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TOWARDS A CELLULAR AND MOLECUIAR UNDERSTANDING OF IMPUTATION IN THE HUMAN: IMPLICATIONS FOR ASSISTED REPRODUCTIVE TECHNOLOGIES Christos Coutifaris, M.D., Ph.D., Jerome F. Strauss, III, M.D.,Ph.D., Harvey Kliman, M.D., Ph.D. INTRODUCTION Successful in vitro fertilization programs report fertilization rates, of 60-75% yet clinical pregnancy rates approximate 20-30% per transfer (Ben Rafael et al., 1986, 1987; Laufer et al, 1984; Lopata 1983~. The large gap between fertilization and pregnancy rates has generally been attributed to failure in the pert-implantation period, a time of wastage following natural conception. In spontaneously conceived pregnancies early emb Tonic wastage may be as high as 31% (Wilcox et al, 1988~. It is estimated that 20-25% of human embryos derived from in vitro fertilization have recognizable chromosomal anomalies which are incompatible with life (Plachot et al., 1987~. It can be assumed that an additional group of embryos may have other lethal genetic defects that are not detectable by current techniques. Nevertheless, at least 50% of the embryos transferred to the uterus may be entirely normal, and yet their transfer does not result in a pregnancy. There are undoubtedly multiple causes of pregnancy wastage following embryo transfer, some of which may relate to technical difficulties. The method of transcervical embryo transfer routinely employed could contribute to embryo loss because of malplacement or trauma. However, it is also likely that subtle biochemical abnormalities in the embryo and the uterus affect implantation competency. At this point, we cross into an unchartered area since implantation is one of the most poorly understood processes in reproductive biology. The initial steps of embryo implantation involve fundamental issues of cell-cell and cell-substratum recognition and adhesion prior to and during the invasion of the trophoblast into the endometri~m. The composition of the endometrial cell surface and extracellular matrix is rigorously controlled such that implantation can take place only after structural and biochemical changes have occurred under the influence of gonadal steroids and, perhaps, other factors (Aplin et al., 1988; Carpin et al., 1985; Faber et al., 1986; Yamaguchi et al., 1985~. Although the hormonal conditions required for uterine receptivity have been extensively studied(Glasser, 1986; Glasser and McCormack, 1980; Glasser and Clark, 1975; Psychoyos, 1973, Weitisuf, 1988), much of the valuable information assembled from the study of laboratory and domestic animals cannot be directly applied to subhuman primates and humans since there are major differences in the morphology, and apparently the mechanisms by which 33o
the morphology, and apparently the mechanisms by which implantation occurs among various species. Several different modes of implantation have been identified including 1) fusion implantation where trophoblasts fuse with uterine epithelial cells, as occurs in the rabbit (Schlafke and Enders, 1975~; 2) displacement implantation where uterine epithelial cells are dislodged and the trophoblast interacts with the basal lamina, which occurs in rodents (Finn and Bredl, 1973: El-Shershaby and Hinchliffe, 1978; and Pijnenborg et al., 1981; Sherman and WudI, 1976; and 3) intrusive implantation where trophoblasts insinuate between epithelial cells prior to initiating frank invasion. The latter is the apparent mechanism by which human implantation is initiated based upon the few specimens of early human implantation sites available for study (Enders et al., 1983; Hata et al., 1981 a, b; Pierce et al., 1964~. A major obstacle in the study of implantation in the human has been the lack of adequate in vitro systems, since it is presently not possible to explore the process in a refined manner in viva. Human endometrial tissue is frequently available, but human zygotes are rarely accessible for experimentation. However, methods to study the function of purified human trophoblasts in culture have been developed in several laboratories (Hall et al., 1977; Kliman et al., 1986; Stromberg et al., 1978; ). These techniques now make possible the exploration of the interactions between trophoblast and endometrium in vitro. Here we will review our observations on the characterization of placental trophoblasts, which we propose as a substitute for the human blastocyst in in vitro studies on implantation. We also will present preliminary data from studies in which the interactions of purified cytotrophoblasts with extracellular matrix and endometrial tissue have been examined. These findings are interpreted in light of the existing information on implantation and a working model for nidation in the human is offered. Preparation and Characterization of Human Cytotrophoblasts By modifying a previously reported method for the enzymatic dispersion of term placental tissue with trypsin-DNase, by addition of a Percoll gradient centrifugation step, we have been able to prepare highly purified cytotrophoblasts (Figure 1, Kliman et al., 1986; Kliman et al., 1987~. This procedure can also be used with first trimester placental tissue, although contamination of the cytotrophoblasts with non-trophoblastic cell types is greater. The isolated cytotrophoblasts undergo striking morphological changes in culture resulting in the formation of functional syncytial trophoblasts. - 337
