Human Reproduction Vol.17, No.5 pp. 1351–1357, 2002
Ultrastructure of the early human feto-maternal interface
co-cultured in vitro
M.O.Babawale1,3, M.A.Mobberley2, T.A.Ryder2, M.G.Elder1 and M.H.F.Sullivan1,4
1Institute
of Reproductive and Developmental Biology, Wolfson & Weston Research Centre for Family Health, Hammersmith
Hospital, Du Cane Road, London W12 0NN, 2Electron Microscopy Unit, Department of Histopathology, 6th Floor, Laboratory
Block, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK
3Present
address: Division of Pathology, School of Clinical Laboratory Sciences, University of Nottingham Medical School, Queens’
Medical Centre, Nottingham NG7 2UH, UK
4To
whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: The study was designed to investigate the ultrastructural features of the early human fetomaternal interface when generated by in-vitro co-culture, and compare these with findings reported previously
from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8–12 week pregnancies
were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for
electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for
trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column
formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the
4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen
granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion.
CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation
of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a
process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no
damage to the surrounding decidual cells, resemble in-vivo data.
Key words: co-culture in vitro/EVT/feto-maternal interface/fusion of plasma membranes
Introduction
Although interest in human development in utero dates back
to antiquity, there is still a dearth of knowledge of the early
events of human placentation. Hence the search continues
for the better understanding of these events, using in-vitro
co-culture models of early human placental villous and
decidua parietalis tissues (Kliman et al., 1990; Lewis et al.,
1993; Vicovac et al., 1993, 1995; Babawale et al., 1996)
or of cells from these tissues. In early human development,
a local breakdown of the syncytial layer of the placenta
occurs as it comes in contact with maternal decidua, whilst
the underlying cytotrophoblast cells proliferate and form
columns of invasive cytotrophoblast cells which form the
anchoring villi (Boyd and Hamilton, 1970; Benirschke and
Kaufmann, 1990). The anchoring villi (as the name implies)
physically connect the placenta to the maternal uterine wall.
Although very little is known about the chronology of these
events in man, breakthrough of the cytotrophoblast may
occur as early as day 12 of pregnancy in the rhesus monkey
(Enders et al., 1983).
The invasive cytotrophoblast cells, commonly known as
© European Society of Human Reproduction and Embryology
extravillous trophoblast (EVT) cells, follow two distinct pathways of differentiation (Pijnenborg et al., 1980). In the first
pathway, EVT cells invade the decidualized endometrium, as
well as the inner third of the myometrium. Some of these
EVT cells further differentiate by fusing with one another to
form the giant cells of the placental bed. It is this spatial
confinement of trophoblast cells to the decidualized endometrium and the inner third of the myometrium that distinguish
them from invasive cancer cells (Fisher and Damsky, 1993).
In the second pathway, EVT cells invade the uterine spiral
arteries and adopt a vascular phenotype (Zhou et al., 1997).
This involves replacement of the vascular smooth muscle
cells and endothelium by trophoblast, and the formation of
distensible vessels for the provision of large volumes of blood
to the feto-maternal interface. The initial remodelling of the
uterine spiral arteries seems to involve cells migrating through
the decidua to the target vessels (Pijnenborg et al., 1980), and
occurs within the first trimester of pregnancy. This is limited
to the decidual portion of these vessels. Remodelling of the
inner myometrial portion occurs at 14–18 weeks gestation and
is mediated by intravascular migration of EVT cells (Pijnenborg
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M.Babawale et al.
Figure 1. Placental villus (PV) co-cultured with decidual explant (D) for 24 h. Breakdown of the syncytium (arrow), and invasion by a
leading cytotrophoblast cell (arrowhead) into decidua may be seen. Toluidine Blue stain. Bar ⫽ 100 µm.
Figure 2. Toluidine Blue-stained section of co-cultured placental villi (PV), attached to the decidua (D) after 48 h. Syncytial knot (arrow) is
barely visible with Toluidine Blue stain. Extravillous trophoblast cells (EVT) could not be distinguished from the decidual stroma cells in
this section. Bar ⫽ 100 µm.
Figure 3. From the same tissue as Figure 2, stained with CAM 5.2. A cytokeratin-positive syncytial knot (arrow) and trophoblast cells
(arrowheads) within the decidua are clearly seen. Cytokeratin immunostaining with Toluidine Blue counterstain on a semi-thin section.
Bar ⫽ 100 µm.
