasion of the trophoblast by the decidual reaction. Indeed, in human pathologies such as placenta accreta and ectopic pregnancies, poor decidualization is associated with a higher degree of trophoblast invasion [5]. In species where there is no breach of the uterine epithelium, there is no decidualization of the endometrium. In humans, decidualization is extensive, occurring throughout the stroma, while in rats, mice and certain non-human primates, it is localized more to the area of the implanted embryo. Furthermore, in rats and mice, where decidualization occurs early in implantation,

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trophoblast invasion is less extensive than in humans, where decidualization becomes evident later [77]. The decidua secretes factors such as the TIMPs, transforming growth factor-b (TGF-b) and LIF which are involved in invasion control (Fig. 3). (1) TIMPs. Two natural proteinase inhibitors, TIMP-1 and -2, have been found to completely inhibit trophoblast invasion in vitro by forming complexes with the active forms of collagenases [62]. Both inhibitors are expressed in human endometrium [5]. In addition, TIMP-3 mRNA expression was observed in vitro in human endometrial stromal cells cultured in the presence of progesterone and in situ in maternal decidual cells [78]. (2) TGF-b. This multifunctional growth factor is expressed by the decidua at the feto–maternal interface from the first trimester until the end of the pregnancy. Decidual TGF-b seems to be released mainly in its latent form and may be activated by plasmin or cathepsin D (reported in Ref. [49]). TGF-b blocks both trophoblast proliferation and invasion: (a) it inhibits proliferation of first trimester human EVTs in culture and induces their differentiation into non-invasive multinucleated cells which are probably the equivalents of murine placental bed giant cells (reported in Ref. [49]). (b) TGF-b hinders trophoblastic invasion via several mechanisms: (i) it inhibits production of the serine proteinase, uPA (reported in Ref. [56]) and induces PA inhibitor-1 (PAI-1) expression (reported in Ref. [62]). It induces the expression of TIMP-1 in both the decidua and the trophoblast, thus inhibiting metalloproteinase (reported in Ref. [49]); (ii) it reduces migration of the EVT [49]. This reduction is linked to overexpression of the a5b1 integrin (a fibronectin receptor) which renders the trophoblast more adhesive to the extracellular matrix and thereby blocks trophoblast progression (reported in Ref. [56]); and (iii) it favors the formation of ECM components, in particular, collagen and fibronectin, by increasing the peri-cellular deposition of fibronectin (reported in Ref. [62]) and stimulating, in vitro, trophoblast oncofetal fibronectin, which may promote trophoblast anchorage to the uterine wall (reported in Ref. [56]). (3) LIF. A cytokine, LIF, has also been reported to reduce proteinase activity of trophoblast (reported in Ref. [56]). Trophoblastic factors. Trophoblasts may autoregulate their own invasiveness. They express several protease inhibitors: (1) TIMP-3. Expression of TIMP-3 mRNA has been reported in EVT cells that have invaded the maternal decidua, where this metalloproteinase inhibitor may limit ECM degradation [78]. (2) TGF-b. TGF-b is also produced, to a minor extent, by EVT cells which release it in its active form [49]. It is present throughout gestation in the villous syncytiotrophoblast and in the EVT of the cytotrophoblast shell, where it may play an autocrine role in blocking invasion (reported in Ref. [56]).

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(3) hCG. By its inhibitory action on the activity of collagenases, hCG may retard the invasion of trophoblastic cells [79]. ECM components play a crucial role in the regulation of trophoblast invasion. Trophoblast cell functions may be modulated by their interactions with the ECM. Binding to laminin has been reported to stimulate trophoblast type IV collagenase activity, while binding to fibronectin by a5b1 integrins facilitates EVT migration (reported in Ref. [56]). Some ECM components can bind and / or store a number of regulatory factors. For example, decorin, an ECM proteoglycan, may bind and inactivate TGF-b. Decorin may also store and present this regulatory factor to uPA for activation (reported in Ref. [56]).

6. Factors involved in an inflammatory reaction Animal studies have shown that at the implantation site, edema and an increase in vascular permeability are signs of a local inflammatory reaction which prepares this part of the endometrium for implantation. In humans, there is no inflammatory response to seminal fluid and remarkably little tissue destruction and necrosis accompanying the early stage of implantation [1]. However, angiogenesis and enlargement of decidual blood vessels have been observed, predominantly around the implantation site (reported in Ref. [40]). Further, inflammatory mediators with potent vasoactive properties, proinflammatory cytokines, MMPs and leucocyte recruitment have been found in the early decidua and may be involved in an inflammatory reaction (Fig. 5).