Trophoblast Aggregation Using the method described above, it has been possible to establish that mononuclear cytotrophoblasts are precursors of the terminally differentiated syncytial trophoblast. Cytotrophoblasts isolated from term placentae initially aggregate and then fuse to form large multinucleated syncytia in vitro. Time lapse cinematography has unequivocally documented this two step sequence. It can be hypothesized that this homotypic recognition is mediated by unique glycoprotein cell adhesion molecules (CAMs, Edelman, 1988~. Using both cytotrophoblasts and JEG-3 choriocarcinoma cells, we have started to characterize the factors responsible for the specificity of trophoblast aggregation. Freshly isolated cytotrophoblasts aggregate in suspension culture over a 24 to 48 h period. Dispersed JEG-3 cells also aggregate in suspension culture, forming tissue-like masses. The extent of aggregation of both cytotrophoblasts and JEG-3 cells in suspension is related to cell density, with greater aggregation occurring with increasing cell concentrations (Kliman et al., 1989~. These findings suggest that one determinant of aggregation is the frequency of random cell contact. The aggregation of trypsinized cells requires the synthesis of proteins since JEG-3 cells fail to coalesce when incubated in the presence of cycloheximide. However, if cycloheximide is removed by washing the cells, aggregation occurs during a subsequent incubation (Kliman et al., 1989~. Calcium is required as well since JEG-3 aggregation is impeded when cells are incubated in calcium and magnesium-free medium (Babalola et al., 1989~. These results suggest that trypsin-EDTA treatment cleaves calcium-dependent CAM s from the cell surface. We speculate that a trophoblast CAM must be synthesized and inserted into the plasma membrane in order for aggregation of dispersed cytotrophoblasts or JEG-3 cells to occur. Trophoblast Fusion The mechanism of fusion of cytotrophoblasts to form syncytial structures has not been elucidated. It is a specif~ process in that cytotrophoblasts will not fuse with non-trophoblastic cell types (e.g., fibroblasts or fetal liver and kidney cells). The specificity could be imparted by the CAM s mediating cell aggregation. Several fundamental questions regarding the fusion competency of trophoblastic cells remain unanswered. What prevents cytotrophoblasts, which lie directly under syncytial trophoblasts in the chorionic villi, from fusing? Why do JEG-3 cells aggregate in culture but rarely fuse to form syncytia? One - 338
possible explanation may be the existence of factors in cytotrophoblasts and JEG-3 cells which restrain membrane fusion. A 34 kilodalton protein, once thought to be a growth factor but now known to be a member of the lipocortin family, is elaborated by cytotrophoblasts (Choudhury-Roy et al., 1988). Lipocortins bind to phospholipids and are inhibitors of phospholipase A2 (Hayashi et al., 1987; Tait et al., 1988). Since phospholipases have been implicated in the process of membrane fusion, it is possible that the lipocortin-like 34 kd protein plays a role in controlling membrane union. Trophoblast Interactions With Extracellular Matrix Cytotrophoblast aggregation and fusion occurs when the cells are cultured under standard conditions in serum-supplemented medium (Kliman et al., 1986~. In contrast, if cytotrophoblasts are cultured in serum-free medium without coating of the culture surface with an extracellular matrix protein, the cells remain solitary and do not aggregate or fuse (Feinman et al., 1986; Kao et al., 1988). However, if the culture surface is pre-coated with a matrix protein including types I, IV, and V collagen, fibronectin, or laminin, but not albumin, cytotrophoblast aggregation and fusion take place, even in serum-free medium (Kao et al., 1988~. It should also be noted that cytotrophoblasts aggregate in suspension culture in both the absence or presence of serum (Babalola et al., 1989). Thus' the requirement for serum in standard tissue culture appears, at least in part, to be for the attachment factors such as fibronectin. In the absence of these factors, cytotrophoblasts are incapable of firm attachment, spreading and motion. This limits opportunities