Figure 4. Photomicrograph of a decidual explant (D) used as a control. The only cytokeratin-positive cells were in the uterine gland
epithelium (arrows) within the decidual matrix, and no trophoblast cells could be seen. Cytokeratin immunostaining with Toluidine Blue
counter-stain on a semi-thin section. Bar ⫽ 100 µm.
et al., 1980). In some abnormalities of pregnancy such as preeclampsia, trophoblast invasion of the uterine spiral arteries
does not proceed beyond the decidual portions of the spiral
arteries (Brosens et al., 1972; Moodley and Ramsaroop, 1989).
Conversely, in other pathological conditions of pregnancy,
trophoblast invasion of the decidua and the myometrium
becomes excessive, resulting in placenta accreta (Hustin
et al., 1990).
The fate of cytotrophoblast cells in the first trimester ‘floating
villi’ differs from those of the anchoring villi. In the floating
villi, cytotrophoblast cells exist as polarized epithelial monolayers anchored to a basement membrane surrounding a
mesenchymal stromal core containing fetal blood vessels (Boyd
and Hamilton, 1970; Benirschke and Kaufmann, 1990; Aplin,
1991). These cytotrophoblast cells are highly proliferative and
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differentiate to form the outer syncytial covering of the villous
tree (Boyd and Hamilton, 1966; Muhlhauser et al., 1993).
Unlike the anchoring villi, the floating villi do not come in
contact with the uterine wall but are bathed in maternal blood,
thus forming the placental barrier where the exchange of
oxygen, nutrients and waste products occurs between the
mother and the fetus.
Models of the feto-maternal interface (Vicovac et al., 1995;
Babawale et al., 1996) have shown that floating villi can
readily form structures resembling anchoring villi when placed
in contact with appropriate substrates, and these villi retain
the potential to differentiate. This study has assessed the
ultrastructural features of the interface formed in vitro, and
compared these findings with previous studies on the fetomaternal interface of human pregnancy.
Ultrastructure of the early feto-maternal interface
Materials and methods
Tissues
First trimester chorionic placental villi and uterine decidual tissues
were obtained from six patients who had therapeutic termination of
pregnancy at 8–12 weeks gestation. The study was approved by the
ethics committee at Hammersmith Hospital, and informed consent
was obtained from all the patients prior to operation. The products
were collected from the operating theatre, washed in phosphatebuffered saline (PBS) solution, pH 7.2, to remove blood clots and
were transported to the laboratory in 150 ml pots containing warm
sterile culture medium, RPMI 1640 (Sigma-Adrich Company Ltd,
Poole, UK). Both tissues were examined under a dissecting microscope
to ensure that the pieces selected were free from contamination: the
decidua parietalis, for evidence of villous attachment and villous
tissue for evidence of damage or of blood clots. Tissues that appeared
unsatisfactory were not used in the study.
Incubation method
All incubations were carried out in 24-well culture plates (Corning Ltd,
High Wycombe, UK) using RPMI 1640 culture medium supplemented
with 10% fetal calf serum, 2 mmol/l L-glutamine, 100 IU/ml penicillin
and 100 µg/ml streptomycin (Sigma-Adrich). From each of six
patients, four co-cultures were set up, corresponding to 24, 48, 72
and 96 h of culture. Cultures were incubated in an atmosphere of 5%
CO2:95% air at 37°C and set up as follows: a piece of villous tissue
(0.5 mm3) was placed on decidual explant (2 mm3) already plated
on a few drops of culture medium left undisturbed for ~30 min. Both
tissue samples were from the same patient. Thereafter, 2 ml culture
medium was added carefully to each culture chamber. Tissue samples
were removed for processing at 24 h intervals and the culture medium
in the remaining culture chambers was changed daily. Four control
cultures of decidual explants alone (without the placental villi) were
set up from each of the six patients, corresponding to 24, 48, 72 and
96 h of culture as per the experiment.
Tissue processing for transmission electron microscopy (TEM)
Co-cultured samples (24 samples) and control samples (24 samples)
were fixed in 3% glutaraldehyde in cacodylate buffer (pH 7.2) for
30 min, post-fixed in 1% osmium tetroxide in cacodylate buffer (pH
7.2), dehydrated through ascending grades of alcohol and embedded
in Araldite. Semi-thin sections were cut at 1 µm and stained in 1%
Toluidine Blue in order to identify areas of interest for viewing on
the electron microscope. Ultra-thin sections (60–80 nm) were taken
from the selected areas, stained with uranyl acetate and lead citrate.
The stained sections were then examined and areas of interest
photographed using a Hitachi electron microscope, model HU12A,
at an accelerating voltage of 75 kV.