6.1. Inflammatory mediators: PAF and prostaglandin E2 In humans, PAF originating from the embryo or endometrial stromal cells (reported in Ref. [21]) appears to contribute to the inflammatory process in the endometrium either directly through its potent vasodilating activity or indirectly by increasing the local release of PGE 2 by the secretory endometrium. PGE 2 may cause increased vascular permeability at this site and seems to play a crucial role. Indeed, inhibition by indomethacin of the cyclooxygenase pathway involved in the synthesis of prostanoids (prostaglandins and thromboxanes) reduces the implantation rate in mice (reported in Ref. [21]).

6.2. Proinflammatory cytokines Cytokines, in particular IL-1 and IL-6, are involved in inflammatory reactions. In the mouse, implantation engenders an acute inflammatory reaction with uterine production of IL-1, IL-6 and tumor necrosis factor (TNF)-a. Maximal production of IL-1 coincides with the peak in estrogens that occurs at the time of implantation on days 4–5. IL-6 activity is highest on days 5–6, while that of

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Fig. 5. Scheme of inflammatory and immune reactions at the implantation site. Edema and an increase in vascular permeability are brought about by platelet activating factor (PAF), prostaglandin E 2 (PGE 2 ) and pro-inflammatory IL-1 and IL-6. Colony-stimulating factors (CSF-1, G-CSF and GM-CSF) are principally expressed by decidual cells, macrophages and LGLs.

TNF-a is highest on day 8 [80]. In the mouse, blockage of IL-1 receptors with an antagonist prevents blastocyst implantation by interfering with attachment (reported in Ref. [6]). In humans, IL-1 appears to interact with endometrial glandular epithelial cells which express type I IL-1 receptors in the luteal phase of the menstrual cycle [81]. Endometrial epithelial cells respond in vitro to recombinant IL-1 by an increase in the production of PGE 2 [82] and in the expression of an MHC class II antigen, HLA-DR [83]. IL-1 has also been shown to stimulate the expression of IL-6 in human endometrial stromal cells [84].

6.3. Matrix metalloproteinases MMPs are also secreted by endometrial cells and are involved in the remodelling of endometrial tissue. Human endometrial stromal cells in primary culture secreted MMP-1, MMP-3 and two gelatinases (MMP-2 and -9). MMP-1, -3 and -9 production (but not MMP-2) is stimulated by both IL-1a and TNF-a [85].

6.4. Leucocyte recruitment The initial phase of implantation is accompanied by the infiltration of large numbers of bone marrow-derived cells in decidua. There is an accumulation of innate immune response effectors such as uterine natural killer (NK) cells and macrophages at the embryo–maternal interface, while

there are only small numbers of T cells and virtually no B cells or granulocytes. In the late secretory phase and in early pregnancy, large granular lymphocytes (LGLs) with the distinctive CD56 1 CD16 2 phenotype are present in great quantities in the decidua in close apposition to the invading trophoblast which expresses the non conventional Class I HLA-G antigen. HLA-G antigen may interact with uterine CD56 1 NK cells which express receptors for HLAG, and may protect cytotrophoblasts against natural killer cytolysis [86].

7. Cytokines and the immune reaction During pregnancy, survival of the semi-allogenic fetus occurs in an immunologically regulated site, the uterus. According to Wegmann’s hypothesis, a proper maternal immune response can affect development of the feto– placental unit by local production of cytokines, such as the macrophage growth factor also known as CSF-1, GM-CSF (a growth factor of both macrophages and granulocytes) and IL-3 (in mice), which can promote the growth and / or differentiation of trophoblast [87].