for cell contact and, thusl aggregation. It is worth mentioning that cytotrophoblasts synthesize fibronectin (Ulloa-Aguirre et al., 1987) but they clearly cannot utilize the endogenously produced protein for effective interaction with the culture surface under serum-free conditions. Fibronectin produced by placental tissue differs both chemically and functionally from plasma fibronectin: placental fibronectin has a different carbohydrate composition and binds to gelatin with lesser affinity than plasma fibronectin (Zhu et al., 1984; Zhu and Laine, 1985~. These differences may account for the failure of cytotrophoblasts to undergo the normal morphologic changes in serum-free culture in the absence of exogenous matrix proteins. The fact that a variety of extracellular matrix proteins support the morphologic differentiation of human cytotrophoblasts in vitro, as well as the outgrowth of mouse blastocysts (Armant et al., 1986), indicates that the trophoblast expresses a variety - 339
of substrate adhesion molecules (SAM's). Since these receptors are frequently coupled to the cellular cytoskeleton (Edelman, 1988), they may have functions other than simply serving as anchors for the cytotrophoblasts (Horwitz et al., 1986~. Indeed, these receptors could be part of a transducing system which signals to the cell the nature of its immediate environment. Preliminary immunocytochemical studies suggest that the extracellular matrix protein on which cytotrophoblasts are cultivated affects the immunocytochemical distribution of fibronectin and laminin (Kliman and Strauss, unpublished observations). These findings raise the possibility of the extracellular matrix influencing cellular synthesis and/or secretion of matrix components, presumably through the intermediacy of cell surface substrate adhesion molecules. Relationship Between Morphologic and Functional Differentiation of The Trophoblast Cytotrophoblasts do not normally express endocrine activities characteristic of the syncytial trophoblast including chorionic gonadotropin (hCG) and placental lactogen secretion. The morphologic differentiation of cytotrophoblasts to form syncytial structures can be dissociated from functional (i.e., endocrine) differentiation. Agents which stimulate adenylate cyclase or the cAMP analog, B-bromo-cAMP, stimulate hCG production by cytotrophoblasts by increasing accumulation of the hCG subunit mRNAs (Feinman et al., 1986; Ulloa-Aguirre et al. 1987; Nulsen et al., 1988; Ringler et al., 1989~. This stimulation is observed in mononuclear cytotrophoblasts as well as multicellular aggregates. Moreover, it is also seen when cytotrophoblasts are cultured under conditions which prevent aggregation and fusion (e.g., culture in serum-free medium) (Feinman et al., 1986; Kao et al., 1988~. Interactions Between Trophoblast And Endometrium Several laboratories have proposed in vitro models for implantation in which blastocysts from laboratory animals have been cultured on extracellular matrices, endometrial tissue and lens capsule (Table I). We are developing systems to explore the interactions of human trophoblasts with human endometrial explants and purified endometrial glandular epithelial cells in culture (Coutifaris et al., 198S, 1989; Kishimoto et al., 1987; X1iman et al., 1988, 1989~. The explant system has the advantage of maintaining intact the cellular architecture of the endometrium, but the "urvival of the endometrial explants is limited to several days. The isolated endometrial cells in - 340
standard culture offer the opportunity of examining interactions between specific cell types. However, the absence of stromal-epithelial relationships may affect the way trophoblasts associate with the endometrial glandular epithelium. Our in vitro models for human implantation assume that isolated cytotrophoblasts, obtained either from first trimester or term placentae, behave like trophectoderm of the blastocyst and that endometrial explants or isolated endometrial cells can retain the characteristics of pert-implantation endometrial tissue in vivo. While it is far too early to know whether these assumptions are legitimate, preliminary data discussed below encourage us to press on with these studies. Cytotrophoblast Interactions With Endometrial Explants Cytotrophoblasts isolated from term placentae and first trimester placental tissue bind to endometrial explants in suspension co-culture during a 24 hour incubation. In preliminary observations, cytotrophoblasts attach to the epithelial cells of secretory endometrium, but they do not associate with the epithelial surface of peritoneum or fallopian tube. The cytotrophoblasts do, however, attach to cut surfaces of proliferative end secretory endometrium and fallopian tube, where stroma and extracellular matrix proteins are exposed. After 24-48 hours