Immunocytochemistry/cytokeratin immunodetection
Immunolabelling of trophoblast cells was carried out with the anticytokeratin antibody CAM 5.2 (International Cancer Research Fund,
London, UK) using the mouse avidin–biotin indirect immunoperoxidase detection method on Araldite-embedded co-cultured placental tissue blocks sectioned at 1 µm thickness. Slides were placed
Figure 5. Electron micrograph showing the syncytial layer of
trophoblast cells (SL) overlying a layer of cytotrophoblast cells
(CT) after 24 h of culture. Areas of apposition are seen between
the CT and SL, which are represented by (* and d) and magnified
as 5* and 5d respectively. Bar ⫽ 5 µm.
Figure 6. A higher magnification of the asterisked area shown in
Figure 5 showing what appears to be an early stage of cell
membrane fusion or a combination of the two, dissolution,
(arrows) between cytotrophoblast cell and the syncytial layer.
Bar ⫽ 500 nm.
Figure 7. A higher magnification of Figure 6 showing what
appears to be an early stage of cell membrane fusion, dissolution,
or a combination of the two (arrows) between cytotrophoblast cell
and the syncytial layer. Bar ⫽ 200nm.
Figure 8. Electron micrograph showing fusion, breakdown or a
combination of the two in the intercellular membranes
(arrows) between cytotrophoblast cell (CT) and the syncytium
(SL). Remnants of the dissolved membranes are seen (*s).
Bar ⫽ 200 nm.
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M.Babawale et al.
Figures 9–14.
Ultrastructure of the early feto-maternal interface
in sodium ethoxide for 30 min to remove the Araldite (etching
procedure) after which endogenous peroxidase activity was blocked
using 3% hydrogen peroxide in tap water for 30 min at room
temperature. The slides were then placed in 0.1% trypsin (SigmaAdrich) in 0.1% CaCl2, pH 7.8 at 37°C for 10 min and then rinsed
in PBS.
Serum blocking was carried out with 5% normal goat serum
for 15 min to eliminate non-specific binding. The tissue sections
were then incubated in CAM 5.2 at 1/10 dilution overnight at
4°C. Non-immune mouse serum was substituted for CAM 5.2 in
tissue sections that served as the negative control. The sections
were thereafter rinsed in 3⫻5 min changes of PBS and incubated
with biotinylated goat anti-mouse secondary antibody (Dako, High
Wycombe, UK) at 1/500 dilution for 1 h. After 3⫻5 min further
changes of PBS, the sections were incubated with peroxidaselabelled streptavidin 1/500 (Boehringer, Lewes, UK) for 1 h. A
final rinse of 3⫻5 min changes of PBS was carried out. Peroxidase
activity was demonstrated by developing the sections in 0.05%
3,3⬘diaminobenzidine solution (DAB; Dako), with 0.03% hydrogen
peroxide as substrate.
Results
Semi-thin histology
After 24 h of co-culture, absence of the syncytium at the
tip of the anchoring villus, cytotrophoblast cell proliferation
and differentiation into EVT cell columns were observed
(Figure 1). The syncytial layer was missing at the interface
between placental villi and decidua, and the syncytial margins
were displaced laterally as the ‘leading’ cytotrophoblast cells
appeared to have commenced invasion into the adjacent
decidual stroma (Figure 1). Cytotrophoblast cells at the
invading front were cytokeratin-positive (micrograph not
shown). No zone of matrix degradation was observed around
the invading trophoblast cells and distant migration of the
EVT cells into the decidual explant had not commenced at
this stage.
After 48 h of co-culture there was a clear column of cells
connecting the placental villi to the decidua (Figure 2),
thus mimicking the anchoring villi seen in vivo in human
placentation. EVT cells were identified in serial sections of
Figure 2 by cytokeratin immunostaining (Figure 3), which
revealed some EVT cells on the outer surface of the placental
villi and within the decidua (arrowheads) and a syncytial knot
(arrow). Fewer cytotrophoblast cells were seen in this location
than are normally present in vivo. This might be because of
the plane of cut of the section, or loss of some of the
cytotrophoblast cells in the placental villi during experimentation, even though blunt forceps were used in placing the villi
and decidual tissues together. The decidual stroma appeared
less dense after 48–96 h of culture than it was after 24 h of
co-culture.
Sections of the decidual explant that had been in culture for
96 h served as controls and were stained for anti-cytokeratin
CAM 5.2. Only cytokeratin-positive cuboidal uterine epithelial
glandular cells were observed. There were no contaminant
EVT cells within the decidual matrix (Figure 4).