7.1. Colony-stimulating factors CSF-1, GM-CSF and granulocyte colony-stimulating factor (G-CSF) are expressed at high levels at the embryo–

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maternal interface, principally by decidual cells, macrophages and decidual LGLs (Fig. 5): (1) CSF-1. Expression of CSF-1 in human decidua and placenta is higher in the first trimester of pregnancy than in the second and third. It has been localized by immunohistochemical staining to glandular epithelial cells, endothelial cells lining the endometrial blood vessels and CTBs in villi and cell columns [88]. Analysis of first trimester placental and decidual cell populations shows that most of the CSF-1 present at the placental uterine interface appears to be produced by decidual stromal cells and decidual LGLs. CSF-1 synthesis may be regulated by ovarian steroids (as in mice) and by IL-2. The expression of the CSF-1 receptor (c-fms) by EVT situated in cell columns and by fetal (Hofbauer cells) and decidual macrophages is consistent with a CSF-1 role in the development and differentiation of these EVT cells as well as in the functions of both fetal and decidual macrophages [89]. (2) GM-CSF. A diverse array of cell lineages are known to produce GM-CSF. In mice, the epithelium is a major source of GM-CSF, production of which is stimulated by seminal factors and regulated by estrogens, bacterial lipopolysaccharide (LPS) and IFN-g (reported in Ref. [24]). In vivo, GM-CSF restores normal gestation in CBA / J3DBA / 2 mating mice which exhibit a high level of spontaneous abortions. In vitro, GM-CSF is able to stimulate ectoplacental cone proliferation (reported in Ref. [90]). In human first trimester pregnancy, decidual LGLs are a major source of this cytokine. Co-culture with decidual stromal cells or stimulation with cytokines (IL-1b or especially IL-2) increases GM-CSF secretion by LGLs. GM-CSF is also detected in first trimester trophoblast cultures (24 h). Thus, given that its receptor is expressed by all trophoblast populations, GM-CSF may act on placental growth and development in both a paracrine and autocrine manner [90]. Both maternal and fetal macrophages are potential targets of GM-CSF as well as CSF-1. GM-CSF and CSF-1 may act in synergy (reported in Ref. [1]). (3) G-CSF. In the human uterus, G-CSF has been shown to be produced by maternal decidual LGLs and macrophages (reported in Ref. [24]). (4) Stem cell factor (also known as kit ligand, or KL). KL is produced by EVT; its targets are decidual macrophages, fetal Hofbauer cells and a subset of decidual NK cells [1].

7.2. Preferential secretion of T-helper type 2 cytokines In murine decidua, the production of T-helper 2 (Th2) cytokines (IL-4, IL-5 and IL-10) is favored over the production of Th1 cytokines (IL-2, IFN-g, lymphotoxin). Successful pregnancy in mice is linked to a shift from predominantly Th1-driven cell-mediated immunity to predominantly Th2-driven humoral immunity [91]. In

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humans, a dominant Th1 cytokine profile has been observed in cases of recurrent spontaneous abortion [92].

7.3. Regulation of hCG secretion by cytokines Production and release of placental hCG has been reported to be regulated by hormones [gonadotropin-releasing hormone (GnRH) and gonadal steroids] as well as by trophoblast-derived cytokines such as IL-6, LIF and IFN-a. IL-1 stimulates the release of hCG by activating IL-6 production and the IL-6 receptor-mediated signal transduction pathway, as shown in first trimester human trophoblasts [93]. Decidual tissue in culture produces LIF which plays a role similar to that of IL-6 in the stimulation of hCG production by first trimester trophoblasts [94]. IFN-a is also able to induce hCG production in vitro [95].

7.4. Interferons Species with non-invasive placentation. During the periimplantation period, constitutive IFN expression has been demonstrated in the trophoblast, but only in species exhibiting non-invasive placentation. However, the nature and timing of IFN expression differ slightly according to the species in which IFNs are expressed (Fig. 6). After the elongation phase, during the peri-attachment period, pig trophoblast produces IFN-g in great quantities as well as a type I IFN of a new family, recently called IFN-d [96,97]. In pig species, the trophoblast does not penetrate beyond the uterine epithelium and the placenta is epitheliochorial. In the horse, the placenta is also epitheliochorial, with endometrial cups. In equine species, a not yet identified IFN is produced by the developing embryo at the preimplantation stage [98] followed by TGF-b production in the endometrium at the time of implantation around day 33 [99]. In ruminants, where there is some fusion of the uterine and chorial epithelia (synepitheliochorial placenta) in placentomes [100], IFN-t is expressed during expansion and elongation of the blastocyst. Then, expression of IFN-t decreases with the adhesion of the blastocyst to the endometrium [101] and the appearance of TGF-b secretion [102]. In addition to its well-known antiluteolytic properties, IFN-t is able to inhibit the expression of proMMP-1 and proMMP-3 in cultured ovine endometrial stromal cells [103]. Species with hemochorial placenta. There is currently no convincing evidence for significant IFN production by the blastocyst before implantation in humans [104] or in mice [24], species in which the hatched blastocyst rapidly invades the endometrium. However, from the first third of gestation until term, type I IFNs have been found in human (reviewed in Ref. [1]) and murine [105] species. In human term placenta, we have shown IFN-a-like transcripts which correlate with functional IFN-a proteins [106]. Furthermore, an IFN-a mRNA showing, respectively, 70% and 89% deduced