of co-incubation, a zone of tissue necrosis can be observed at the junction between the attached trophoblastic elements and the endometrium (Fig. 2; Kliman et al., 19881. Histologically, this zone resembles Nitabuch's layer, which is made up of fibrinoid material and separates the cytotrophoblast cell columns seen in normal human implantation sites. Moreover, some cytotrophoblasts penetrate into the endometrial explants and can be clearly identified in tissue sections by the use of immunocytochemistry with antibodies against hCG subunits. Unlike cytotrophoblasts, melanoma cells, endothelial cells and amniocytes do not induce a zone of necrosis. Cytotrophoblast Interactions With Endometrial Glandular Epithelium And Stroma Gurpide and colleagues (Schatz et al., 1986; Schatz et al., 1984; Satyaswaroop et al., 1979) developed methods to prepare human endometrial cells and maintain them in culture. These techniques have permitted us and others to examine the interaction of cytotrophoblasts with enriched endometrial cell types. Isolated endometrial glands form nests of epithelial cells after being established in culture. Cytotrophoblasts bind to the collections of glandular epithelial cells and then penetrate into the islands ultimately causing them to detach from the culture surface (Figure 3; Coutifaris et al., 1988; Kishimoto - 3~1
et al, 19871. In contrast, melanoma cell" surround the nests of glandular epithelium but do not interact or invade them. Cytotrophoblasts, JEG-3 choriocarcinoma cells and melanoma cells, all adhere to established cultures of endometrial stromal cells and co-minq~e with them without causing cellular detachment (Coutifaris et al., 1989~. Recently, Lindenberg and co-workers (1986) incubated hatched human blastocysts onto established monolayer cultures of human endometrial epithelial cells and observed trophoblastic cell attachement and outgrowth. Ultimately, the endometrial cells were displaced and allowed the trophoblasts to come in contact with the culture dish. Taken together, the findings reviewed above suggest that cytotrophoblasts have a proclivity for endometrial epithelial cells and are capable of penetrating through them in a process which appears to resemble intrusive implantation. Cytotrophoblasts are also capable of associating with stromal cells and a variety of extracellular matrix proteins. As discussed earl ier , a number of matrix proteins permit trophoblast attachment, flattening and syncytium formation. We speculate that the specificity (both spatial and temporal) of attachment of trophoblast could be determined by the endometrial epithelium while the underlying extracellular matrix is always permissive. Therefore, under normal circumstances, implantation might occur only in the presence of a receptive endometrial epithelium. However, if the epithelium is eroded, exposing stroma and extracellular matrix, implantation could occur in a variety of locations (e.g., sites where the oviductal epithelium is denuded). Our speculations raise several key questions. Does endometrial epithelial receptivity result from the expression of unique CAM s recognized by the trophoblast? Is the "window" for human implantation determined by the temporal pattern of expression of these CAM's? The existing literature suggests that the answers to these two questions is affirmative. Studies in laboratory animals are consistent with a role for CAM-like molecules in blastocyst attachment. Cell surface charge is known to be important in blastocyst attachment in rodents (Morris and Potter, 1984; and Nilsson and Hjerten, 1982~. In addition, inhibition of glycoprotein synthesis impedes trophoblast binding to the uterus and the lectin, concanavalin A, blocks blastocyst adhesion in the mouse (Surani, 1979; Wu and Chang, 1978~. Moreover, a calcium dependent CAM, p-cadherin, has been identified in mouse implantation sites (Nose and Takeichi, 1986~. These data are all consistent with a role for CAMs in the implantation process. Recent analyses of endometrial histology and pregnancy rates in women with premature ovarian failure treated with exogenous hormones to prepare the uterus for transfer of a fertilized donor oocyte suggest a discrete period 342 -