Transmission electron microscopy
After 24 hours of co-culture, normal placental villous structures
were still preserved. An outer syncytial layer containing
a nucleus showing perinuclear chromatin condensation was
observed overlying an inner layer of cytotrophoblast cells
(Figure 5). Some areas of the underlying cytotrophoblast cell
plasma membrane were observed showing early signs of fusion
to the overlying syncytium (5* and 5d). Higher magnification
of the interfaces of the plasma membranes of cytotrophoblast
cells and the overlying syncytium (Figures 6, 7 and 8) showed
variable extents of contact and apparent fusion between the
plasma membranes. Areas of apparent membrane degradation
could also be seen (Figure 8).
Cytotrophoblast cells rest on the basal lamina overlying the
fetal mesenchymal core (Figure 9). The fetal mesenchymal
core consisted of a heterogeneous cell population, collagen
fibrils, reticular, mesenchymal and Hofbauer cells and new
blood vessels (micrograph not shown).
EVT cells at the interface with the decidua generally showed
a similar structure throughout the co-culture period of 96 h.
Typical TEM images are shown to demonstrate these points.
EVT cells at the leading edge of the cell column had invaded
the decidual matrix (Figure 10), and showed a pointed morphology. A remnant of the syncytium (S) appears to be in
poor condition, and may have been be damaged during the
remodelling processes of EVT invasion.
Figure 9. Electron micrograph of the same tissue block as in Figure 5 showing the basement membrane (*s) and a cytotrophoblast cell (CT)
under the main layer of cytotrophoblast cells. The basement membrane separates the CT cells from the fetal mesenchymal core (FM).
Bar ⫽ 3.6 µm.
Figure 10. Electron micrograph showing leading cytotrophoblast cells (CT) which had commenced invading the decidual matrix (D) after
48 h of co-culture. The invading front of the leading trophoblast cells appears to be pointed (*s) and the adjacent syncytial cell (S) at the
trophoblast–decidual interface appears to have been damaged as the invading front of CT advanced into the decidua. Bar ⫽ 3 µm.
Figure 11. Electron micrograph showing adjacent extravillous trophoblast (EVT) cells in a cytotrophoblast column and a decidual cell
apparently attached to each other by desmosomal junctions. Strands of intermediate filaments (arrowheads) are associated with the
intercellular desmosomes in adjacent cells, after 48 h of co-culture. These cells contain healthy looking mitochondria (*s). Bar ⫽ 0.2 µm.
Figure 12. Electron micrograph showing the trophoblast–decidual stroma junction (arrowheads), after 72 h of co-culture. The intact
extravillous trophoblast (EVT) cell membrane, surrounding stroma (*s) and the decidual matrix (D) appeared normal. Bar ⫽ 1.0 µm.
Figure 13. Electron micrograph showing a migratory extravillous trophoblast cell (EVT) that had invaded deep into the decidual matrix (D)
after 96 h of culture. A lamellipodic protrusion of the EVT cell (L) into the decidual matrix (D) was observed. Bar ⫽3 µm.
Figure 14. Electron micrograph showing an extravillous trophoblast (EVT) cell with Golgi saccules (g), dilated rough endoplasmic
reticulum (*s), an auto or phagolysosome (central region) and the nucleus (N) after 96 h of co-culture. Bar ⫽1.2 µm.
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M.Babawale et al.
There was also extensive cell–cell contact between the
trophoblast cell column and decidual cells (Figure 11), which
seems to show desmosomes and the associated intermediate
filaments connecting trophoblast and decidual cells. There
were no signs of cellular degeneration in the decidual matrix
(Figure 12), suggesting that the processes of trophoblast
invasion are tightly regulated to prevent this and that the cocultured tissues are viable in culture. Some trophoblast cells
appeared to be very active, with large nucleoli (Figure 13)
indicative of intense biosynthetic activity. The lamellopodic
processes (locomotor apparatus) of some of the active trophoblast cells were observed to have invaded the adjacent decidual
matrix (Figure 13).
The ultrastructural integrity of the EVT cells remained intact
for up to 96 h of co-culture (Figures 11–14), with dilated Golgi,
rough endoplasmic reticulum and healthy looking mitochondria
(Figures 11, 12 and 14). Inclusions of lipid, auto or phagolysosomes and remnants of glycogen granules could also be
seen (Figure 14), all of which suggest that these cells were
metabolically very active.