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Fig. 6. Simplified representation of the feto–maternal barrier in different species and comparative IFN production in the peri-implantation period. Type I IFNs include IFN-a, IFN-v, IFN-t and IFN-d. Type II IFN is represented by IFN-g.

amino acid homology with ovine IFN-t and ovine IFN-v [107] but only 62% with human IFN-v is primarily expressed in the CTBs and syncytiotrophoblast during the first trimester of pregnancy [108]. These IFNs appear to be expressed in the absence of any apparent induction. We hypothesize that they may be induced by endogenous retroviruses which are selectively expressed at the basal border of the syncytiotrophoblast (reported in Ref. [1]). At the present time, the role that placental IFNs may play during gestation is not clear. These proteins are potentially immunosuppressive, given that IFN-a inhibits the proliferation of lymphocytes in response to the action of IL-2. The role of type I IFN in antiviral defense is essential, as can be seen by the high susceptibility to viral infections in homozygous mice lacking the type I IFN receptor gene [109]. Furthermore, IFN-a expression appears to be related to developmental and differentiation processes since IFN-a has been shown to be produced constitutively in diverse fetal epithelia during murine development [105].

8. Conclusions Unlike tumor invasion or inflammatory reactions, implantation in the human species is a strictly controlled process in both space and time. Any disequilibrium between the factors favoring the trophoblast’s invasive properties and the factors limiting its invasion can ultimately lead to a pathologic state. Uncontrolled invasion is characteristic of ectopic pregnancies, placenta accreta, hydatiform moles and choriocarcinomas, while insufficient invasion may be implicated in severe hypertensive diseases such as preeclampsia. Little data is currently available concerning implantation and invasion in humans. An

improved knowledge of growth factors and cytokines involved in implantation and the processes associated with it may lead to more successful pregnancies.

9. Abbreviations ATPase BP CSF-1 CTB ECM EGF EPF ET-1 EVT G-CSF GM-CSF GnRH HB-EGF hCG HLA-G hPL IFN IGF IL-1 IVF KL LDL LGL LH LIF MHC

Adenosine triphosphatase Binding protein Colony-stimulating factor-1 Cytotrophoblast Extracellular matrix Epidermal growth factor Early pregnancy factor Endothelin-1 Extravillous trophoblast Granulocyte colony-stimulating factor Granulocyte and macrophage colonystimulating factor Gonadotropin-releasing hormone Heparin binding-epidermal growth factor Human chorionic gonadotrophin Histocompatibility-linked antigen-G Human placental lactogen Interferon Insulin-like growth factor Interleukin-1 In vitro fertilization Kit-ligand Low density lipoprotein Large granular lymphocyte Luteinizing hormone Leukemia inhibitory factor Major histocompatibility complex

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MMP NK PA PAI PAF PGE 2 PGF 2 a PIF TGF Th2 TIMP TNF uPA VEGF

Matrix metalloproteinase Natural killer Plasminogen activator Plasminogen activator inhibitor Platelet activating factor Prostaglandin E 2 Prostaglandin F 2 a Preimplantation factor Transforming growth factor T-helper 2 Tissue inhibitor of metalloproteinases Tumor necrosis factor Urokinase-type plasminogen activator Vascular endothelial growth factor

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Acknowledgements ´ We wish to thank L. Cedard, J. Doly, K. Imakawa, C. La ` P. Lebon, F. Lefevre, ` Bonnardiere, J. Martal and J.R. Zorn for their helpful advice and comments; C. Poirot and J.P. Wolf for morula and blastocyst photographs; S. Allman for manuscript assistance and M. Verger for secretarial assistance.

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