of endometrial receptivity (Rosenwaks, 1987;Navot et al., 1988) In these series, the optimal time of embryo transfer was day 17-19 while no pregnancies were achieved when embryos were transferred on or after day 20. These observations are consistent with a discrete period of endometrial receptivity. Trophoblast Invasion Into The Endometrium The mechanism by which trophoblastic elements penetrate _ the human endometrium are not known. Studies in laboratory animals suggest that a variety of proteases are involved in implantation, including plasminogen activator (Dabich and Andray, 1974; Denker, 1981; Glass, 1983; Strickland et al., 1976~. Axelrod (1985) reported that the to mouse produces blastocysts which are less invasive than controls and have an associated diminished production of plasminogen activator activity. Other proteases which degrade extracellular matrix such as stromelysin and collagenases probably participate in trophoblast invasion, since a variety of proteins must be hydrolyzed in concert or sequence during implantation. However, documentation that these enzymes play critical roles in nidation is scant. Since trophoblast invasion must be controlled, mechanisms to localize the site of protease action and to limit the activity of the enzymes are also expected to play a key role in implantation. Purified human cytotrophoblasts elaborate several proteases capable of digesting gelatin, among them being urokinase (Martin and Arias-Stella, 1982; Queenan et al., 1987). Urokinase may have a direct role in the degradation of fibronectin, as well as activating other enzymes, including plasmin, which hydrolyze matrix proteins (Fisher et al., 1985). The expressed activity of urokinase is determined not only by the amount of enzyme protein but also by levels of plasminogen activator inhibitors which covalently bind to and inhibit the enzyme (Blast et al., 1987~. The trophoblast produces at least two different plasminogen activator inhibitors, plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2). PAI-1 is localized by immunocytochemistry to primarily trophoblasts invading into the endometrium whereas PAI-2 is found predominantly in the syncytial trophoblast of the chorionic villi (Feinberg et al., 1989~. The significance of this differential production of PAI-] and PAI-2 by trophoblastic elements remains to be determined. The coordinated regulation of urokinase and PAIs provides one means by which trophoblast invasion could be tightly regulated (Feinberg et al., 1988~. Furthermore, urokinase is produced as an inactive proenzyme which must be activated by limited proteolysis (Blast, et al., 1987~. Urokinase is also known to bind to cell surface receptors, fixing the site of its action. Thus, there are several levels at which urokinase activity can be 343 -
modulated including I) enzyme protein; 2) enz ~ e activation; 3) cell surface receptors for enzyme and 4) inactivation of enzyme by specific inhibitors. Urokinase is not the only protease with multiple loci for control. Stromelysin and collagenases are also produced as proenzymes and a specific inhibitor of these metalloproteinases (TIMP: Tissue inhibitor of metalloproteinases) is present in the uterine environment (amniotic fluid, gunning et al., 1984; Chin et al., 1985~. There are many apparent similarities between the process of implantation and the degradation of the extracellular matrix that occurs in association with tumor invasion (Tryggvason et al., 1987~. However, in the former case penetration is restricted. Hence, the elucidation of the ways in which trophoblast invasion is controlled will undoubtedly provide insight into the abnormal behavior of choriocarcinoma cells as well as other malignancies. Paracrine Interactions During Implantation There is compelling evidence that paracrine dialogue occurs between the blastocyst and endometrium in the pert-implantation period. Physical contact of the blastocyst with the endometrium and carbon dioxide produced by the conceptus have been proposed as "signals" to the endometrium in rodents, but more compelling evidence indicates that histamine, prostanoids and locally generated steroids play the critical roles in the initial events in nidation (Weitiauf, 1988~. It remains to be determined whether these substances have similar functions in human pregnancy. We do know from our in vitro studies that progesterone produced by the trophoblast can have a local effect on the adjacent endometrium. Co-incubations of cytotrophoblasts and endometrial explants, there is local induction of morphological changes in the glandular epithelium indicative of secretory activity (e.g., subnuclear vacuolization) by cytotrophoblasts (Kliman and Strauss, unpublished observations). The conceptus also elaborates polypeptide factors, including monokines and growth factors, which effect endometrial function (Adamson, 1987; Flint et al., 1988; see Roberts et al. in this volume for additional references). Finally, the endometrium, both epithelium and decidua, are capable of producing growth factors, monokines, and growth factor binding proteins, which can either act directly on the trophoblast or modify the action of available growth factors on the trophoblast (Bartocci et al., 1986; Han et al., 1987; Bell and Keyte, 1988; Ringler et al., 1989~. These paracrine relationships may modulate the immune system, endometrial activity and trophoblast function and growth (Mogil and Wegman, 1988~. - 344 -
