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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
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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.
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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
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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
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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
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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
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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 -
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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 -
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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~.
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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 -
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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
OCR for page 351
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.i .
- 3~7
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OCR for page 358
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Figure 1 Isolation of human cYtotropboblasts
- 35S
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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 —
OCR for page 360
.
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
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OCR for page 361
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361 -
Representative terms from entire chapter:
human endometrial