Discussion
There are many previous studies on human anchoring villi and
the decidua, so the in-vivo structures and their likely functional
correlates have been well described (Enders, 1968; Boyd and
Hamilton, 1970; Jones and Fox, 1991; Jones and Jauniaux,
1995). Our observations generally agree well with the earlier
studies, in that similar features are seen. A critical point is
that the interface in our study was generated by in-vitro coculture of placental villi with decidual explants, whereas in
earlier investigations the same interface formed in vivo. The
similarities will be considered in more detail below, but
the broad agreement indicates that this model is valid for
investigating the feto-maternal interface of human pregnancy.
The validity of these in-vitro models has already been suggested
on the basis of light microscopy studies of co-cultures (Vicovac
et al., 1995; Babawale et al., 1996) and by electron microscopy
of the development of trophoblast cell columns on model
matrices (Aplin et al., 1999).
The tissues comprising the feto-maternal interface showed
the anticipated structures in this study. The decidua contained
no low-molecular weight cytokeratin-positive cells in the
stromal compartment (Figure 4), and the placental villi showed
a multi-layered structure of mesenchymal core, basal lamina,
cytotrophoblast and syncytium (Figures 5 and 9). Differentiation of cytotrophoblast into syncytium appears to involve
fusion, dissolution, or both of the intervening plasma membranes between the cells (Figures 7 and 8). These findings are
consistent with earlier work, and indicate that they occur
in vitro as a dynamic process to maintain the syncytial structural
integrity and function (Enders, 1965; Boyd and Hamilton,
1966; Morrish et al., 1997; Mayhew et al., 1999).
Previous studies have shown that desmosomal proteins are
expressed in human placenta and trophoblast, and these may
be involved in the functional differentiation of human and
guinea pig trophoblast (Firth et al., 1980; Douglas and King,
1990; Winterhager et al., 1999, 2000; Cronier et al., 2001).
1356
The presence of desmosomes within the cell column (Figure
11) suggests that cell adhesion was well maintained between
the cells in culture. There was extensive cell–cell contact
between the trophoblast cell column and decidual cells (Figures
1–3, 11,13). This was particularly apparent in Figure 13, and
seems to show desmosomes and the associated intermediate
filaments connecting trophoblast and decidual cells. Further
studies are needed to confirm this unexpected finding but, if
correct, this is a clear indication of the lack of reaction between
these genetically distinct cells. The expression of integrins
changes within trophoblastic cell columns as they differentiate
and invade either the decidua or the blood vessels (Damsky
et al., 1994; Vicovac et al., 1995; Vicovac and Aplin, 1996)
and it is possible that the desmosomes, in their role in
maintaining adequate cell–cell adhesion, may be involved in
this process.
Previous work has shown that all cytotrophoblast cells
express low-molecular weight cytokeratins (Khong et al., 1986;
Aplin et al., 1999). Not all cytotrophoblast expressed CAM
5.2 in the semi-thin sections used for immunohistochemistry
(Figure 3), which was unexpected, as our earlier work on this
model showed general positive staining (Babawale et al.,
1996). We attribute the apparent incompatibilities to the more
rigorous procedures needed for semi-thin methodology, which
affects the detection limit so that only cells with high levels
of cytokeratin can be visualized.
Differentiation of cytotrophoblast to form cell columns
and invasive EVT was clearly documented (Figures 1–3 and
10–11). The process of trophoblastic cell invasion does not
seem to damage the decidual structures (Figures 10–13), but
rather there is continued healthy contact between invading
trophoblast, decidual cells and decidual matrix. Some of the
factors implicated in regulation have already been identified
(matrix metalloproteinase-2 and -9, tissue inhibitors of metalloprotineases-1 and -2) (Librach et al., 1991; Pollete et al., 1994;
Babawale et al., 1995; Bischof et al., 1998) and the co-culture
model may be a useful technique to explore the roles of these
factors further. Invasion is obviously an active process, and is
consistent with the depletion of glycogen storage as well as
the presence of an active nucleoli and lamellipodic processes
in some of the trophoblast cells observed (Figures 11 and 14).
The nature of the lamellae observed (Figure 12) requires
further investigation, as such structures are not common in
connective tissue.
In summary, our findings indicate that this model has
features at the cellular and sub-cellular levels that resemble
those observed in vivo in human pregnancy, and support our
observations that this model is useful for the study of human
placentation.
Acknowledgements
This work was supported by medical research grants from The
Sir Halley Stewart Trust (Cambridge, UK) to M.O.B. and The Robert
McAlpine Foundation (Kent, UK) to M.H.F.S. The authors express
their thanks to Ms Ann Kane for her assistance with photoimaging
and Trevor Gray for help with some of the electron micrographs.
Ultrastructure of the early feto-maternal interface
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Submitted on July 12, 2001; resubmitted on November 19, 2001; accepted on
January 7, 2002
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