A Working Model For Implantation In The Human And Future Directions For Research From our studies and the existing literature, we propose a two step model for implantation in the human which entails 1) the binding of trophoblast to a specific endometrial epithelial CAM, the expression of which governs the implantation window; and 2) trophoblast penetration through the epithelial layer with attachment and outgrowth on extra-cellular matrix, the invasion being mediated by proteases checked by the action of specific inhibitors. It remains to be determined whether the in vitro systems introduced in this paper are adequate to characterize the sequence of events in human nidation. The primary assumptions regarding the equivalency of cytotrophoblasts to the blastocyst trophectoderm and the maintenance of normal endometrial function under in vitro conditions are still open to question. Perhaps hybrid systems in which endometrial explants and cytotrophoblast interactions occur in an animal host (i.e., nude mouse or rat) may also be useful. The evaluation of our working model will most certainly require refinements in the in vitro techniques with which the cell and molecular biology of implantation are studied. None-the-less, should further investigation prove that in vitro systems do indeed reflect the events of implantation, investigators will be in an excellent position to explore the fundamental aspects of nidation. It should be recognized, however, that in vitro systems will not permit experimental approaches to questions relating to the apposition of the blastocyst to the uterine lining (i.e., spatial considerations). This would require the maintenance of the geometry of a normal uterine cavity. Thus, the identification of a suitable sub-human primate model for implantation should be a major goal for future research so that emerging concepts can be evaluated using in viva approaches. Our knowledge of the biochemistry of the human endometrium during the pert-implantation period is deficient. We are still in a descriptive phase of research in which the morphology and various biochemical parameters such as steroid receptor levels and distribution are being defined (Jacobs et al., 1987; Garcia et al., 1988; Lessey et al, 1988~. A detailed knowledge of the endometrial epithelial cell, the endometrial glandular secretions and the composition of the endometrial stroma are needed as part of the definition of the "receptive" endometrium. Whether stromal-epithelial interactions are critical to the "receptive" state and the extent to which dysynchrony in stromal-epithelial function can perturb the capacity of the endometrium to receive the conceptus remain to be explored. - 345 -
Severa ~ strateg ies are available to identify putative CAM s involved in blastocyst binding to the endometrium including the generation of monoclonal antibodies to endometrial epithelial cells which could be screened for ability to interfere with cytotrophoblast attachment to endometrial explants or isolated epithelial cells. Full length c DNA clones could be isolated from expression libraries prepared from human endometrial epithelial cell RNA using these immunologic probes. In this way the amino acid sequence of the CAMs could be deduced and additional antibodies prepared from knowledge of the p rim-e ry amino acid sequences. These antibodies could provide important information in the evaluation of endometrial biopsies, as they could help identify the appropriate hormonal regimens for preparation of the uterus for embryo transfer. A knowledge of the distribution of these CAMs throughout the uterine cavity could provide important clues as to the spatial specificity of implantation, which usually occurs on the posterior fundal endometrium. The identification of molecules involved in placental morphogenesis and the factors regulating protease and protease inhibitor production by trophobla=ts may shed light on pregnancy failure during the pert-implantation period. Moreover, toxemia of pregnancy is associated with a failure of trophoblastic invasion and remodeling of uterine spiral arteries (Rushton, 1984; Thong et al., 1986~. Thus, an understanding of the factors controlling trophoblast invasion may provide new insight into the pathophysiology of a major pregnancy-associated disorder. Such knowledge may also shed light on other pathologic conditions including placenta accreta and gestational trophoblastic disease. Immunocytochemistry and in situ hybridization for detection of protease and protease inhibitor protein and mRNAs in normal and abnormal implantation sites will provide useful descriptive information which could yield clues as to the functions of these proteins. With specific enzyme inhibitors in conj unction with the co-culture systems described here, it may be possible to define the actions of each of these proteases in nidation. The discovery of the various paracrine substances involved in implantation, the elucidation of their sequence of action and fee~back relationships represent formidable challenges for future research. Central issues relating to how the early conceptus, particularly the trophoblast, grows and differentiates remain to be addressed. Although it is recognized that the trophoblast is enriched in growth factors and growth factor receptors, the specific functions of~many of these proteins are still unknown. [n vitro ~;ystems appear to be the most amenable experimental approach for clarifying the situation. Finally, important data relating to the process of human implantation could be obtained from a national or perhaps an international registry of in vitro fertilization/embryo transfer programs. The incidence of abnormal implantation, including ~ 346
early wastage (e.g., "chemical pregnancies"), and its correlation with various regimens for follicular recruitment and post-retrieval endocrine support as well as the mode of embryo transfer needs to be determined. These data coupled with emerging concepts from in vitro experimentation will advance our knowledge of this critical and still mysterious event in human reproduction. - 34'
ACKNOWLEDGEMENTS · ; ~ · The authors wish to acknowledge the assistance of their colleagues Drs. Lee-Chuan Kao, Ronald F. Feinberg, G. O. Babalola, John T. Queenan, Jr., Guy E. Ringler, Michael A.. Feinman, and Emiliano A. Soto who have contributed to various aspects of the work described in this paper. Special thanks are offered to Julia Haimowitz and Peter Wu for technical assistance and Mrs. Barbara McKenna for their help in the preparation of this manuscript. REFERENCES Adamson, E. D. (1987~. Expression of proto-oncogenes in the placenta. Placenta B: 449-466. Aplin, J. D., A. K. CharIton, S. Ayad (19881. An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy. Cell and Tissue Research 253: 231-240. Armant, D. R., H. A. Kaplan, W. J. Lennarz (1986~. Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts. Dev. Biol. ll6, 510. Axeirod, H. R. (1985) Altered trophoblast functions in implantation-defective mouse embryos. Dev. Biol. 108: 185-190. Babalola, G. O., E. A. Soto, C. Coutifaris, H.~. Kliman, J. F. Strauss ITI (1989~. Morphogenesis of the human placenta: characterization of the aggregation of cytotrophoblastic cells 36th Annual Meeting of the Society for Gynecologic Investigation , San Diego, CA. Abstract 45. Bartocci, A., J. W. Pollard and E. R. Stanley (1986~. Regulation of colony-stimulating factor ~ during pregnancy. J. Exp. Med. 164: 956-961. Bell, S. C. and J. W. Keyte (1988~. N-terminal amino acid sequence of human pregnancy-associated endometrial a~-globulin, an endometrial insulin-like growth factor (IGF) binding protein-evidence for two small molecular weight IGE binding proteins. Endocrinology 123: 1202-1204. 348 -
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^ #, lance ~ ~~ ~ \ 'a ~ T~psin-DNaseO~esllon _^ Aid- ~~ ~ _ ~ ~ _ ~ ~ CuIlure l _ : Pelle11hrough Call Serum l Godly Cen141ugation Figure 1 Isolation of human cYtotropboblasts - 35S
-A s~.~-~-- a i. ~ S #' . ~ ~ . ·~t,~;~l, ,- ·~ ~^ I- ma.<. ~ "~;` ` ·,, 1,~,,. r_ . ~ ' 'i ~ . .' _~ `;. ~ · Figure 2 Contact necrosis. Cytotrophoblasts from a term placenta were co-cultured with mid-proli ferative phase endometrium (E) for 24 h, the tissue was fixed in Bouin's solution, processed for light microscopy and stained with ~ ~ hematoxyl in and eosin . The trophobIasts ATE have attached to the surface of the endometrium and have induced a zone of necrosis (arrow heads). This zone was only present at points of trophoblast attachment. Note that some of the cytotrophoblasts have formed syncytial structures (arrows) during the 24 hour culture period. The bar represents 20 um. 350
. J . - - Figure 3 Interactions of trophoblasts with human endometrial glands in vitro. Purified human endometrial glands were co- cultured with human cytotrophoblasts for 24 h, fixed with Bouin's solution and immunocytochemically stained with antibodies against alpha- hCG using DAB as chromagen. Several darkly stained - - ~ ~ ~ ~ (E). Note a trophoblast group making initial contact with the gland (arrow head). Two large syncytial trophoblast groups can be seen adherent to the gland's edge (large arrows). In addition, several trophoblasts have penetrated the glandular group (small arrows). The bar represent 50 um. tronhoblast arouns can be seen around the enuometr~at Ghana - 360 -
x= - x oN ~ ~ - ~ x ~ ~ x x x ~ To ~ x x To ~ x x x Do x or or ~ ~ cry ~ or or - ~ ~ - - - - - ~ - - ~ l - - - - lo; - - is c - - ·~ - A o 9 Vet - c C c C a . E ~ C ~ ~ ~ C ~ ~ e C: A E o ~ 5; ~ a I at, I I la Hi | i i a _~ ' t~ ~~ . ~ ~ ~ |E a l ~ j C a ~ a ~ a ~ A: ~ ~ ~ ~ ~ ~ ~ ~ Cal 361 -
